Repair of thymine · guanine and uracil · guanine mismatched base-pairs in bacteriophage M13mp18 DNA heteroduplexes

J. Mol. Biol. (1987) 197, 617-626

Repair of Thymine Guanine and Uracil Guanine Mismatched Base-pairs in Bacteriophage Ml 3mp 18 DNA Heteroduplexes l

Suresh Shenoy I, Kenneth

l

C. Ehrlich2

and Melanie

Ehrlich’

y

‘Department of Biochemistry Tulane Medical School New Orleans, LA 70112, U.S.A. ‘Southern Regional Research Center U.S. Department of Agriculture New Orleans, LA 70179, U.S.A. (Received 23 December 1986, and in revised form 8 June 1987) Repair of thymine. guanine (T . G) and uracil . guanine (U * G) mismatched base-pairs in bacteriophage M13mp18 replicative form (RF) DNA was compared upon transfection into repair-proficient or repair-deficient Escherichia coli strains. Oligonucleotide-directed mutagenesis was used to prepare covalently closed circular heteroduplexes that contained the mismatched base-pair at a restriction recognition site. The heteroduplexes were unmethylated at dam (5’-GATC-3’) sites to avoid methylation-directed biasing of repair. In an E. coli host containing uracil-DNA glycosylase (ung+), about 97% of the transfecting U *G-containing heteroduplexes had the U residue excised by the uracil-excision repair system. With the analogous T. G mispair, mismatch repair operated on almost all of the transfecting heteroduplexes and removed the T residue in about 75% of them when the mismatched T was on the minus strand of the RF DNA. Similar preferential excision of the minus-strand’s mismatched base was observed whether the heteroduplex RF DNA molecules had only one or both strands unmethylated at dcm (5’-CC(A/T)GG-3’) sites and whether the RF DNA was prepared by primer extension in vitro or by reannealing mutant and non-mutant DNA strands. Also, the extent and directionality of repair was the same at a U. G mispair in ung- host cells as at the analogous T. G mispair in ung- or ungf cells. Only in a mismatch repair-deficient (mutH-) host was the plus strand of the transfecting M13mp18 heteroduplex DNA preferentially repaired. It is suggested that the plus strand nick made by the M13-encoded gene II protein might be employed by a mutH- host to initiate repair on that strand.

excision repair system that is normally functional in E. coli. Wild-type E. coli K12 strains contain m5C as a minor base (Hattman, 1981). Methylation of C residues can increase the frequency of compared to the spontaneous mutagenesis unmethylated isogenic E. coli DNA (Coulondre et al., 1978). This increased mutagenesis may result from spontaneous deamination of m5C residues, thereby converting m5C *G base-pairs to T. G mispairs (Coulondre et al., 1978; Duncan & Miller, 1980). This conversion occurs at a higher frequency (Wang et al., 1982; Ehrlich et aZ., 1986) and should be subject to less faithful repair than is characteristic of the C. G to U. G transition (Lindahl, 1979; Ehrlich & Wang, 1981). Although E. coli can correct a T. G mispair by DNA mismatch repair (Claverys & Lacks, 1986),

1. Introduction

Spontaneous deamination of cytosine (C) and 5-methylcytosine (m5C) residues in DNA can cause the formation of uracil . guanine (U. G) and thymine. guanine (T . G) mispairs (Lindahl, 1979; Ehrlich & Wang, 1981; Wang et al., 1982; Ehrlich et al., 1986) at low but biologically significant rates. Escherichia coli mutants deficient in uracil-DNA glycosylase (ung- cells) have a higher frequency of G * C to A . T transition mutations (Duncan et al., 1978; Duncan & Weiss, 1978; Duncan & Miller, 1980). This has been interpreted as the result of spontaneous deamination of C residues yielding U. G mispairs that are not repaired by the uracilt Author to whom correspondence should be sent. 0022%2836/87/200617-10$03.00/O

