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P1 plasmid replication
J. Mol. Biol. (1986)
192, 275-285
Pl Plasmid Replication Role of Initiator
Titration in Copy Number Control
Subrata K. Pal, Rebecca J. Mason and Dhruba K. Chattorajt Laboratory of Genetics and Recombinant DNA LBI-Basic Research Program NCI-Frederick Cancer Research Facility Frederick, MD 21701, U.S.A. and Laboratory of Biochemistry National Cancer Institute National Institutes of Health Bethesda, MD 20892, U.S.A. (Received 24 March 1986) The copy number control locus incA of unit copy plasmid Pl maps in a region containing nine 19 base-pair repeats. Previous results from studies in vivo and in vitro indicated that incA interacts with the plasmid-encoded RepA protein, which is essential for replication. It has been proposed that the repeat sequences negatively control copy number by sequestering the RepA protein, which is rate-limiting for replication. Our results lend further support to this hypothesis. Here we show that the repeats can be deleted completely from Pl miniplasmids and the deletion results in an approximately eightfold increase in plasmid copy number. So, incA sequences are totally dispensable for replication and have only a regulatory role. The copy number of incA-deleted plasmids can be reduced if incA sequences are present in tram or are reincorporated at two different positions in the plasmid. This reduction in copy number is not due to lowered expression of the repA gene in the presence of incA. We show that one repeat sequence is sufficient to bind RepA and can reduce the copy number of incA-deleted plasmids. When part of the repeat was deleted, it lost its ability to bind as well as influence copy number. These results show a strong correlation between the capacity of incA repeats to bind RepA protein both in vivo and in vitro, and the function of incA in the control of copy number.
1, Introduction A 1.5 kb$ segment of phage Pl specifies a replicon (mini-Pl) that appears to be under the same stringent replication control as the intact 90 kb plasmid prophage Pl (Chattoraj et al., 1985a,b,c). The segment contains a 245 bp replication origin, a 959 bp region that encodes a protein, RepA, required for replication and, finally, a 285 bp
incA, which is responsible for keeping the copy number at the low value characteristic of Pl (Fig. 1). The striking feature of the replicon is the presence of a 19 bp repeat sequence that occurs nine times in the incA locus and five times in the in& locus within the origin (Fig. 1). Extra copies of these repeat sequences can inhibit replication originating in a mini-P1 plasmid, apparently by
t Present address of the authors: Bldg 37, Rm 4D-18, XIH, Bethesda, MD 20892, U.S.A. $ Abbreviations used: kb, 10’ bases or base-pairs; bp, base-pair(s); CmR, chloramphenicol resistant; AmpR, ampicillin resistant; SDS, sodium dodecyl sulfate. &A refers to the region of Pl DNA that contains a part or all of the 9 repeats within 285 bp (see Fig. 1). The region was isolated within a 306 bp fragment having
4 and 17 extra flanking base-pairs (Chattoraj et al.. 1984). The fragment is present in I copy in all our intact incA clones, pALA18, pALA and pALA323, and phage 1DKC177. Unless otherwise indicated, many of our &A-deleted plasmids still retain repeat 9 and 15 out of 19 bp of repeat 8. These include pALABS, p.&LA96, pALA139, pALA169, pALA316, pALA and pRJM362.
region,
275 0022-2836/86;220275-11
X03.00/0
0
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Academic Press Inc. (London)
Ltd.
276
S. K. Pal et al.
I 0
HidI
RI
1
1
Hinlll 1
1
ori
2 kb
I I fepAincI h
inc C
19 bp
repeats
@ e---e
@
2
spacer brd
@
@
2
2
@
12
3
21
22
12
12
12
11
12
Figure 1. The structure of the basic mini-P1 replicon. in& and incA (black rectangles) denote the sets of 5 and 9 repeats. They flank the gene for the RepA protein (stippled arrow). The individual 19 bp repeats (small arrows) are identified by circled numbers 1 to 14. The sizes (in bp) of the spacer sequences between the repeats are shown between the arrows. ori marks the minimal origin region. H&III, HindIII; RI, EcoRI.
sequestering the essential RepA protein (Chattoraj et al., 1984). Binding of RepA to repeat sequences has been demonstrated in vitro (Abeles, 1986). Although
the 14 repeats
are largely
homologous
IllCC
to
each other and all bind RepA, the two sets of repeats apparently play very different roles. Deletion of two of the five incC repeats inactivates the origin, whereas deletion of seven of the nine incA
repeats
leads to a mini-plasmid
with
increases
maximally.
Indeed,
incA is totally
dispensable but the deleted plasmids remain sensitive to incA in tram. Our results are most consistent with the RepA protein being the component of RepA
that interacts
with incA and show a role
binding to incA in the copy number control. Preliminary reports of some of this work have been presented (Chattoraj et al., 1985a,c).
2. Materials and Methods (a) Con&w&m
of incAdeleted
H2
H
R
P2B
H
R
P2 B* X
elevated
copy number (Austin et al., 1985; Chattoraj et al., 1984). So, unlike in&, incA appears to be nonessential for replication but required for its control. It has been postulated that RepA binding to in& initiates replication, whereas binding to incA effectively reduces the availability of RepA for incC. Also, one extra copy of incA but not in& can inhibit replication. Only the incA set of repeats appear to control copy number of the wild-type plasmid. These results encouraged us to ask whether the dispensable for incA sequences are totally replication, and, in that case, whether the copy number
incA
r.?pA
plasmids
Repeats 1 to 7 and a part of repeat 8 of incA were previously deleted by taking advantage of a unique BglI site in repeat 8 (Fig. 2; Chattoraj et al., 1984). From the resultant plasmid pALA139, the remaining incA sequences were deleted with S, nuclease following the
pALA
4.3kb
pALA
4.3kb
P pALA
pALAS
44kb
IH
RH
P2P
.. ,. .,,,,
-a-
',
\
4Okb
Figure 2. Map of Pl DNA involved in plasmid DNA replication and in different plasmids used in this work. The positions of genetic elements incC and incA (black rectangles) and the repA open reading frame (stippled arrow) are shown in the top map. Restriction enzyme sites are abbreviated as follows: B, BgZI; H, HindIII; H2, HincII; P, P&I; PI, PvuI; P2, PvuII; R, EcoRI; X, XmaIII. The asterisk represents an inactivated site. The locations of the sites are given with respect to the arbitrarily assigned 5’ guanosine of the EcoRI site as bp 1999. The thin lines represent pBR322 sequences. Circular plasmid maps are broken at the P&I site at coordinate 3698 of the pBR322 map and drawn as linear maps progressing from left to right instead of clockwise. Plasmids pALA139, pALA and pALAO have been described (Chattoraj et al., 1984). pALA was derived from pALA by converting the XmaIII (X) site to BanzHI site (Barn), see Materials and Methods. (---A---) denotes a deletion.
Control of DNA Replication monolysogens was at least 2 orders of magnitude than from polylysogens.
method of Mizuuchi & Mizuuchi (1980). First, the unique XmaIII site of pALA was converted to a BumHI site with synthetic linkers (Maniatis et al., 1982). The resultant plasmid pALA (Fig. 2) was linearized with HnmHI and digested with S, nuclease at 25°C for 60 min in the presence of 40 mnr-sodium acetate (pH 5-O), 50 mM-NaCl, 1 mM-znc12. About 50 units of S, were added per pg of linear DNA. Any protruding ends that might have been generated were filled in with the Klenow fragment of Pol I and dNTPs before adding BarnHI linkers (Maniatis et aE., 1982). The extent of the deletions was determined by Sanger dideoxy sequencing (P-L Riochemicals, 1981). In order to make the sequences downstream from &A similar in all cases to those in pALA318. the smaller HindIII-BamHI fragment from each of the deleted plasmids was ligated to the larger (3.4 kb) HindIII-BamHI fragment of pALA318. In this way. any possible effect of deletion outside the incA locus was eliminated (see Fig. 3).
(c) Determination
The unique PwuI and XmaIII sites of the &A-deleted plasmid pALA were first converted to BarnHI sites with synthetic linkers; the derivatives were called pALA and pALA318, respectively. The 306 bp incA fragment with BumHI ends was introduced into the BrzmHI sites of the 2 plasmids. Derivatives of pALA bearing 1 copy of the incA fragment in the 2 orientations were called pALA and pALA183, and of pALA were called pALA and pALA185. &A-carrying plasmids were unstable because of low copy number (see below) and absence of partition function (Austin & Abeles, 1983). However, it was possible to characterize the number and orientation of the incA fragments using a radioactive probe by the method of Southern (Maniatis et al., 1982). One copy of incA was introduced into the chromosomal attB site via, a Aim-A phage, 1DKC208, a ~1857 derivative of the previously described AincA phage, 1DKC177 (Chattoraj et al.. 1984). Since the phage was Int-, a i attint+ im.m434 helper phage (G281 of R. A. Weisberg, personal communication) was used to supply the Int protein. Only monolysogens of LineA phage were used and they were detected by the ter assay (Gottesman $ Yarmolinsky. 1968). The yield of phage from 1520 CTC CCC GM
leu pro glu pSPlOa
1540 TAA GTGTGTGCTGGAGGW 9+
lower
of incA sensitivity
Instability of mini-P1 plasmids in the presence of extra copies of incA in trans wss taken as the criterion for incA sensitivity. Extra copies of incA were provided through AmpR vectors, pBR322 or pMF3 (unit copy mini-F vector; Manis k Kline, 1977). DHI, a recA strain carrying the CmR mini-P1 plasmids (pALA or its deletion derivatives, Fig. 3) were transformed with pMF3 or pBR322 carrying incA (pALA and pALA18, respectively) and grown overnight under Amp select,ion to allow segregation of the unselected mini-P1 plasmids in the colony. To determine the fraction of the cells in the colony that retained the mini-P1 plasmids, 8 independent colonies were streaked out for individual cells under Amp selection only. Colonies that grew from these individual cells were then transferred with sterile toothpicks to L agar and L agar containing chloramphenicol. Growth on the 1st plate, but not on the 2nd, indicated loss of the mini-P1 plasmid, and growth on both the plates indicated retention of the mini-P1 plasmid in the presence of incA.