617

(Q 1987 Academic Press Limited

8. A’henoy et al

618

previous studies suggest that this mismatch repair generally has little or no intrinsic bias for excising the T residue rather than the G residue (Kramer et al., 1984; Dohet et al., 1985). This is in contrast to uracil-DNA glycosylase, a virtually ubiquitous enzyme that specifically excises any Ir residue including one in a U .G mismatch (Lindahl, 1979). In E. coli, t#he DNA mismatch repair pathway is normally directed towards one strand or the other by dam (DNA adenine methylase) methylation (GAT(’ to Gm6ATC; m6A. ilr6-methyladenine: Nevers &, Spatz. 1975; Rydberg, 1978; Wildenberg & Meselson, 1975). It is much reduced when both DNA strands are dam-methylated. as is usually the case in mature E. coli DNA (Pukkila et al., 1983; repair Dohet et al., 1985). E. coli DNA mismatch operates on misincorporated bases at or near the replication fork using asymmetrically methylated dam sit,es (Glickman & Radman, 1980: Pukkila et al., 1983; Lu rt al., 1983) to specifically repair mismatched nucleotides on the newly synthesized, transiently unmethylated DNA strand (Gold et al., 1963; Marinus, 1976). Tn order to direct,ly determine the fate of a U. G mismatch and compare it, to that of an analogous T. G mismatch, we have analyzed DNA mismatch repair and uracil-excision repair in E. coli transfected with bacteriophage M13mp18 DNA heteroduplexes creat,ed by oligonucleotide-directed mutagenesis (Zoller & Smith, 1983). Previously, studies of DEA heteroduplex repair upon t,ransfection have utilized mismatches created by annealing nonidentical DNA strands (Kramer et a,l., 1984; Pukkila et al.; 1983). The heteroduplex M 13mpl8 replicative form (RF?) DNA used in the present study was unmethylated at its 5’-GATC’-3’ sites, so that the E. coli DNA mismatch repair system could operate without, dam methylation-directed repair (Kramer et al.. 1984; Pukkila et al., 1983; Dohet, et al., 1985). mismat)ch repair b? oligonucleotideStudying directed mutagenesis, purification of covalently t Abbreviations used: RF, replicatire form; C(!C. covalently closed circular; p.f.u.. plaque-forming units.

cllosed

circular

((‘(‘c’)

l)Siz

2. Materials

E. coli strain JMlOl GM 1674 (:M 1675 l(!R540 ES553

F4652 II ES914 ES1484 ES1481 RH721 IiLl 1sw310

x ICR36

and

(a) Bacterial

strains,

and Methods

growth conditions,

and enzymes

The sources and the relevant genotypes of the E. co/i strains used in t,his study are listed in Table 1. These cultures were grown from single colonies in EC medium (10 g tryptone. 5 g yeast extract, 5 g NaCl per liter) at 37°C. Restriction enzymes, phage T4 polynucleotide kinase, phage T4 DNA ligase. E. coli dam methylase. and the Klenow fragment of E. coli polymerase 1 were purchased from New England Biolabs, Bethesda Research Labs, or International Biotechnologies. Inc. and were used with buffers recommended by the suppliers. (b) Preparation

of single mismatch-containing DNA

heteroduplexes

Phage X13mp18 (Yanisch-Perron et al.. 1985) viral strand DNA derived from a dam- E’. coli strain (GM167.5: Table 1) or a dam- dcm- E. coli strain (GM1674: Table 1) was annealed to previously described Sphosphorylated oligonucleotides, SGGTCGATTCTAGAG-3’ or 5’-GGTCGAUTCTAGAG-3’ (Shenoy it al., 1986). The underlined residues are transition-type mutant bases at the unique HincIT-AccI-SaZJ site (Yanisch-Perron et al.. 1985). The template-primers were converted to 32P-labeled. VW‘ DNA (Fig. l(a)) in 200-~1 reaction mixt,ures containing 1 unit of Klenow fragment. 1 unit of T4 DNA ligasr and the resulting T. G- or LT.G-containing CCC DNA (sprc. acat. - lo6 cts/min per pg) purified and concentrated as described (Shenoy it al.. 1986: Shenoy & Ehrlich. 1985)

Table 1 E. coli strain

moleculrs,

of gerrotvpe by restriction analysis has allowed us t)o p&iselg target I’. (: and T . (; mismatches, quantify the relative amount of uracilexcision and mismatch repair. and determine the timing of these processes relative t,o Ml3 1)N.l replication and to each ot,her. The methodolog) employed in this study for obtaining and analyzing pure preparations of unnicked DNA heteroduplexes by oligonucleotide-directed mutagenesis ca,rl TV adapted to studies of DNA repair in other prokaravotic cells or in eukaryotic cells t~ransfec%ctl with DGA heteroduplexes. assessment

designation

and sourced

Genotype .zupE, thi, A(lac-proAB), [F’. traD36, pro4B. ZacZ?ZAM15] dam-3, dcm-6, A(lac-pro),,,, tax-78, eupE44, galK2, galT22. thi-1. IF-‘. laePZAM15, pro+] dam-4, A(lac-pro),,,,, thi-1, supE44, (r&41)!, [I!‘, lacPZMA15, pro+] F-3 lacZ(ICR36), trpA540 F-, mutL25, lacZ(ICR36), trpA540, dmpd F-, mutS3, lacZ(ICR36), trpA540 F-. mutR34 ( = mutH34), lacZ(ICR36), trpA540 F-. mutL2IX: : TnlO, thy-, metB1, lacY14, thi-? F-. mutS216: : T&O, thy-, metB1, lacY14, thi-? HfrH, Am&R1 (= AmutHl), lacZlJl31, thi-? Hfr, thi-I, &Al, spoT1, 1Hfr, ung-I. tki-1, relA1, spoT1, 1-

Source .J. Messing 111.Q. Marinus bl. G. Marinus E. (‘. Siegel E. C’. Siegel E. C. Siegel E. C. Siegel E. (‘. Siegel E. (‘. Siegel E. (!. Siegel U. 13achmatm I%. 13achmann

DNA Hismatch Repair and Uracil-Excision

(a)

Hmc’

Repair

619

Hint’

-73% (minus Primer

.

extension

’

Transfect

Into

Mut+ E co11

Hint’ phage strand repaired)

_ /

+ -27% Hint’ phage (plus strand repaired)

MN&

strand

(b) Y3’-

Hint’ GTCGAC CAGCTG Hint’

HincS d&3 .j/-5 -78% Hint’ phage (minus strond repaired) Transfect

Denature +

Anneal

into

Mutt Ecoli

>

+ -22% Hint’ phoge ( plus strard rewired)

Minus strand

Figure 1. A summary of DNA repair in m&+ E. coli transfected with heteroduplex M13mp18 CCC DNA containing a mismatched base-pair at the unique H&c11 site. (a) Heteroduplex M13mp18 RF DNA with a T. G mismatch at the HincII site prepared by synthesis in vitro. The 14-base, synthetic oligonucleotide with a C to T transition at the H&c11 site was annealed to Dam- M13mp18 plus strand DNA. This template-primer was used for synthesis in vitro of a CCC heteroduplex, purification of the CCC DNA, transfection of E. wli, and analysis of progeny phage as described in Materials and Methods. (b) Heteroduplex M13mp18 RF DNA with a C. A mismatch at the H&II site prepared by the denaturation and annealing procedure. In this experimental protocol, a heteroduplex with a G to A transition mutation at its mismatch was constructed without DNA synthesis in vitro. The plus strand of HincII-resistant (Hint’) M13mp18 DNA containing a G to A transition was annealed to the minus strand of HindIII-linearized HincII-sensitive (Hines) M13mp18 RF DNA and incubated with T4 DNA ligase as described in Materials and Methods. Then, pure heteroduplex CCC DNA that was resistant to H&II (Shenoy et al., 1986) was isolated and used for transfection and restriction analysis as in (a). The enlarged base-pairs in the Figure are the mismatches.