(b) Reincorporation of incA repeats (1 to 9) into irwA-tkleted plavmids or into the chromosome
pAlA
277
(d) Measurement of copy number The copy number of the various ineA-deleted CmR plasmids was determined with respect to a compatible AmpR pBR322-derived plasmid, pSPl0 (described below), in the same cell. Cells were grown to log phase in L broth containing 2Opg Cm/ml and 100 pg Amp/ml and equal A 590 units of cells from each culture were lysed by the method of Holmes & Quigley (1981). The DNA preparations were purified with RNase and phenol, linearized with HindIII, which cuts the plasmids once. and run on 1% (w/v) agarose gel. The bands were stained with ethidium bromide, photographed and negatives were densitometrically traced. The linearity of densitometric measurements was verified using 3 different known concentrations of DNA. Relative amounts of DNA were obtained by weighing the plasmid bands from the densitometric plot. The internal standard. pSPl0, was derived from pBR322 by deleting the EcoRV-h’rlr1 fragment. The copy number of pSP10 was found to be identical to that of pBR322 using another compatible plasmid. pST52. as an internal standard (Som & Tomizawa. 1982). 1590
AACCGCATTAAWUGATGTC
---mm-
TGCTGCCGGCGGCC
cm----
I
CCGGATCCGG
,
GGCCGACGCG
1520 CTC CCC GAA TM
leu pro glu
l
1520
pSP111
CTC CCC GAA TAA GTGTGTGCTGGA
leu pro glu
psP102
1520 CTC CCC GM
leu pro glu psP115
CTC CCC
+----
Y-----
CCGGATCCGG
TAA GTGT CCGGATCCGG
GGCCGACGCG
GGCCGACGCG
l --9’--
CCGGATCCGG
GGCCGACGCG
leu proI Figure 3. Map of sequences around the incA locus of &CA-deleted plasmids. The amino acids at the carboxy terminus of repA, the termination codon (*) and identification number of incA repeats underlie the sequences. Arrows represent the intact repeat 9 and broken lines represent incomplete repeat sequences 8’ or 9’. BarnHI linker sequences are shown in boxes. Also shown (in italics) are pBR322 sequences following the BarnHI linkers. In the case of pALA318, there are also 5 bases of pBR322 DNA (C-G-G-C-C) p receding the linker. Pl map co-ordinates appear at the top of sequences. Sequence hyphens are omitted for clarity.
278
S. K. Pal et al.
The copy number of low copy plasmids was determined by blotting cell lysates and using a radioactive probe. Log phase cultures were exhaustively lysed as follows. Cells were first resuspended in 50 mw-Tris. HCI (pH 8-O). 50 mM-EDTA at a density of about 109/ml and were frozen at -70°C. After thawing, lysozyme was added to 1 mgjml and the suspensions were incubated at room temperature for 10 min, then SDS was added to 1% (w/v). The viscosity of the solution was reduced by sonication at 50 W for 5 a. Next, proteinase K was added to 0.25 mg/ml and the solution, after incubation for 1 h at 45”C, was extracted once with an equal volume of phenol, once with phenol/chloroform (1 : 1, v/v) and finally with chloroform. The aqueous phase was removed to a fresh tube to which NaOH was added to 0.3 M and incubated at 65°C for 1 h to hydrolyze RNA and denature DNA. The solution was neutralized by adding an equal volume of 2 M-ammonium acetate. The solution was diluted &fold for loading into the blotting apparatus (Manifold II; Schleicher t Schuell) or 30-fold for measuring absorbance at 270nm, the absorbance maximum, to compare recovery among different cultures. All dilutions were made with 1 M-ammonium acetate, 0.02 iv-Pu'aOH before use: 100~1 of diluted solutions (equivalent to about 5 x lo6 cells) was loaded per slot. Following filtration, the filter (BA85) was baked at 80°C for 2 h and prehybridized for 30 min at 42°C in a solution containing 50% (v/v) 50 m&r-HEPES (pH 7-O), 1 x Dendhardt’s formamide, solution (Denhardt, 1966) 3 x SSC (SSC is 0.15 M-N&l, 0.015 M-trisodium citrate, pH 7), 15 ,ug of denatured salmon sperm DNA/ml and 20 pg of yeast tRNA/ml. The nick-translated HindIII-BumHI fragment of pSP102 containing the repA gene was added at 1 x 10’ to 2 x 10’ cts/min per ml and hybridization was continued for 16 h at 42°C with rocking. The filters were then washed at 65°C following Maniatis et al. (1982, p. 388). Dried filters were autoradiographed with Kodak XAR-2 film for 12 h at room temperature. The intensity of the bands was quantitated with a Zeineh soft laser scanning densitometer.
3. Results (a) incA repeats are totally dispensable for mini-P1 replication We
previously
obtained
a high
copy
number
mutant of mini-P1 by deleting most of the incA repeats and argued that incA is not essential for replication but required for its control (Chattoraj et al., 1984). The plasmid, pALA139, still retained repeats 9 and part of 8. One could, therefore, expect of the incA it to retain residual characteristics locus. Here we show that these remaining incA repeats are not essential for mini-P1 replication by constructing deletions further into the repeats (Fig. 3). We show also that, although not required for replication, the sequences are still able to control replication. The four deletions described in Figure 3 either retain only the repeat 9 (pSPlOS), or parts of repeat 9 (pSP111 and pSPlO2) or no incA
(e) RepA-DNA binding assay in,cA DNA fragments were obtained from the various ineil-deleted plasmids by restricting at the PwuII site at co-ordinate 1376 (Fig. 2) and at the BumHI linkers at the end of incA (Fig. 3). The fragments were end-labeled with [y-32P]ATP according to Maxam & Gilbert (1980). Increasing amounts of purified RepA (a generous gift from A. Abeles) were added to a mixed amount of labeled DNA. Binding reactions were carried out in 20 1.11of binding buffer (20 mM-Tris. HCl (pH 8*0), 40 mm-KCl, 10 mM-MgCl,, 1 mu-dithiothreitol, 0.1 mru-EDTA, and 7.5ng of bovine serum albumin/ml). Protein was added to the side of the Eppendorf tubes and the reaction was started by briefly spinning the tubes in a microfuge. The binding reaction was allowed to proceed at room temperature for 15 min and was stopped by addition of 4 ~1 of gel loading buffer (20 mr&-Tris. HCl (pH %O), 40 m&r-KCl, 10 m&r-MgCl,, 0.1 mM-EDTA, 0.1% (w/v) bromophenol blue, 50% (v/v) glycerol): 10-/d portions were immediatley loaded on a 5% (w/v) polyacrylamide gel that had been equilibrated overnight in Peacock buffer. A volume (50~1) of a 20 mM-NaCl solution in Peacock buffer was added to each gel slot prior to loading. The gel was run at 10 mA for 4 h, dried and autoradiographed with Kodak XAR-5 film without intensifying screen for 4 to 5 h. Other details of the binding reactions have been described (Abeles, 1986).
Figure 4. Copy number measurement of &A-deleted plasmids. DNA was isolated from different plasmidcarrying cells as described in Materials and Methods, linearized with Hind111 and run on a 1% agarose gel at 75V for 8 h. Linearized pALA DNA was run in lane a as a length marker. Lanes b to-f represent DNA from doubly transformed cells, the upper band representing different mini-P1 plasmids as identified in the top of the gel and the lower band pSPl0 DNA used here as an internal standard. The gel was stained with ethidium bromide, photographed and the negative was scanned in a densitometer to determine copy number.
Control of DNA Replication repeat sequences at all (pSPl15). In case the deletions affected RepA production in cis, the plasmids were initially obtained by transformation of DHl cells (Maniatis et al., 1982) carrying a RepA complementing plasmid pALA (Chattoraj et al., 1984). However, all the four deletion derivatives of pALA were found to be capable of transforming DHl cells equally efficiently without pALA69. It should be noted that pSP115 lacks the entire incA repeat sequences and the carboxy-terminal amino of RepA and is replication acid, glutamine, proficient. One can, therefore, conclude that incA is dispensable for the replication of mini-P1 plasmids.
279
(b) Complete deletion of incA leads to maximal increase in copy number The copy number of pALA with more than seven of the nine incA repeats deleted was already elevated several-fold over that of mini-P1 plasmid with the intact incA locus, l-P1 : 5R (Chattoraj et al., 1984). In order to determine whether further deletions into incA repeats can raise the copy number even more, we compared the copy number of each of the deleted plasmids to that, of pALA (Fig. 4). Within limits of experimental error, the copy number of pSP108 that retains only repeat 9
(a) Figure 5. Autoradiograph of slot-blots to determine relative plasmid copy numbers. Identically treated crude lysates from various plasmid-carrying cells were loaded in duplicate in adjacent columns. Where indicated, dilutions have been made with extracts from cell cultures without any plasmids, so that the cell mass loaded per slot remains unchanged. (a) Cell extracts (NlOO) without any plasmids do not hybridize with the mini-P1 repA fragment probe under the experimental condition (row 1). Cell extracts contained l-P1 : 5R in row 2, I-P1 : 5R at chromosomal ZozB site in row 3, PI Cm in row 4, pALA in rows 5 to 7 with 0, 2 and 4-fold further dilutions, respectively, and pSP102 in rows 8 to 11 with 0, 2. 4 and &fold dilutions, respectively. Integration of I-P1 : 5R at the ZmB site has been described (Chattoraj et al., 1984). I-P1 : 5R, rl-Pl : 5R at ZozB and PlCm are used here as standards for “unit” copy plasmid. The relative copy numbers were 1, 1.4kO.3, l-2+0*3, 3.4kO.4 and 84f 1.6 for I-P1 : 5R, I-P1 : 5R at ZOZB,PlCm, pALA and pSP102, respectively. from at least 4 independent measurements. (b) Reduction in copy number of pALA by incA in cis or in trans. Blots of the pALA derivative with a BumHI linker at the XmaIII site (pALA318) are shown with 2 and 4-fold dilutions in rows 12 and 13, respectively. Derivatives of pALA (pALA derivative with a BamHI linker at the PvuI site) bearing an intact incA are called pALAl and pALA (rows 14 and 15, respectively) and of pALA are called pALA and pALA in the 2 orientations (rows 16 and 17, respectively). Row 18 is a blot of pALA but is derived from cells that had a single integrated AincA prophage. Row 19 is a 2-fold dilution of row 18, and row 20 is a repeat of row 12. Before DNA isolation, the cultures were assayed for mini-P1 plasmid-free cells because, even under selection, the &CA-carrying plasmids could not be maintained stably (see Results). After accounting for the plasmid-free cells, the reduction in copy number due to incA in cis was at least 5-fold (rows 14 to 17) and about P-fold with incA in tram (rows 18 to 19).
280
S. K. Pal et al.
was the same as that of pALA318. However, when the integrity of the repeat 9 was affected as in pSPl11 and pSP102, the copy number increased at least twofold in either case. Thus with respect to the wild-type plasmid I-P1 : 5R, the increase in copy number due to partial and total incA deletion was 3.4 and S-4-fold, respectively (Fig. 5). These observations indicate that even a single repeat or, to be more precise, repeat 9 can have an effect on the control of copy number of mini-Pl. Also, it appears that in order to have an effect on the copy number, the repeat needs to be intact. (c) incA-deleted plasmids are still sensitive to incompatibility exerted by incA
derivatives were about equally unstable, even in the presence of selection (loss frequency was about 55% in overnight cultures). Under selection, the copy number of the plasmids was reduced due to incA insertions at least fivefold (Fig. 5(b)). The reduction in copy number in cells carrying one copy of integrated AincA prophage was about twofold (Fig. 5(b)) and the loss frequency was only about 15% in overnight cultures. So, the incA fragment was more active in cis than it was in trans. Similarly, a 1.3 kb PvuII fragment from pALA containing repeats 9 and part of 8 was inserted at the PvuI site of pSP102 in either orientation to give plasmids pSP121 and pSP122. Such insertions brought down the copy number of pSPlO2 to that of pALA318, i.e. about twofold (Fig. 6(c)). On the
Like the intact Pl, mini-P1 plasmids are destabilized by one or more extra copies of incA of incA(Chattoraj et al., 1984). The viability deleted plasmids permitted us to inquire whether incA itself is the site of action of an incAdetermined regulation. Sensitivity to incA was determined as described in Materials and Methods, section (c). Briefly, the cells carrying the various incA-deleted plasmids were transformed with incAcarrying plasmids and the presence of only the transforming plasmid was selected. Loss of the unselected (incA-deleted) plasmids indicated incA sensitivity. The primary conclusion from these studies is that the incA-deleted plasmid was still sensitive to incA in trans, independent of the incA effector used (Table 1). Thus, an effect of incA is exerted on a component of the replicon that is outside the incA region. One copy of the incA fragment was also active in cis. When inserted in pALA at two novel locations of the plasmid (Fig. 2, Pl and X), the
Table 1 Effect of incA in trans on the stability of incA-deleted plasmids Mini-P 1 plasmids pALA
Incompatibility exerting plasmids pMF3 pALA
Fraction of cells retaining mini-P plasmids after 25 generationst 96/96
= pMF3 +incA(l-9)S
pBR322 pALA = pBR322 + &A( 1-9)
O/96 96/96 O/96
pSPIO8
pMF3 pALA pBR322 pALA
96/96 O/96 96/96 O/96
pSP102
pMF3 pALA pBR322 pALA
96/96 O/96 96/96 O/96
t Twelve colonies from 8 independent transformants were examined in each experiment. 2 pALA is derived from the unit copy vector pMF3 by inserting 1 copy of intact &A. pALA is a pBR322 derivative containing 1 copy of intact incA (Chattoraj et al., 1984).
Figure 6. Reduction in copy number of pSPlO2 by incorporation of &A repeats 9 and 8’ in cis. These repeats are naturally present in pALA (lane a) and are inserted into the PvuI site of pSP102 in either orientation to form pSPl21 and pSP122 (lanes b and c). The lower band is the internal standard pSPl0 in all cases. The copy
number of the plasmids was determined described
in the legend
to Fig. 4.
using the method
281
Control of DNA Replication other hand, the same repeat sequences when present in trans in the multicopy plasmid pALA (Fig. 2) reduced the copy number of both pALA and pSP102 by about 20% only (data not shown). This indicates that incA is more active in cis than in trans. In summary, it appears that incA sequences in bans and, somewhat more effectively, in cis can lower the copy number of mini-P1 plasmids and cause their eventual loss. (d) Binding of RepA protein to a single incA repeat in vitro Specific binding of RepA to incA repeat sequences have been demonstrated earlier in vitro and the evidence was used to support the model that RepA binding to incA effectively reduces the concentration of the free protein, which in turn limits replication. Since affecting the integrity of repeat 9 increased copy number, we expected that
(a) b
pSPlll
pSPlO8
pALA
a
an intact repeat 9 would bind RepA in vitro but the partially deleted repeat would not. Figure 7 shows the results of such an experiment. Binding of protein to DNA was assayed as described in Materials and Methods. This assay relies on the different electrophoretic mobilities of protein-DNA complexes and free DNA in a polyacrylamide gel. The BarnHI-PvuTI fragment from pALA containing repeat 9 and 15 out of 19 bp of repeat 8 gave rise to two discrete retarded bands and a band at the top of the gel in the presence of RepA (Fig. 7(a), lane b). At higher protein to DNA ratios, more DNA remained at the top of the gel, indicating that more non-specific binding or aggregation was occurring (Fig. 7(a), lanes c and d). The corresponding fragment from pSP108, which contains only repeat 9? gave rise to only one retarded band. In the case of pSPll1 and pSPlO2, which do not have an intact repeat, no discrete retarded bands appeared. It appears that a single
(cl
(b) C
d
a
b
pSPIO2
c
d
0
b
(d) c
d
a
b
c
d
Figure 7. Binding of RepA protein to &CA DNA. A Ba?nHI--Pv~11 fragment was obtained from the &A region of the plasmids identified in the top of the Figure (see also Fig. 3) and labeled with [y-“P]ATP (see Materials and Methods). The size of the fragment varied from 202 to 156 bp depending on the extent of incA deletion. RepA-DNA complexes were formed by adding purified protein to a final concentration of 0, 0.2, 0.4 and 0.7 pg/ml (lanes a to d, respectively) of the binding buffer containing a fixed amount of the DNA fragment, run on a 5% polyacrylamide gel and autoradiographed. RepA concentration in each case increases from left to right.
S. K. Pal et al.
282
repeat is capable of specific binding to RepA in the absence of any possibility of co-operative DNAprotein interactions and that when the last repeat was reduced by 6 bp, specific RepA binding was severely reduced. (e) incA does not affect repA expression in trans In this section we consider an alternative hypothesis that incA could limit replication by interfering with repA expression. By fusing different regions of mini-P1 to easily assayable genes galK or la&, we previously localized the promoter of repA between co-ordinates 473-598 (around repeat 11 of incC, Fig. 1). We showed also that repA expression could be reduced severely in the presence of RepA protein supplied in trans from compatible plasmids (Chattoraj et al., 1985b,c). We take advantage of the fusion strains again to check whether incA has any regulatory activity on repA expression. Table 2 shows that, in contrast to RepA, there is no significant effect of incA in trans on repA transcription as seen from galK fusion plasmid pALA and/or translation as seen from 1acZ fusion plasmids pALA326. It should be noted that pALA has the promoter, translation initiation signals and the first 62 NH,-terminal codons of repA fused in-frame to la&, and that the activity of the repA-la& fusion gene is dependent on both the transcription and translational signals of repA (Chattoraj et al., 1985a,b,c; Abeles, 1986). In order to rule out any artifact that might have resulted from studying the regulation in multicopy vectors, the fusion was transferred from pALA to a 1 vector, RRZ5 (vector constructed by R. Zagursky & M. Berman,
Transcription/translation
Plasmid Name
Cloned gene
pST52 pALA pALA
incAl repAS
personal communication), and integrated into the chromosome at attB. Once again, while RepA from unit copy mini-Pl, I-P1 : 5R, caused a marked decrease in fi-galactosidase activity, the presence of incA in multiple copies as in pALA or in a single copy as an integrated prophage I-P1 : 5R repA at 1ozB (Chattoraj et al., 19853) produced no alteration in repA expression (data not shown). So, binding to RepA seems to be the only function of incA so far. It remains to be seen whether incA has any retroregulatory activity on repA in its natural context. (f) An assay for RepA-incA
interaction in vivo
The efficient regulation of repA-1acZ fusion gene by RepA in trans provided us with a means of analyzing RepA-incA interaction in vivo. We argued that if RepA binds to both the repA promoter PrepA and incA, as has been found in vitro, then extra copies of incA repeats should compete with RepA binding to PrepA and hence increase the activity of the fusion gene. The design of the experiment is shown in Figure 8. The fusion gene is integrated into the chromosome via a A vector, 1RZ5. In the presence of a constitutive source of RepA from pRJM362, the activity of the fusion gene was reduced from 169 to 62 Miller units. when various amounts of incA In addition,
-
PrepA 1 t-1
Table 2 of repA is not affected by incA
Galactokinaae units from NlOO(pALA174)t 192 192 3
/3-Galactosidase units from SE5OOO(pALA326)? 3870 4200 2
t A 245 bp fragment of Pl (co-ordinates 366-610) containing the promoter for repA was cloned into pKO6 to generate the plasmid pALA174. A ReaI-P&I fragment of Pl (co-ordinates 287-849) was fused in-frame to k&Z of pMLB1034 to generate the plasmid pALA326. Other details of plasmid construction have been described (Cbattoraj et al., 1985b). Galactokinase measurements were made in gaZKtreeA3 strain NlOO (McKenny et al., 1981) and /%galactosidase in A(avF-Zac) U169 recA56 strain SE5000 (Silhavy et al., 1984). $ The incA fragment (co-ordinates 15051811) was obtained from pALA (Chattoraj et al., 1984) after digestion with BarnHI and the repA fragment (co-ordinates 606-1569) from pALA (Chattoraj et al., 198%) after digestion with BamHI and H&II (the enzyme sites are present in flanking pBR322 sequences). Addition of BamHI linkers to the HincII end permitted cloning of the 2 fragments individually to the BglII site of pST52 (Som & Tomizawa, 1982) to generate pALA and pALA169, respectively.
Figure
8. Representation in
of the experiment
RepA-incA
interaction
vivo.
experiment
is the observation
The
that
b&sis
to test of
the
RepA protein
efficiently shuts off the activity of PrepA and, hence, of the repA-1acZ fusion gene. The fusion gene was integrated into the chromosome via the vector IRZ-5 (R. Zagursky 6 M. Berman, unpublished results). RepA was produced from pRJM362, which is similar to pALA (Table 2), except that repA transcription from the bla-p2 promoter was reduced by insertion of 2 transcription terminators T2 (hatched areas) between the promoter and the gene (stippled arrow; Chattoraj et al., 19853). Use of a low constitutive source of RepA was necessary to increase the sensitivity
of the test.
In order
to check
whether
RepA
binding to PrepA (located in in& repeats) can be competed with incA repeat sequences (black areas), they were introduced with a compatible plasmid pBR322. The cloning
was done at the BamHI
site in all cases.
Control of DNA Replication
Table 3 Relief by incA of RepA repression of PrepA pBR322 derivatives carrying ineA Identification no. of repeatsin the clone Name pALA pALA pALA pRJM354 pRJM355 pRJM356 pBR322
fi-Galactosidase activity from hepA-lacZ in the presence of pRJM362
l-9 449 7-9 ‘8-9 9 ‘9
131.6& 4.5t 116.1510.9 113.0f 6.5 95.2* 5.0 92.5+ 8.0 57.75 3.6 61.9& 6.4
pALA18. pALA and pALA have been described (Chattoraj et al., 1984). pRJM354, pRJM355 and pRJM356 are identical, except that the &A region carried on a Sau3A fragment was obtained from pALA318, pSPlO6 and pSP111, respectively (see Fig. 3). One end of the Sau3A fragments was at co-ordinate 1177 and the other at the BamHI linker at the end of ine.A, and the fragment sizes were 401, 372 and 362 bp, respectively.
t Mean f standard deviation from 6 measurements. The derepressed activity of the fusion gene (i.e. in the absence of the RepA producer plasmid pRJM362) was 169 units.
sequences were present in pBR322, there was an expected increase in /l-galactosidase activity (Table 3). The magnitude of the increase was dependent on the concentration of incA repeats. Some activity of incA was seen even from the clone with a single incA repeat but not from the partially deleted repeat (as in pRJM355 versus pRJM356). As stated in the previous section, incA clones do not change repA promoter expression in the absence of RepA, and none of the incA clones studied in this work number of pBR322 within changed copy experimental error (about 8%). These results are consistent with our finding that the integrity of a single repeat was required for it to have an effect on copy number control (see section (b), above).