except that 2 stacked nitrocellulose membranes were used for the filtration. This CCC DNA was > 96 To resistant to linearization by H&II or AccI because of its mismatch (Shenoy et al., 1986). An analogous heteroduplex was prepared with a T. G mismatch at the unique KpnI site by using as a primer, 5’-GATCCCCGGGTGCCGAGCT-3’, which contains the underlined A to G transition in the KpnI site. The resulting CCC DNA had a T. G mismatch with the opposite strand orientation to that synthesized with the aforementioned oligonucleotide. Heteroduplex M13mp18 CCC DNA with a C. A mismatch in the HincII site (Fig. l(b)) was prepared by a denaturation-renaturation procedure similar to that described by Kramer et al. (1984), except that CCC DNA molecules were produed. First, plaque-purified, HincIIresistant M13mp18 phage with a G to A transition mutation at the viral strand’s HincII site were obtained from E’. coli transfected with the heteroduplex M13mp18

RF DNA containing the oligonucleotide primer with the C to T transition. These phage were used to infect E. coli GM1674 and then M13mp18 viral strand DNA was isolated (Messing, 1983). This DNA (1 pg) was incubated at 100°C for 3 min and 58°C for 20 min in 40 ~1 of 20 mMTris. HCl (pH 8.0), 50 mnr-NaCl, 10 mM-MgCl, with 1 pg of deproteinized, HindIII-linearized, wild-type M13mp18 RF DNA also derived from E. coli GM1674. It was precipitated with ethanol and then incubated at 23°C for 2 h with 5 units of T4 DNA ligase, 66 mnr-Tris. HCl (pH 7.5), 6.6 mM-MgCl,, 10 mM-dithiothreitol, 1 mMATP. After precipitation with ethanol, the mixture of heteroduplex and homoduplex DNA was incubated at 37°C for 2 h with 10 units of HincII in order to linearize the homoduplex CCC DNA. As an internal control, 1 pg of pUC19 DNA (Yanisch-Perron et al., 1985) was present and was completely digested. The HincII-resistant, heteroduplex CCC DNA was purified away from the

linearized J)NA. Ehrlich. 1985). (d) Analysis

concentrated.

and desalted

ut dam and

of methylation

of hetwodupbr

(Shrnoy

dcm

&

sitrs

IILYA

Methylation at dam sites was analyzed by incubating portions of radiolabeled heteroduplex DNA and 1 pg of unlabeled, in-&o-derived Ml3mp19 RF DNA with IO units of IMboI or Dpnl for 2 h at 37°C” (Lacks & Greenberg, 1977: Fishel it al.. 1986). The unlabeled RF DNA from JMIOI host cells contained 5’%m6ATC-3’ and 5’-Cm5C(A/T)GG3’ sequences in their TINA; that is. they (Yanisrh-Perron ut (~1.. are Dam+ and J>cm+, respectively 1985; Hatt,man. 1981). The products were rlectrophoresrd polyacrylamide gel and in a 5y0 to ZOO/, (w/v) gradient autoradiographed. As a cont,rol. in-l’itro-synthesized heteroduplex DNA (-I ng) was methylated in the presence of 30 units of K. co/i dam met,hylase for 1 h at 37 Y”, and then digested and analyzed as described above. The methylation status at dcm sites was determined by similar incubation with 10 units of EcoKIT (Roberts, 1985) and electrophoresis in a 1 q0 (w/v) agarose gel.

of E. coli with

(P) Transfection

MldmplR

Single-stranded

from

progeny

of progeny

phagr

after reinfection

viral DN4 was prepared obtained after reinfection and

converted to 32P-JabeIed CCC DNA (Shenoy & Ehrlich, 1985), which was mostly non-superhelical (RFIV DNA). Purified, radiolabeled CCC DNAs were incubated under standard conditions for 2 h with 5 to 10 units of HincII or KpnI in 50-p] reaction mixtures containing, as an internal control, 1 pg of unlabeled. in-&o-derived, homoduplex M13mp19 RF DNA. RFIV DNA, nicked circular DNA, and linearized DNA were resolved by agarose gel electrophoresis in the presence of ethidium bromide (Shenoy & Ehrlich, 1985). The control DNA was detected by fluorescence and the sample DNA by autoradiography (Shenoy et al.. 1986) followed by determination, to 55% counting error, of Cerenkov radiation in DNA bands excised from the dried gel. The

of’ irtfwtiw

centers

Total RF DNA ((0 and nicked circular forms) was isolated (Holmes 8L Quigle?. 1981 ) from individual obbained from mfectivr crnt,ers plaques J’lated immediately after transfection. This unlabeled 1)iY.A (- 1 pg) was incubated with .5 t,o IO units of Hiflc*IJ and. - 1000 cts/min of in-llitm-synthesized. 32 I’as a control, labeled Ml3mpl8 homoduplex (‘(Y’ RF DNA (- lOh cts/min per pg). The samples were analyzed by agarose gel electrophoresis as abovr. Mixed progeny plaques wrrc’ those that, gave generally similar amounts of restrictionresistant and restriction-sensitive unlabeled. sample DNA in contrast to purr burst plaques. which gave only linear DNA or circular DNA (CCC plus nicked circular forms). The nicked circular DNA represented pre-existing nicked circles in the DNA preparations. In all cases. the internal control DNA was fully linearized.

heteroduplez

M13mp18

phage

(g) A nalysis

3. Results

DNA4

A single 6. coli colony was inoculated into EC medium and incubated until it reached an absorbance of -0.3 at 600 nm. The cultures were chilled on ice for 20 min, centrifuged at 4°C and 8OOOg and then resuspended in 10 ml of cold 50 mM-CaCl, (Messing. 1983). After 20 min on ice, the calcium-shocked cells were sedimented and resuspended in 1.3 ml of cold 50 mM-CaCl,. Then. 200 ~1 of the suspension was left on ice for 2 h with 100 ~1 of heteroduplex M13mp18 DNA (IO to 100 rig/ml H,(j). After 2 min at 42°C (heat-shock), 100 ~1 of exponentially growing E. coli JMlOl was added and the mixture plated on EC agar for analysis of infective cent,ers. Approximately 25 to 100 plaques (and generally -60 to obtained. The remaining 200 ,ul of the 80) were transfection mixture was incubated in 4 ml of EC medium at 37°C for 12 h with agitation to release progeny phage. Supernatant)s from these cultures (containing - 10’ p.f.u./ml) were used to infect 5-ml cultures of exponentially growing E. coli ,JMlOl t,o increase the phage titer to between IO” and 10” p.f.u./ml. The final supernatants (progeny phage after reinfection) were obtained from the latter cult.ures for anal,vsis as described below. (f) Analysis

csontrol RF 1)X;\ \vah ;tl\\ay~ fully linearizrcl. ‘J’htl amount,> of radioactivci. rrstric? ion-resistant 1)X:2 itn(l restriction-sensitive, 11X.4 w-as assessed as radioacativity in persisting circular molecules and in Iinearizrcl molrcult~s. respectively.