4. Discussion We attempted to establish two aspects of replication control of mini-P1 plasmids: (1) the control locus incA has only an inhibitory role in DNA replication; and (2) incA binding of the initiator protein RepA has a role in the negative regulation of replication. We have shown earlier that extra copies of incA destabilize mini-P1 plasmids apparently by interfering with DNA replication. On the other hand, partial deletion of the incA locus increased plasmid copy number (Chattoraj et al., 1984). Here we show that complete deletion of the locus is possible and this leads to even further increase in copy number. Thus incA appears to have only an inhibitory role in the plasmid replication. We further provide physical evidence in support of inhibition of replication. Because of the availability of incA-deleted high copy number plasmids we could directly show decrease in plasmid copy number in the presence of
incA. Earlier studies were done with wild-type unit copy plasmids, and inhibition of replication was argued from decreased plasmid stability or cell survival when the latter was made dependent upon mini-P1 plasmid replication (Austin & Abeles, 1983; Chattoraj et al., 1984). Development of the blotting method in this study greatly facilitated measurement of the copy number of low copy number plasmids (see Materials and Methods). RepA apparently binds to all incA repeats (Abeles, 1986). Here we have shown that even a single repeat can bind RepA and influence copy number. When the single repeat was partially deleted to the extent that it lost its binding proficiency, the incomplete repeat also lost its ability to influence copy number. Thus RepA binding to incA was shown to be significant for the copy number control. If incA controls replication by sequestering RepA, then it is easily understood why incA is not essential for incA sensitivity in trans. Since PI plasmids are sensitive to one extra copy of incA, a criterion perhaps expected of a unit copy plasmid, we wanted to check whether in.cA-deleted multicopy plasmids also follow the same rule. One extra copy of in.cA was introduced into cells carrying mini-P1 plasmid, pALA139, by a AincA (Chattoraj et al., 1984). phage , ADKC177 Replication of the phage was blocked by a susP80 mutation and the multiplicity of infection was 0.01, ensuring that most of the cells never contained more than a single llincA copy. When the cells were grown under immunity selection only, at least 90% of the cells in the colony had lost the resident plasmid. Thus, together with the experiments in Table 1, we can be confident that the target of incA maps outside of the locus itself. In this respect, the behavior of plasmids derived from the unit-copy sex factor F is very similar. The organization of the plasmid maintenance region of PI and F is strikingly similar, although the DNA sequence of the two plasmids show little homology (Abeles et al., 1984). In mini-F also, one set of repeats (called incC) could be deleted entirely without impairing its autonomous replication ability, and such deletion also elevated plasmid copy number (Tsutsui et al., 1983; Tolun & Helinski, 1981). Moreover, the target of the control locus in.& has been argued in mini-F to be the plasmid-encoded initiator protein E (Rokeach et al., 1985). Thus there seems to be functional similarity of the plasmid-encoded elements as well. In fact, a similar situation appears to exist in several other replicons that have repeat sequences and plasmid-specified replication proteins in them, like Rtsl (Terawaki t Itoh, 1985), R6K (Germino & Bastia, 1983; McEachern et al., 1985), RK2 (Stalker et al., 1981), pSClO1 (Locke & Bastia, 1985). In contrast, there are other well-studied plasmids like ColEl (Tomizawa & Itoh, 1982), Rl (Light & Molin, 1983), NRl (Womble et al., 1985), pT181 (Kumar & Novick, 1985), where the control of copy number is primarily mediated by one or two small RNA molecules. These small RNA molecules
S. K. Pal et al. interact with complementary sequences of longer RNA molecules involved with primer formation (ColEl) or expression of an initiator protein in the other cases. In other words, the target of the regulatory molecules is encoded by its own site of synthesis. Thus there appears to be two rather diverse mechanisms of achieving the same goal. Our results help define one of the boundaries of the incA locus. From previous studies it appeared that the repeats alone constitute the incA locus (Chattoraj et al., 1984). Here we show that, in order to be effective, repeat 9 needs to be intact. At the other end, the in.eA clones had only four extra basepairs beyond repeat 1. So, the bounds of incA may not extend beyond the repeat sequences. Although the activity of incA in vivo was dependent on the concentration of the repeats, the exact relation between the number of repeats and copy number remains to be determined. Also, it is not obvious why incA sequences are more active in cis. If RepA binding to repeats alters the physical state of DNA, as has been demonstrated in the case of R6K (Mukherjee et aZ., 1985), we expect to see some phenotype of incA only when present in cis. In that case, simple titration may not be the only role of incA. It should be noted that, even in the total absence of &A, plasmid copy numbers are still controlled, although at an elevated level. According to the titration model, at the high copy number, incC titrates RepA and makes it rate-limiting. Although not as active as incA in one copy, in&! inhibitory activity can be seen when present in only three extra copies (unpublished observations). Since the incA-deleted plasmids have a copy number higher than three, incC could very well be the control element. The apparent weakness of in.& is not understood. As noted earlier, incC has fewer repeats than incA. The two loci also differ markedly in the length of the spacer sequences between the repeats the repeats (Abeles et al., 1984). In addition, themselves can differ among each other by a few base-pairs. An alternative mechanism for the control of replication in the absence of incA could be the negative regulatory role of the RepA protein when present in high concentration (Chattoraj et al., 1985a,b,c). Indeed, there is considerable increase in RepA concentration in cells carrying incA-deleted plasmids (unpublished observation). It remains to be seen what actually limits the rate of replication in the absence of incA. Although we provide additional evidence in this paper in favor of sequestration of RepA as the incA function, a major difficulty remains. These results are difficult to reconcile with the fact that RepA is an autoregulated protein (Chattoraj et aZ., 19856). Depletion of free RepA by sequestration should trigger new RepA synthesis, as shown in Results, the effect of section (f), and would nullify sequestration. A resolution of this dilemma involving two forms of RepA has been proposed (Trawick & Kline, 1985), and we are in the process of studying whether this solution is applicable to Pl
The authors are particularly indebted to Ann Abeles for help with the DNA sequencing and the gel binding experiments, and to our other colleagues, Michael Yarmolinsky, Stuart Austin and Egon Hansen for many helpful discussions. Ann Abeles also helped us write the paper. Research at Frederick was sponsored in part by the National Cancer Institute, DHHS, under contract no. l-CO-23909 with Litton Bionetics, Inc.
References Abeles, A. L. (1986). J. Biol. Chem. 261, 3548-3555. Abeles, A. L., Snyder, K. M. & Chattoraj, D. K. (1984). J. Mol. Biol. 173, 307-324. Austin, S. & Abeles, A. (1983). J. Mol. Biol. 169, 353-372. Austin, S. J., Mural, R. J., Chattoraj, D. K. & Abeles. A. L. (1985). J. Mol. Biol. 183, 195-202. Chattoraj, D., Cordes, K. & Abeles, A. (1984). Proc. Nat. Acad. Sci., LJ.S.A. 81, 6456-6460. Chattoraj, D. K., Abeles, A. L. & Yarmolinsky, M. B. (1985u). In Plasmids in Bacteria (Hollaender, A., ed.), pp. 355381, Plenum Press, New York. Chattoraj, D. K.. Snyder, K. M. t Abeles, A. L. (19856). Proc. Nat. Acad. Sci., Lr.S.A. 82, 2588-2592. Chattoraj, D. K., Pal, S. K., Swack, J. A., Mason, R. J. & Abeles, A. L. (1985c). In Sequence Specificity in Transcription and Translation. UCLA Symp. Mol. Cell. Biol. Ilreu! Ser. (Calendar, R. & Gold. L., eds). vol. 30, pp. 271-280, Alan R. Liss, Inc., New York. Denhardt, D. T. (1966). Biochem. Biophys. Res. Commun. 23, 641-646. Germino, J. & Bastia, D. (1983). Cell, 34, 125-134. Gottesman, M. E. & Yarmolinsky, M. B. (1968). Cold Spring Harbor Symp. Quant. Biol. 33, 735-748. Holmes, D. & Quigley, M. (1981). Anal. B&hem. 114, 193-197. Kumar, C. C. & Novick, R. P. (1985). Proc. iVat. Acad. Sci., U.S.A. 82, 638-642. Light, J. & Molin, S. (1983). EMBO J. 2, 93-89. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Manis, J. J. & Kline, B. C. (1977). Mol. Gen. Genet. 152, 175-182. Maxam. A. & Gilbert, W. (1980). Methods Enzymol. 65, 499-560. McEachern. M. J.. Filutowicz, M. & Helinski, D. R. (1985). Proc. Nat. Acad. Sci., LT.S.A. 82, 14861484. McKenny, K., Shimatake, H., Court, D.. Schmeissner. LJ.. Brady, C. & Rosenberg, M. (1981). In Gene Ampli&ation a& Analysis (Chirkijian, J. G. &. Papas, 2, pp. 384-417. T. S.. eds) . vol. Elsevier/North-Holland, Amsterdam. Mizuuchi. M. & Mizuuchi, K. (1980). Proc. Nat. Acad. Sci., U.S.A. 77, 32263224. Mukherjee, S.. Patel. J. & Bastia. D. (1985). Cell, 43, 189197. P-L Biochemicals (1981). Manual for the M13mp7 Cloning and Sequencing Kit, Catalog no. 1510. P-L Biochemicals, Milwaukee, Wis. Rokeach, L. A., Sogaard-Anderson, L. & Molin, S. (1985). J. BacterioE. 164, 1262-1270. Silhavy. T. J., Berman, M. L. & Enquist, L. W. ( 1984). Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Som. T. & Tomizawa. J. (1982). Mol. Gen. Genet. 187, 375-383. Stalker, D. M., Thomas, C. M. & Helinski, I). R. (1981). Mol. Gen. Genet. 181, 8-12.
Control of DNA Replication Terawaki, Y. & Itoh, Y. (1985). J. Bacterial. 162, 72-77. Tolun, A. & Helinaki, D. R. (1981). Cell, 24, 687494. Tomizawa, J. & Itoh, T. (1982). Cell, 31, 575-583. Trawick, J. D. & Kline, B. C. (1985). Plasmid, 13, 59-69. Tsutsui, H.. Fujiyama, A., Murotsu, T. & Matsubara, K. (1983). J. Bmtrriol. 155. 337-344.
Vocke, C. 6 Bastia,
285 D. (1985).
hoc.
Nat.
ilcud.
Ssci.,
U.S.A. 82, 2252-2256. Womble, D. D., Sampathkumar, P., Easton, A. M.. Luckow, V. A. & Rownd. R. H. (1985). J. Mol. Biol. 181, 395-410.
Edited by K. Matsubara
192, 275-285
Pl Plasmid Replication Role of Initiator
Titration in Copy Number Control
Subrata K. Pal, Rebecca J. Mason and Dhruba K. Chattorajt Laboratory of Genetics and Recombinant DNA LBI-Basic Research Program NCI-Frederick Cancer Research Facility Frederick, MD 21701, U.S.A. and Laboratory of Biochemistry National Cancer Institute National Institutes of Health Bethesda, MD 20892, U.S.A. (Received 24 March 1986) The copy number control locus incA of unit copy plasmid Pl maps in a region containing nine 19 base-pair repeats. Previous results from studies in vivo and in vitro indicated that incA interacts with the plasmid-encoded RepA protein, which is essential for replication. It has been proposed that the repeat sequences negatively control copy number by sequestering the RepA protein, which is rate-limiting for replication. Our results lend further support to this hypothesis. Here we show that the repeats can be deleted completely from Pl miniplasmids and the deletion results in an approximately eightfold increase in plasmid copy number. So, incA sequences are totally dispensable for replication and have only a regulatory role. The copy number of incA-deleted plasmids can be reduced if incA sequences are present in tram or are reincorporated at two different positions in the plasmid. This reduction in copy number is not due to lowered expression of the repA gene in the presence of incA. We show that one repeat sequence is sufficient to bind RepA and can reduce the copy number of incA-deleted plasmids. When part of the repeat was deleted, it lost its ability to bind as well as influence copy number. These results show a strong correlation between the capacity of incA repeats to bind RepA protein both in vivo and in vitro, and the function of incA in the control of copy number.
1, Introduction A 1.5 kb$ segment of phage Pl specifies a replicon (mini-Pl) that appears to be under the same stringent replication control as the intact 90 kb plasmid prophage Pl (Chattoraj et al., 1985a,b,c). The segment contains a 245 bp replication origin, a 959 bp region that encodes a protein, RepA, required for replication and, finally, a 285 bp
incA, which is responsible for keeping the copy number at the low value characteristic of Pl (Fig. 1). The striking feature of the replicon is the presence of a 19 bp repeat sequence that occurs nine times in the incA locus and five times in the in& locus within the origin (Fig. 1). Extra copies of these repeat sequences can inhibit replication originating in a mini-P1 plasmid, apparently by
t Present address of the authors: Bldg 37, Rm 4D-18, XIH, Bethesda, MD 20892, U.S.A. $ Abbreviations used: kb, 10’ bases or base-pairs; bp, base-pair(s); CmR, chloramphenicol resistant; AmpR, ampicillin resistant; SDS, sodium dodecyl sulfate. &A refers to the region of Pl DNA that contains a part or all of the 9 repeats within 285 bp (see Fig. 1). The region was isolated within a 306 bp fragment having
4 and 17 extra flanking base-pairs (Chattoraj et al.. 1984). The fragment is present in I copy in all our intact incA clones, pALA18, pALA and pALA323, and phage 1DKC177. Unless otherwise indicated, many of our &A-deleted plasmids still retain repeat 9 and 15 out of 19 bp of repeat 8. These include pALABS, p.&LA96, pALA139, pALA169, pALA316, pALA and pRJM362.
region,
275 0022-2836/86;220275-11
X03.00/0
0
1986
Academic Press Inc. (London)
Ltd.
276
S. K. Pal et al.
I 0
HidI
RI
1
1
Hinlll 1
1
ori
2 kb
I I fepAincI h
inc C
19 bp
repeats
@ e---e
@
2
spacer brd
@
@
2
2
@
12
3
21
22
12
12
12
11
12
Figure 1. The structure of the basic mini-P1 replicon. in& and incA (black rectangles) denote the sets of 5 and 9 repeats. They flank the gene for the RepA protein (stippled arrow). The individual 19 bp repeats (small arrows) are identified by circled numbers 1 to 14. The sizes (in bp) of the spacer sequences between the repeats are shown between the arrows. ori marks the minimal origin region. H&III, HindIII; RI, EcoRI.
sequestering the essential RepA protein (Chattoraj et al., 1984). Binding of RepA to repeat sequences has been demonstrated in vitro (Abeles, 1986). Although
the 14 repeats
are largely
homologous
IllCC
to
each other and all bind RepA, the two sets of repeats apparently play very different roles. Deletion of two of the five incC repeats inactivates the origin, whereas deletion of seven of the nine incA
repeats
leads to a mini-plasmid
with
increases
maximally.
Indeed,
incA is totally
dispensable but the deleted plasmids remain sensitive to incA in tram. Our results are most consistent with the RepA protein being the component of RepA
that interacts
with incA and show a role
binding to incA in the copy number control. Preliminary reports of some of this work have been presented (Chattoraj et al., 1985a,c).
2. Materials and Methods (a) Con&w&m
of incAdeleted
H2
H
R
P2B
H
R
P2 B* X
elevated
copy number (Austin et al., 1985; Chattoraj et al., 1984). So, unlike in&, incA appears to be nonessential for replication but required for its control. It has been postulated that RepA binding to in& initiates replication, whereas binding to incA effectively reduces the availability of RepA for incC. Also, one extra copy of incA but not in& can inhibit replication. Only the incA set of repeats appear to control copy number of the wild-type plasmid. These results encouraged us to ask whether the dispensable for incA sequences are totally replication, and, in that case, whether the copy number
incA
r.?pA
plasmids
Repeats 1 to 7 and a part of repeat 8 of incA were previously deleted by taking advantage of a unique BglI site in repeat 8 (Fig. 2; Chattoraj et al., 1984). From the resultant plasmid pALA139, the remaining incA sequences were deleted with S, nuclease following the
pALA
4.3kb
pALA
4.3kb
P pALA
pALAS
44kb
IH
RH
P2P
.. ,. .,,,,
-a-
',
\
4Okb
Figure 2. Map of Pl DNA involved in plasmid DNA replication and in different plasmids used in this work. The positions of genetic elements incC and incA (black rectangles) and the repA open reading frame (stippled arrow) are shown in the top map. Restriction enzyme sites are abbreviated as follows: B, BgZI; H, HindIII; H2, HincII; P, P&I; PI, PvuI; P2, PvuII; R, EcoRI; X, XmaIII. The asterisk represents an inactivated site. The locations of the sites are given with respect to the arbitrarily assigned 5’ guanosine of the EcoRI site as bp 1999. The thin lines represent pBR322 sequences. Circular plasmid maps are broken at the P&I site at coordinate 3698 of the pBR322 map and drawn as linear maps progressing from left to right instead of clockwise. Plasmids pALA139, pALA and pALAO have been described (Chattoraj et al., 1984). pALA was derived from pALA by converting the XmaIII (X) site to BanzHI site (Barn), see Materials and Methods. (---A---) denotes a deletion.
Control of DNA Replication monolysogens was at least 2 orders of magnitude than from polylysogens.
method of Mizuuchi & Mizuuchi (1980). First, the unique XmaIII site of pALA was converted to a BumHI site with synthetic linkers (Maniatis et al., 1982). The resultant plasmid pALA (Fig. 2) was linearized with HnmHI and digested with S, nuclease at 25°C for 60 min in the presence of 40 mnr-sodium acetate (pH 5-O), 50 mM-NaCl, 1 mM-znc12. About 50 units of S, were added per pg of linear DNA. Any protruding ends that might have been generated were filled in with the Klenow fragment of Pol I and dNTPs before adding BarnHI linkers (Maniatis et aE., 1982). The extent of the deletions was determined by Sanger dideoxy sequencing (P-L Riochemicals, 1981). In order to make the sequences downstream from &A similar in all cases to those in pALA318. the smaller HindIII-BamHI fragment from each of the deleted plasmids was ligated to the larger (3.4 kb) HindIII-BamHI fragment of pALA318. In this way. any possible effect of deletion outside the incA locus was eliminated (see Fig. 3).
(c) Determination
The unique PwuI and XmaIII sites of the &A-deleted plasmid pALA were first converted to BarnHI sites with synthetic linkers; the derivatives were called pALA and pALA318, respectively. The 306 bp incA fragment with BumHI ends was introduced into the BrzmHI sites of the 2 plasmids. Derivatives of pALA bearing 1 copy of the incA fragment in the 2 orientations were called pALA and pALA183, and of pALA were called pALA and pALA185. &A-carrying plasmids were unstable because of low copy number (see below) and absence of partition function (Austin & Abeles, 1983). However, it was possible to characterize the number and orientation of the incA fragments using a radioactive probe by the method of Southern (Maniatis et al., 1982). One copy of incA was introduced into the chromosomal attB site via, a Aim-A phage, 1DKC208, a ~1857 derivative of the previously described AincA phage, 1DKC177 (Chattoraj et al.. 1984). Since the phage was Int-, a i attint+ im.m434 helper phage (G281 of R. A. Weisberg, personal communication) was used to supply the Int protein. Only monolysogens of LineA phage were used and they were detected by the ter assay (Gottesman $ Yarmolinsky. 1968). The yield of phage from 1520 CTC CCC GM
leu pro glu pSPlOa
1540 TAA GTGTGTGCTGGAGGW 9+
lower
of incA sensitivity
Instability of mini-P1 plasmids in the presence of extra copies of incA in trans wss taken as the criterion for incA sensitivity. Extra copies of incA were provided through AmpR vectors, pBR322 or pMF3 (unit copy mini-F vector; Manis k Kline, 1977). DHI, a recA strain carrying the CmR mini-P1 plasmids (pALA or its deletion derivatives, Fig. 3) were transformed with pMF3 or pBR322 carrying incA (pALA and pALA18, respectively) and grown overnight under Amp select,ion to allow segregation of the unselected mini-P1 plasmids in the colony. To determine the fraction of the cells in the colony that retained the mini-P1 plasmids, 8 independent colonies were streaked out for individual cells under Amp selection only. Colonies that grew from these individual cells were then transferred with sterile toothpicks to L agar and L agar containing chloramphenicol. Growth on the 1st plate, but not on the 2nd, indicated loss of the mini-P1 plasmid, and growth on both the plates indicated retention of the mini-P1 plasmid in the presence of incA.