(a) 8trategy For

analysis

of DNA

repair

experiments. CCC MlSmplt) DNA T. (‘r or IT. G mismatch within a unique restriction site in the non-essential polylinker region was synthesized by extension of a mutation-cont’aining, synthetic oligonucleotide primer. Before transfection, all DNA molecules that, were not covalently closed circles were removed t’o eliminate unused template and nicked or gapped molecules that might bias mismatch repair (Claverys & Lacks, 1986). The purified CCC DNA. which lacked dam methylation (see below), was used to transfect repair-proficient (mut+ ungf ), mismatch repair-deficient (mut-), or uracil-DNA (ung-) E. coli. The host. glycosylase-deficient bacteria were dam’ but this has no effect on the mismatch repair of transfecting phage DNA heteroduplexes (Pukkila rt al., 1983). Repair of the mutant T or 1: residue within the mismatched basepair of the transfect’ing heteroduplexes yields progenv phage whose J)NA has the wild-t’ype restriction site (Fig. l(a)). Repair of the nonmutated G residue of the mismatch yields restriction-resistant progeny phage. After transfection, progeny phage for restriction analysis were obtained by two procedures. Firstly, transfected cells were plated on E. coli JMlOl immediately after the heat-shock step (infective centers after transfection) to distinguish cells that repaired the heteroduplex (yielding pure bursts) from those that replicated the heteroduplex before it could be repaired (yielding mixed bursts). In the second method, after transfection, the cells were incubated overnight and the released progeny phage used to reinfect E. coli JMlOl in liquid culture (progen? phage after reinfection). Analysis of the genotype of the large number of pooled progeny phage particles obtained by reinfection in liquid medium allows the containing

most

for

a single

DNA

Mismatch

Repair

.7249

‘4032

.2129

,507 ,332

‘153 96

Figure 2. Demonstration of the lack of adenine methylation at 5’-GATC-3’ sites (dam methylation) in the in-vitro-synthesized, 32P-labeled heteroduplex M13mp18 RF DNA used for transfection. The heteroduplex DNA was radiolabeled in the presence of [cr-32P]dATP by the extension of the C to T transition-containing oligonucleotide annealed to Dam- Ml3mp18 viral DNA as described (Shenoy et al., 1986); therefore, the specific activity of each DNA fragment was proportional to its size. Autoradiography was performed after electrophoresis of the following samples: lanes A and B, unmethylated M13mp18 heteroduplex DNA; lanes C and D, in-vitro dam-methylated M13mp18 heteroduplex DNA. The enzymes used for digestion were as follows: lanes A and C, MboI; lanes B and D, DpnI. The number of base-pairs in each fragment is indicated on the right. From a comparison of our M13mp18 RF DNA preparation and the known sequence of M13mp18 DNA (Yanish-Perron et al., 1985), an alteration in the Mb01 restriction pattern was noticed. The missing restriction site was a silent mutation at position 6935 and resulted in the absence of the 434 base-pair fragment in the MboI digest of unmethylated M13mp18 RF DNA and in the DpnI digest of the analogous dam-methylated RF DNA.

rapid and sensitive measurement of strand bias during DNA repair. To eliminate the bias in mismatch repair caused by dam-methylation, the M13mp18 viral DNA that was used as a template in the above-described invitro synthesis of transfecting heteroduplex DNA had been obtained from phage propagated on damhost cells (Dam- plus strand DNA). The in-vitro-

synthesized minus strand totally lacked m6A residues. We checked that the Dam- viral strand DNA was devoid of methylation at 5’-GATC-3’ sites (Lacks & Greenberg, 1977; Bale et al., 1979; Marinus et al., 1983; Smith et al., 1985) because methylation of any residual sites on only one strand (hemi-

and Uracil-Excision

Repair

621

methylation) could strongly influence the directionality of DNA mismatch repair (Glickman & Radman, 1980; Lu et al., 1983; Pukkila et aE., 1983; Kramer et al., 1984). Hemimethylated dam sites can be detected by their resistance to MboI and DpnI (Lacks & Greenberg, 1977; Fishel et al., 1986). Figure 2 shows that the in-vitro-synthesized RF DNA

prepared

from

the Dam-

template

was

completely digested by MboI, which will digest only bifilarly unmethylated 5’-GATC-3’ sites (Fishel et al., 1986). As expected, when this heteroduplex DNA was dam-methylated in vitro, the DNA was rendered resistant to MboI and virtually completely susceptible to DpnI (Fig. 2). The similarity of the MboI digest of the unmethylated DNA and the DpnI digest of the in-vitro-methylated DNA and the absence of smearing of the MboI bands (Fig. 2) argues against the presence of residual methylation of 5’-GATC-3’ sites in the template DNA molecules. (b) Repair