(b) Reincorporation of incA repeats (1 to 9) into irwA-tkleted plavmids or into the chromosome
pAlA
277
(d) Measurement of copy number The copy number of the various ineA-deleted CmR plasmids was determined with respect to a compatible AmpR pBR322-derived plasmid, pSPl0 (described below), in the same cell. Cells were grown to log phase in L broth containing 2Opg Cm/ml and 100 pg Amp/ml and equal A 590 units of cells from each culture were lysed by the method of Holmes & Quigley (1981). The DNA preparations were purified with RNase and phenol, linearized with HindIII, which cuts the plasmids once. and run on 1% (w/v) agarose gel. The bands were stained with ethidium bromide, photographed and negatives were densitometrically traced. The linearity of densitometric measurements was verified using 3 different known concentrations of DNA. Relative amounts of DNA were obtained by weighing the plasmid bands from the densitometric plot. The internal standard. pSPl0, was derived from pBR322 by deleting the EcoRV-h’rlr1 fragment. The copy number of pSP10 was found to be identical to that of pBR322 using another compatible plasmid. pST52. as an internal standard (Som & Tomizawa. 1982). 1590
AACCGCATTAAWUGATGTC
---mm-
TGCTGCCGGCGGCC
cm----
I
CCGGATCCGG
,
GGCCGACGCG
1520 CTC CCC GAA TM
leu pro glu
l
1520
pSP111
CTC CCC GAA TAA GTGTGTGCTGGA
leu pro glu
psP102
1520 CTC CCC GM
leu pro glu psP115
CTC CCC
+----
Y-----
CCGGATCCGG
TAA GTGT CCGGATCCGG
GGCCGACGCG
GGCCGACGCG
l --9’--
CCGGATCCGG
GGCCGACGCG
leu proI Figure 3. Map of sequences around the incA locus of &CA-deleted plasmids. The amino acids at the carboxy terminus of repA, the termination codon (*) and identification number of incA repeats underlie the sequences. Arrows represent the intact repeat 9 and broken lines represent incomplete repeat sequences 8’ or 9’. BarnHI linker sequences are shown in boxes. Also shown (in italics) are pBR322 sequences following the BarnHI linkers. In the case of pALA318, there are also 5 bases of pBR322 DNA (C-G-G-C-C) p receding the linker. Pl map co-ordinates appear at the top of sequences. Sequence hyphens are omitted for clarity.
278
S. K. Pal et al.
The copy number of low copy plasmids was determined by blotting cell lysates and using a radioactive probe. Log phase cultures were exhaustively lysed as follows. Cells were first resuspended in 50 mw-Tris. HCI (pH 8-O). 50 mM-EDTA at a density of about 109/ml and were frozen at -70°C. After thawing, lysozyme was added to 1 mgjml and the suspensions were incubated at room temperature for 10 min, then SDS was added to 1% (w/v). The viscosity of the solution was reduced by sonication at 50 W for 5 a. Next, proteinase K was added to 0.25 mg/ml and the solution, after incubation for 1 h at 45”C, was extracted once with an equal volume of phenol, once with phenol/chloroform (1 : 1, v/v) and finally with chloroform. The aqueous phase was removed to a fresh tube to which NaOH was added to 0.3 M and incubated at 65°C for 1 h to hydrolyze RNA and denature DNA. The solution was neutralized by adding an equal volume of 2 M-ammonium acetate. The solution was diluted &fold for loading into the blotting apparatus (Manifold II; Schleicher t Schuell) or 30-fold for measuring absorbance at 270nm, the absorbance maximum, to compare recovery among different cultures. All dilutions were made with 1 M-ammonium acetate, 0.02 iv-Pu'aOH before use: 100~1 of diluted solutions (equivalent to about 5 x lo6 cells) was loaded per slot. Following filtration, the filter (BA85) was baked at 80°C for 2 h and prehybridized for 30 min at 42°C in a solution containing 50% (v/v) 50 m&r-HEPES (pH 7-O), 1 x Dendhardt’s formamide, solution (Denhardt, 1966) 3 x SSC (SSC is 0.15 M-N&l, 0.015 M-trisodium citrate, pH 7), 15 ,ug of denatured salmon sperm DNA/ml and 20 pg of yeast tRNA/ml. The nick-translated HindIII-BumHI fragment of pSP102 containing the repA gene was added at 1 x 10’ to 2 x 10’ cts/min per ml and hybridization was continued for 16 h at 42°C with rocking. The filters were then washed at 65°C following Maniatis et al. (1982, p. 388). Dried filters were autoradiographed with Kodak XAR-2 film for 12 h at room temperature. The intensity of the bands was quantitated with a Zeineh soft laser scanning densitometer.
3. Results (a) incA repeats are totally dispensable for mini-P1 replication We
previously
obtained
a high
copy
number
mutant of mini-P1 by deleting most of the incA repeats and argued that incA is not essential for replication but required for its control (Chattoraj et al., 1984). The plasmid, pALA139, still retained repeats 9 and part of 8. One could, therefore, expect of the incA it to retain residual characteristics locus. Here we show that these remaining incA repeats are not essential for mini-P1 replication by constructing deletions further into the repeats (Fig. 3). We show also that, although not required for replication, the sequences are still able to control replication. The four deletions described in Figure 3 either retain only the repeat 9 (pSPlOS), or parts of repeat 9 (pSP111 and pSPlO2) or no incA
(e) RepA-DNA binding assay in,cA DNA fragments were obtained from the various ineil-deleted plasmids by restricting at the PwuII site at co-ordinate 1376 (Fig. 2) and at the BumHI linkers at the end of incA (Fig. 3). The fragments were end-labeled with [y-32P]ATP according to Maxam & Gilbert (1980). Increasing amounts of purified RepA (a generous gift from A. Abeles) were added to a mixed amount of labeled DNA. Binding reactions were carried out in 20 1.11of binding buffer (20 mM-Tris. HCl (pH 8*0), 40 mm-KCl, 10 mM-MgCl,, 1 mu-dithiothreitol, 0.1 mru-EDTA, and 7.5ng of bovine serum albumin/ml). Protein was added to the side of the Eppendorf tubes and the reaction was started by briefly spinning the tubes in a microfuge. The binding reaction was allowed to proceed at room temperature for 15 min and was stopped by addition of 4 ~1 of gel loading buffer (20 mr&-Tris. HCl (pH %O), 40 m&r-KCl, 10 m&r-MgCl,, 0.1 mM-EDTA, 0.1% (w/v) bromophenol blue, 50% (v/v) glycerol): 10-/d portions were immediatley loaded on a 5% (w/v) polyacrylamide gel that had been equilibrated overnight in Peacock buffer. A volume (50~1) of a 20 mM-NaCl solution in Peacock buffer was added to each gel slot prior to loading. The gel was run at 10 mA for 4 h, dried and autoradiographed with Kodak XAR-5 film without intensifying screen for 4 to 5 h. Other details of the binding reactions have been described (Abeles, 1986).
Figure 4. Copy number measurement of &A-deleted plasmids. DNA was isolated from different plasmidcarrying cells as described in Materials and Methods, linearized with Hind111 and run on a 1% agarose gel at 75V for 8 h. Linearized pALA DNA was run in lane a as a length marker. Lanes b to-f represent DNA from doubly transformed cells, the upper band representing different mini-P1 plasmids as identified in the top of the gel and the lower band pSPl0 DNA used here as an internal standard. The gel was stained with ethidium bromide, photographed and the negative was scanned in a densitometer to determine copy number.
Control of DNA Replication repeat sequences at all (pSPl15). In case the deletions affected RepA production in cis, the plasmids were initially obtained by transformation of DHl cells (Maniatis et al., 1982) carrying a RepA complementing plasmid pALA (Chattoraj et al., 1984). However, all the four deletion derivatives of pALA were found to be capable of transforming DHl cells equally efficiently without pALA69. It should be noted that pSP115 lacks the entire incA repeat sequences and the carboxy-terminal amino of RepA and is replication acid, glutamine, proficient. One can, therefore, conclude that incA is dispensable for the replication of mini-P1 plasmids.
279
(b) Complete deletion of incA leads to maximal increase in copy number The copy number of pALA with more than seven of the nine incA repeats deleted was already elevated several-fold over that of mini-P1 plasmid with the intact incA locus, l-P1 : 5R (Chattoraj et al., 1984). In order to determine whether further deletions into incA repeats can raise the copy number even more, we compared the copy number of each of the deleted plasmids to that, of pALA (Fig. 4). Within limits of experimental error, the copy number of pSP108 that retains only repeat 9
(a) Figure 5. Autoradiograph of slot-blots to determine relative plasmid copy numbers. Identically treated crude lysates from various plasmid-carrying cells were loaded in duplicate in adjacent columns. Where indicated, dilutions have been made with extracts from cell cultures without any plasmids, so that the cell mass loaded per slot remains unchanged. (a) Cell extracts (NlOO) without any plasmids do not hybridize with the mini-P1 repA fragment probe under the experimental condition (row 1). Cell extracts contained l-P1 : 5R in row 2, I-P1 : 5R at chromosomal ZozB site in row 3, PI Cm in row 4, pALA in rows 5 to 7 with 0, 2 and 4-fold further dilutions, respectively, and pSP102 in rows 8 to 11 with 0, 2. 4 and &fold dilutions, respectively. Integration of I-P1 : 5R at the ZmB site has been described (Chattoraj et al., 1984). I-P1 : 5R, rl-Pl : 5R at ZozB and PlCm are used here as standards for “unit” copy plasmid. The relative copy numbers were 1, 1.4kO.3, l-2+0*3, 3.4kO.4 and 84f 1.6 for I-P1 : 5R, I-P1 : 5R at ZOZB,PlCm, pALA and pSP102, respectively. from at least 4 independent measurements. (b) Reduction in copy number of pALA by incA in cis or in trans. Blots of the pALA derivative with a BumHI linker at the XmaIII site (pALA318) are shown with 2 and 4-fold dilutions in rows 12 and 13, respectively. Derivatives of pALA (pALA derivative with a BamHI linker at the PvuI site) bearing an intact incA are called pALAl and pALA (rows 14 and 15, respectively) and of pALA are called pALA and pALA in the 2 orientations (rows 16 and 17, respectively). Row 18 is a blot of pALA but is derived from cells that had a single integrated AincA prophage. Row 19 is a 2-fold dilution of row 18, and row 20 is a repeat of row 12. Before DNA isolation, the cultures were assayed for mini-P1 plasmid-free cells because, even under selection, the &CA-carrying plasmids could not be maintained stably (see Results). After accounting for the plasmid-free cells, the reduction in copy number due to incA in cis was at least 5-fold (rows 14 to 17) and about P-fold with incA in tram (rows 18 to 19).
280
S. K. Pal et al.
was the same as that of pALA318. However, when the integrity of the repeat 9 was affected as in pSPl11 and pSP102, the copy number increased at least twofold in either case. Thus with respect to the wild-type plasmid I-P1 : 5R, the increase in copy number due to partial and total incA deletion was 3.4 and S-4-fold, respectively (Fig. 5). These observations indicate that even a single repeat or, to be more precise, repeat 9 can have an effect on the control of copy number of mini-Pl. Also, it appears that in order to have an effect on the copy number, the repeat needs to be intact. (c) incA-deleted plasmids are still sensitive to incompatibility exerted by incA
derivatives were about equally unstable, even in the presence of selection (loss frequency was about 55% in overnight cultures). Under selection, the copy number of the plasmids was reduced due to incA insertions at least fivefold (Fig. 5(b)). The reduction in copy number in cells carrying one copy of integrated AincA prophage was about twofold (Fig. 5(b)) and the loss frequency was only about 15% in overnight cultures. So, the incA fragment was more active in cis than it was in trans. Similarly, a 1.3 kb PvuII fragment from pALA containing repeats 9 and part of 8 was inserted at the PvuI site of pSP102 in either orientation to give plasmids pSP121 and pSP122. Such insertions brought down the copy number of pSPlO2 to that of pALA318, i.e. about twofold (Fig. 6(c)). On the
Like the intact Pl, mini-P1 plasmids are destabilized by one or more extra copies of incA of incA(Chattoraj et al., 1984). The viability deleted plasmids permitted us to inquire whether incA itself is the site of action of an incAdetermined regulation. Sensitivity to incA was determined as described in Materials and Methods, section (c). Briefly, the cells carrying the various incA-deleted plasmids were transformed with incAcarrying plasmids and the presence of only the transforming plasmid was selected. Loss of the unselected (incA-deleted) plasmids indicated incA sensitivity. The primary conclusion from these studies is that the incA-deleted plasmid was still sensitive to incA in trans, independent of the incA effector used (Table 1). Thus, an effect of incA is exerted on a component of the replicon that is outside the incA region. One copy of the incA fragment was also active in cis. When inserted in pALA at two novel locations of the plasmid (Fig. 2, Pl and X), the
Table 1 Effect of incA in trans on the stability of incA-deleted plasmids Mini-P 1 plasmids pALA
Incompatibility exerting plasmids pMF3 pALA
Fraction of cells retaining mini-P plasmids after 25 generationst 96/96
= pMF3 +incA(l-9)S
pBR322 pALA = pBR322 + &A( 1-9)
O/96 96/96 O/96
pSPIO8
pMF3 pALA pBR322 pALA
96/96 O/96 96/96 O/96
pSP102
pMF3 pALA pBR322 pALA
96/96 O/96 96/96 O/96
t Twelve colonies from 8 independent transformants were examined in each experiment. 2 pALA is derived from the unit copy vector pMF3 by inserting 1 copy of intact &A. pALA is a pBR322 derivative containing 1 copy of intact incA (Chattoraj et al., 1984).
Figure 6. Reduction in copy number of pSPlO2 by incorporation of &A repeats 9 and 8’ in cis. These repeats are naturally present in pALA (lane a) and are inserted into the PvuI site of pSP102 in either orientation to form pSPl21 and pSP122 (lanes b and c). The lower band is the internal standard pSPl0 in all cases. The copy
number of the plasmids was determined described
in the legend
to Fig. 4.
using the method
281
Control of DNA Replication other hand, the same repeat sequences when present in trans in the multicopy plasmid pALA (Fig. 2) reduced the copy number of both pALA and pSP102 by about 20% only (data not shown). This indicates that incA is more active in cis than in trans. In summary, it appears that incA sequences in bans and, somewhat more effectively, in cis can lower the copy number of mini-P1 plasmids and cause their eventual loss. (d) Binding of RepA protein to a single incA repeat in vitro Specific binding of RepA to incA repeat sequences have been demonstrated earlier in vitro and the evidence was used to support the model that RepA binding to incA effectively reduces the concentration of the free protein, which in turn limits replication. Since affecting the integrity of repeat 9 increased copy number, we expected that
(a) b
pSPlll
pSPlO8
pALA
a
an intact repeat 9 would bind RepA in vitro but the partially deleted repeat would not. Figure 7 shows the results of such an experiment. Binding of protein to DNA was assayed as described in Materials and Methods. This assay relies on the different electrophoretic mobilities of protein-DNA complexes and free DNA in a polyacrylamide gel. The BarnHI-PvuTI fragment from pALA containing repeat 9 and 15 out of 19 bp of repeat 8 gave rise to two discrete retarded bands and a band at the top of the gel in the presence of RepA (Fig. 7(a), lane b). At higher protein to DNA ratios, more DNA remained at the top of the gel, indicating that more non-specific binding or aggregation was occurring (Fig. 7(a), lanes c and d). The corresponding fragment from pSP108, which contains only repeat 9? gave rise to only one retarded band. In the case of pSPll1 and pSPlO2, which do not have an intact repeat, no discrete retarded bands appeared. It appears that a single
(cl
(b) C
d
a
b
pSPIO2
c
d
0
b
(d) c
d
a
b
c
d
Figure 7. Binding of RepA protein to &CA DNA. A Ba?nHI--Pv~11 fragment was obtained from the &A region of the plasmids identified in the top of the Figure (see also Fig. 3) and labeled with [y-“P]ATP (see Materials and Methods). The size of the fragment varied from 202 to 156 bp depending on the extent of incA deletion. RepA-DNA complexes were formed by adding purified protein to a final concentration of 0, 0.2, 0.4 and 0.7 pg/ml (lanes a to d, respectively) of the binding buffer containing a fixed amount of the DNA fragment, run on a 5% polyacrylamide gel and autoradiographed. RepA concentration in each case increases from left to right.
S. K. Pal et al.
282
repeat is capable of specific binding to RepA in the absence of any possibility of co-operative DNAprotein interactions and that when the last repeat was reduced by 6 bp, specific RepA binding was severely reduced. (e) incA does not affect repA expression in trans In this section we consider an alternative hypothesis that incA could limit replication by interfering with repA expression. By fusing different regions of mini-P1 to easily assayable genes galK or la&, we previously localized the promoter of repA between co-ordinates 473-598 (around repeat 11 of incC, Fig. 1). We showed also that repA expression could be reduced severely in the presence of RepA protein supplied in trans from compatible plasmids (Chattoraj et al., 1985b,c). We take advantage of the fusion strains again to check whether incA has any regulatory activity on repA expression. Table 2 shows that, in contrast to RepA, there is no significant effect of incA in trans on repA transcription as seen from galK fusion plasmid pALA and/or translation as seen from 1acZ fusion plasmids pALA326. It should be noted that pALA has the promoter, translation initiation signals and the first 62 NH,-terminal codons of repA fused in-frame to la&, and that the activity of the repA-la& fusion gene is dependent on both the transcription and translational signals of repA (Chattoraj et al., 1985a,b,c; Abeles, 1986). In order to rule out any artifact that might have resulted from studying the regulation in multicopy vectors, the fusion was transferred from pALA to a 1 vector, RRZ5 (vector constructed by R. Zagursky & M. Berman,
Transcription/translation
Plasmid Name
Cloned gene
pST52 pALA pALA
incAl repAS
personal communication), and integrated into the chromosome at attB. Once again, while RepA from unit copy mini-Pl, I-P1 : 5R, caused a marked decrease in fi-galactosidase activity, the presence of incA in multiple copies as in pALA or in a single copy as an integrated prophage I-P1 : 5R repA at 1ozB (Chattoraj et al., 19853) produced no alteration in repA expression (data not shown). So, binding to RepA seems to be the only function of incA so far. It remains to be seen whether incA has any retroregulatory activity on repA in its natural context. (f) An assay for RepA-incA
interaction in vivo
The efficient regulation of repA-1acZ fusion gene by RepA in trans provided us with a means of analyzing RepA-incA interaction in vivo. We argued that if RepA binds to both the repA promoter PrepA and incA, as has been found in vitro, then extra copies of incA repeats should compete with RepA binding to PrepA and hence increase the activity of the fusion gene. The design of the experiment is shown in Figure 8. The fusion gene is integrated into the chromosome via a A vector, 1RZ5. In the presence of a constitutive source of RepA from pRJM362, the activity of the fusion gene was reduced from 169 to 62 Miller units. when various amounts of incA In addition,
-
PrepA 1 t-1
Table 2 of repA is not affected by incA
Galactokinaae units from NlOO(pALA174)t 192 192 3
/3-Galactosidase units from SE5OOO(pALA326)? 3870 4200 2
t A 245 bp fragment of Pl (co-ordinates 366-610) containing the promoter for repA was cloned into pKO6 to generate the plasmid pALA174. A ReaI-P&I fragment of Pl (co-ordinates 287-849) was fused in-frame to k&Z of pMLB1034 to generate the plasmid pALA326. Other details of plasmid construction have been described (Cbattoraj et al., 1985b). Galactokinase measurements were made in gaZKtreeA3 strain NlOO (McKenny et al., 1981) and /%galactosidase in A(avF-Zac) U169 recA56 strain SE5000 (Silhavy et al., 1984). $ The incA fragment (co-ordinates 15051811) was obtained from pALA (Chattoraj et al., 1984) after digestion with BarnHI and the repA fragment (co-ordinates 606-1569) from pALA (Chattoraj et al., 198%) after digestion with BamHI and H&II (the enzyme sites are present in flanking pBR322 sequences). Addition of BamHI linkers to the HincII end permitted cloning of the 2 fragments individually to the BglII site of pST52 (Som & Tomizawa, 1982) to generate pALA and pALA169, respectively.
Figure
8. Representation in
of the experiment
RepA-incA
interaction
vivo.
experiment
is the observation
The
that
b&sis
to test of
the
RepA protein
efficiently shuts off the activity of PrepA and, hence, of the repA-1acZ fusion gene. The fusion gene was integrated into the chromosome via the vector IRZ-5 (R. Zagursky 6 M. Berman, unpublished results). RepA was produced from pRJM362, which is similar to pALA (Table 2), except that repA transcription from the bla-p2 promoter was reduced by insertion of 2 transcription terminators T2 (hatched areas) between the promoter and the gene (stippled arrow; Chattoraj et al., 19853). Use of a low constitutive source of RepA was necessary to increase the sensitivity
of the test.
In order
to check
whether
RepA
binding to PrepA (located in in& repeats) can be competed with incA repeat sequences (black areas), they were introduced with a compatible plasmid pBR322. The cloning
was done at the BamHI
site in all cases.
Control of DNA Replication
Table 3 Relief by incA of RepA repression of PrepA pBR322 derivatives carrying ineA Identification no. of repeatsin the clone Name pALA pALA pALA pRJM354 pRJM355 pRJM356 pBR322
fi-Galactosidase activity from hepA-lacZ in the presence of pRJM362
l-9 449 7-9 ‘8-9 9 ‘9
131.6& 4.5t 116.1510.9 113.0f 6.5 95.2* 5.0 92.5+ 8.0 57.75 3.6 61.9& 6.4
pALA18. pALA and pALA have been described (Chattoraj et al., 1984). pRJM354, pRJM355 and pRJM356 are identical, except that the &A region carried on a Sau3A fragment was obtained from pALA318, pSPlO6 and pSP111, respectively (see Fig. 3). One end of the Sau3A fragments was at co-ordinate 1177 and the other at the BamHI linker at the end of ine.A, and the fragment sizes were 401, 372 and 362 bp, respectively.
t Mean f standard deviation from 6 measurements. The derepressed activity of the fusion gene (i.e. in the absence of the RepA producer plasmid pRJM362) was 169 units.
sequences were present in pBR322, there was an expected increase in /l-galactosidase activity (Table 3). The magnitude of the increase was dependent on the concentration of incA repeats. Some activity of incA was seen even from the clone with a single incA repeat but not from the partially deleted repeat (as in pRJM355 versus pRJM356). As stated in the previous section, incA clones do not change repA promoter expression in the absence of RepA, and none of the incA clones studied in this work number of pBR322 within changed copy experimental error (about 8%). These results are consistent with our finding that the integrity of a single repeat was required for it to have an effect on copy number control (see section (b), above).