of U. G and T. G mismatches in E. coli

The repair of U. G and T. G mismatches in Dam- M13mp18 RF DNA transfected into mismatch repair-proficient E. coli was compared; in these mismatches the pyrimidine residue was the mutant base. The former mispair can arise from heat-induced or spontaneous deamination of C residues, and the latter from deamination of m5C residues or from misincorporation of dTTP instead of incorporation of dCTP at the replication fork (Lindahl, 1979; Wang et al., 1982; Ehrlich et al., 1986). When a mutf ung+ E. coli host (KL16) was transfected with M13mp18 CCC RF DNA containing a U. G mismatch at its HincII site, about 97% of the radiolabeled RF DNA derived from progeny phage after reinfection was digested by HineII (Hint”; Table 2). The unlabeled internal control, M13mp19 RF DNA, was completely digested. Nonetheless, to eliminate the possibility of slight residual incomplete digestion accounting for the 3% HincII-resistant (Hint’) DNA, a second digest was made with both HincII and SalI, which has the same recognition site on M13mp18 RF DNA as does HincII. Again, 3% of the radiolabel was observed in undigested RFIV DNA. This suggests that the very small amount of Hint’ DNA came from transfecting heteroduplex molecules that totally escaped DNA repair or that escaped uracil excision and had the mispaired G residue excised by mismatch repair. M13mp18 RF DNA prepared separately from 30 individual plaques derived from the transfection mixture and plated on indicator bacteria (infective center assay) all possessed only Hin? DNA (Table 2). The absence of detected Him’ pure bursts might be due to the sample size not being large enough to detect only 3% Hint’ plaques. The involvement of uracil-DNA glycosylase in this highly efficient, demonstrated with

base-directed DNA repair was an isogenic ung- E. coli host

(BW310). Analysis of infective

centers as well as of

8. Shenoy et, al.

622

Table 2 Repair

of 7’. G and 1,: . C mismatches

RF DXA in Dam- M13mplX repair-projicirnt tc:. coli

i,n ung-- a,nd ung’

Infective

renters after transfectiong Pure bursts

Progeny phage after reinfectionf Total infertive E. coli host uw310 KLl6 BW310 KL16

Mismatch?

y,, Him*” (+) a

U.G LT G T.G T.G

77*3 (5) 97+_% (3) 78&4 (5) 73_ft (4)

ungung+ ungung+

“jO Hi& (-) h 23+3 3+_n PXf4 %7&2

mismatch

centers

a,jb

Ob pure bursts

‘?. mixed bursts

O. Hinr’ (+) a

3.3 32 3.5 2.7

9ti 100 96 96

4 0 4 4

74 loo 81 70

Ratio

Y,, Him’ ‘6’ 26 (I I9 30

Ratio n/h 1.8 >lOO 4.3 “..i

t The mutant base of the unique ffincT1 site of the heteroduplex Ml3mp18 RF DNA is underlined and represents a (1 to IT or (’ to T transition in the minus strand at a I1.G or T .(Z mismatch. This CCC heteroduplex was synthesized as shown in Fig. I(a) with an MlJmplH template DNA prepared from phage propagated in E. coli GM1675. The mutant base of the heteroduplex confers resistance to H&II. The full genotype of the isogenic hosts for this and subsequent tables is given in Table 1. 1 The values reported are mean percentages &standard deviation. The total number of determinations from duplicate transfections is reported in parentheses. Hint” and Him’ represent the respective plus strand and minus strand genotypes. This was assessed by restriction analysis of pooled progeny phage DNA that had been converted in z&o to the radiolabeled RF DNA as described in Materials and Methods. The plus or minus sign designates whether the progeny phage and pure bursts display t,he plus strand or the minus strand genotype of the parental, mismatch-containing RF DNA. $ The NincII-sensitivity of 20 to 30 individual plaques from each transfection mixture was tested as described in Materials and Methods. Plaques with the Hint” phenotype have the plus strand genotype due to repair of the minus strand of the transfecting heteroduplex and, reciprocally, those with the Him’ phenotype have the minus strand genotype due to repair of t,he plus strand (see Discussion).

progeny phage after reinfection showed that, in the absence of uracil-DNA glycosylase, only about 75% of the transfecting heteroduplex molecules containing a IJ. G mismatch gave rise to Hint” phage (Table 2). Analysis of individual plaques revealed only 4% mixed bursts (mixtures of Hint” and Hint’ DNA), which can be attributed to replication of an unrepaired heteroduplex RF DNA upon transfection.