4. Discussion We attempted to establish two aspects of replication control of mini-P1 plasmids: (1) the control locus incA has only an inhibitory role in DNA replication; and (2) incA binding of the initiator protein RepA has a role in the negative regulation of replication. We have shown earlier that extra copies of incA destabilize mini-P1 plasmids apparently by interfering with DNA replication. On the other hand, partial deletion of the incA locus increased plasmid copy number (Chattoraj et al., 1984). Here we show that complete deletion of the locus is possible and this leads to even further increase in copy number. Thus incA appears to have only an inhibitory role in the plasmid replication. We further provide physical evidence in support of inhibition of replication. Because of the availability of incA-deleted high copy number plasmids we could directly show decrease in plasmid copy number in the presence of
incA. Earlier studies were done with wild-type unit copy plasmids, and inhibition of replication was argued from decreased plasmid stability or cell survival when the latter was made dependent upon mini-P1 plasmid replication (Austin & Abeles, 1983; Chattoraj et al., 1984). Development of the blotting method in this study greatly facilitated measurement of the copy number of low copy number plasmids (see Materials and Methods). RepA apparently binds to all incA repeats (Abeles, 1986). Here we have shown that even a single repeat can bind RepA and influence copy number. When the single repeat was partially deleted to the extent that it lost its binding proficiency, the incomplete repeat also lost its ability to influence copy number. Thus RepA binding to incA was shown to be significant for the copy number control. If incA controls replication by sequestering RepA, then it is easily understood why incA is not essential for incA sensitivity in trans. Since PI plasmids are sensitive to one extra copy of incA, a criterion perhaps expected of a unit copy plasmid, we wanted to check whether in.cA-deleted multicopy plasmids also follow the same rule. One extra copy of in.cA was introduced into cells carrying mini-P1 plasmid, pALA139, by a AincA (Chattoraj et al., 1984). phage , ADKC177 Replication of the phage was blocked by a susP80 mutation and the multiplicity of infection was 0.01, ensuring that most of the cells never contained more than a single llincA copy. When the cells were grown under immunity selection only, at least 90% of the cells in the colony had lost the resident plasmid. Thus, together with the experiments in Table 1, we can be confident that the target of incA maps outside of the locus itself. In this respect, the behavior of plasmids derived from the unit-copy sex factor F is very similar. The organization of the plasmid maintenance region of PI and F is strikingly similar, although the DNA sequence of the two plasmids show little homology (Abeles et al., 1984). In mini-F also, one set of repeats (called incC) could be deleted entirely without impairing its autonomous replication ability, and such deletion also elevated plasmid copy number (Tsutsui et al., 1983; Tolun & Helinski, 1981). Moreover, the target of the control locus in.& has been argued in mini-F to be the plasmid-encoded initiator protein E (Rokeach et al., 1985). Thus there seems to be functional similarity of the plasmid-encoded elements as well. In fact, a similar situation appears to exist in several other replicons that have repeat sequences and plasmid-specified replication proteins in them, like Rtsl (Terawaki t Itoh, 1985), R6K (Germino & Bastia, 1983; McEachern et al., 1985), RK2 (Stalker et al., 1981), pSClO1 (Locke & Bastia, 1985). In contrast, there are other well-studied plasmids like ColEl (Tomizawa & Itoh, 1982), Rl (Light & Molin, 1983), NRl (Womble et al., 1985), pT181 (Kumar & Novick, 1985), where the control of copy number is primarily mediated by one or two small RNA molecules. These small RNA molecules
S. K. Pal et al. interact with complementary sequences of longer RNA molecules involved with primer formation (ColEl) or expression of an initiator protein in the other cases. In other words, the target of the regulatory molecules is encoded by its own site of synthesis. Thus there appears to be two rather diverse mechanisms of achieving the same goal. Our results help define one of the boundaries of the incA locus. From previous studies it appeared that the repeats alone constitute the incA locus (Chattoraj et al., 1984). Here we show that, in order to be effective, repeat 9 needs to be intact. At the other end, the in.eA clones had only four extra basepairs beyond repeat 1. So, the bounds of incA may not extend beyond the repeat sequences. Although the activity of incA in vivo was dependent on the concentration of the repeats, the exact relation between the number of repeats and copy number remains to be determined. Also, it is not obvious why incA sequences are more active in cis. If RepA binding to repeats alters the physical state of DNA, as has been demonstrated in the case of R6K (Mukherjee et aZ., 1985), we expect to see some phenotype of incA only when present in cis. In that case, simple titration may not be the only role of incA. It should be noted that, even in the total absence of &A, plasmid copy numbers are still controlled, although at an elevated level. According to the titration model, at the high copy number, incC titrates RepA and makes it rate-limiting. Although not as active as incA in one copy, in&! inhibitory activity can be seen when present in only three extra copies (unpublished observations). Since the incA-deleted plasmids have a copy number higher than three, incC could very well be the control element. The apparent weakness of in.& is not understood. As noted earlier, incC has fewer repeats than incA. The two loci also differ markedly in the length of the spacer sequences between the repeats the repeats (Abeles et al., 1984). In addition, themselves can differ among each other by a few base-pairs. An alternative mechanism for the control of replication in the absence of incA could be the negative regulatory role of the RepA protein when present in high concentration (Chattoraj et al., 1985a,b,c). Indeed, there is considerable increase in RepA concentration in cells carrying incA-deleted plasmids (unpublished observation). It remains to be seen what actually limits the rate of replication in the absence of incA. Although we provide additional evidence in this paper in favor of sequestration of RepA as the incA function, a major difficulty remains. These results are difficult to reconcile with the fact that RepA is an autoregulated protein (Chattoraj et aZ., 19856). Depletion of free RepA by sequestration should trigger new RepA synthesis, as shown in Results, the effect of section (f), and would nullify sequestration. A resolution of this dilemma involving two forms of RepA has been proposed (Trawick & Kline, 1985), and we are in the process of studying whether this solution is applicable to Pl
The authors are particularly indebted to Ann Abeles for help with the DNA sequencing and the gel binding experiments, and to our other colleagues, Michael Yarmolinsky, Stuart Austin and Egon Hansen for many helpful discussions. Ann Abeles also helped us write the paper. Research at Frederick was sponsored in part by the National Cancer Institute, DHHS, under contract no. l-CO-23909 with Litton Bionetics, Inc.
References Abeles, A. L. (1986). J. Biol. Chem. 261, 3548-3555. Abeles, A. L., Snyder, K. M. & Chattoraj, D. K. (1984). J. Mol. Biol. 173, 307-324. Austin, S. & Abeles, A. (1983). J. Mol. Biol. 169, 353-372. Austin, S. J., Mural, R. J., Chattoraj, D. K. & Abeles. A. L. (1985). J. Mol. Biol. 183, 195-202. Chattoraj, D., Cordes, K. & Abeles, A. (1984). Proc. Nat. Acad. Sci., LJ.S.A. 81, 6456-6460. Chattoraj, D. K., Abeles, A. L. & Yarmolinsky, M. B. (1985u). In Plasmids in Bacteria (Hollaender, A., ed.), pp. 355381, Plenum Press, New York. Chattoraj, D. K.. Snyder, K. M. t Abeles, A. L. (19856). Proc. Nat. Acad. Sci., Lr.S.A. 82, 2588-2592. Chattoraj, D. K., Pal, S. K., Swack, J. A., Mason, R. J. & Abeles, A. L. (1985c). In Sequence Specificity in Transcription and Translation. UCLA Symp. Mol. Cell. Biol. Ilreu! Ser. (Calendar, R. & Gold. L., eds). vol. 30, pp. 271-280, Alan R. Liss, Inc., New York. Denhardt, D. T. (1966). Biochem. Biophys. Res. Commun. 23, 641-646. Germino, J. & Bastia, D. (1983). Cell, 34, 125-134. Gottesman, M. E. & Yarmolinsky, M. B. (1968). Cold Spring Harbor Symp. Quant. Biol. 33, 735-748. Holmes, D. & Quigley, M. (1981). Anal. B&hem. 114, 193-197. Kumar, C. C. & Novick, R. P. (1985). Proc. iVat. Acad. Sci., U.S.A. 82, 638-642. Light, J. & Molin, S. (1983). EMBO J. 2, 93-89. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Manis, J. J. & Kline, B. C. (1977). Mol. Gen. Genet. 152, 175-182. Maxam. A. & Gilbert, W. (1980). Methods Enzymol. 65, 499-560. McEachern. M. J.. Filutowicz, M. & Helinski, D. R. (1985). Proc. Nat. Acad. Sci., LT.S.A. 82, 14861484. McKenny, K., Shimatake, H., Court, D.. Schmeissner. LJ.. Brady, C. & Rosenberg, M. (1981). In Gene Ampli&ation a& Analysis (Chirkijian, J. G. &. Papas, 2, pp. 384-417. T. S.. eds) . vol. Elsevier/North-Holland, Amsterdam. Mizuuchi. M. & Mizuuchi, K. (1980). Proc. Nat. Acad. Sci., U.S.A. 77, 32263224. Mukherjee, S.. Patel. J. & Bastia. D. (1985). Cell, 43, 189197. P-L Biochemicals (1981). Manual for the M13mp7 Cloning and Sequencing Kit, Catalog no. 1510. P-L Biochemicals, Milwaukee, Wis. Rokeach, L. A., Sogaard-Anderson, L. & Molin, S. (1985). J. BacterioE. 164, 1262-1270. Silhavy. T. J., Berman, M. L. & Enquist, L. W. ( 1984). Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Som. T. & Tomizawa. J. (1982). Mol. Gen. Genet. 187, 375-383. Stalker, D. M., Thomas, C. M. & Helinski, I). R. (1981). Mol. Gen. Genet. 181, 8-12.
Control of DNA Replication Terawaki, Y. & Itoh, Y. (1985). J. Bacterial. 162, 72-77. Tolun, A. & Helinaki, D. R. (1981). Cell, 24, 687494. Tomizawa, J. & Itoh, T. (1982). Cell, 31, 575-583. Trawick, J. D. & Kline, B. C. (1985). Plasmid, 13, 59-69. Tsutsui, H.. Fujiyama, A., Murotsu, T. & Matsubara, K. (1983). J. Bmtrriol. 155. 337-344.
Vocke, C. 6 Bastia,
285 D. (1985).
hoc.
Nat.
ilcud.
Ssci.,
U.S.A. 82, 2252-2256. Womble, D. D., Sampathkumar, P., Easton, A. M.. Luckow, V. A. & Rownd. R. H. (1985). J. Mol. Biol. 181, 395-410.
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