Transfection of ung+ or ung- E. coli with the analogous T 3G mismatch-containing heteroduplex yielded about 70 to 80% Hint” progeny and 4O/, mixed bursts (Tables 2 and 3). From the similar results with the T. G mismatch in ung+ and ungE. coli, we infer that the heteroduplexes were free of any dUMP residues in the minus strand that might have biased repair. Such residues could have been incorporated from contaminating dUTP during the

Table 3 Repair

of T. G mismatches

in Dam-

M13mp18

RF DNA -

in mismatch

~~~._.

repair-deficient

Infective centers after transfection ~~~~~~~~._ ~~~..~ ...~~~~~

Progeny phage after reinfectiont ‘+, Hint” (+)

ft. coli hosts ES553 (mutL25) ES652 (m~tS3) ES914 (mutH34) ICR540 x ICR36

a

@nut+ )

41_+6 42*9 20*1 73fl

O. Hint’ (-) b

(3) (3) (3) (5)

59k6 5t3+9 8Ok 1 27&l

a/b

0.7 0.7 0.25 2.7

~~

Pure bursts: __

Ratio

H. coli

Total infective .~~__.

renters ~__~

0X pure bursts

96 mixed bursts

47 50 67 97

,53 50 33 3

Y0 Hint’ (+I a -9 Z8 33 66

O0 Him,’ C-1 h

Ratio a/h

41 .52 67 34

I4 0.9 0.5 1.9

The T .G mismatch-containing DNA used in this study is the same heteroduplex with a C to T transition in the minus strand of the M13mp18 RF DNA as that in Table 2 and the genotype analysis are as described in that Table. As in Table 2, Hint’ phage have the plus strand genotype ( + ) and Hint’ phage have the minus strand genotype ( - ). t The differences seen in mut hosts between the a/b ratio for progeny phage after reinfection and that for purr burst infective centers probably reflect a small overrepresentation of minus strand progeny in the former resulting from a replication bias (Kramer ct al.. 1984). $ These data are from the analysis of 41 to 76 infective centers for each type of mutant, which included 30 to 42 pure burst plaques. The results from 3 sets of transfection experiments were pooled in order to increase the sample size. One of these transfection experiments utilized ES1484 (mutL218: : TnlO), ES1481 (m&S215 : : TnZO), and RH721 (AmutHI) as hosts instead of the corresponding point mutants listed in this Table. Similar results were obtained with the point mutants and the corresponding deletion or transposoninsertion mutants. For comparison, data from the ICR540 x ICR36 host,, which is wild-type in its mut genes and isogenir to the point mutants, are included in this Table.

DNA Mismatch Repair and Uracil-Excision

Repair

623

Table 4 Repair of T * G, inverted T. G, and C. A mismatches in Dam- Dcm- or Dam- dcm-hemimethylated Ml3mpl8 RF DNA in mismatch repair-proficient E. coli Progeny phage after reinfection1 DNA modification (minus strand/plus

strand)

Dam-Dcm-/Dam-Dcm+ Dam-Dcm-/Dam-DcmDam-Dcm-/Dam-Dcm+ Dam-Dcm-/Dam-Dcm-

Mismatch?

T.G T.G G.T C.A

Source of minus strand in heteroduplex DNA

Synthesis in vitro Synthesis in vitro Synthesis in vitro Replication in viva

o/0 Plus strand genotype

73+1 (5)Q 72+4 (6) 59, 63 (2) 78+7 (6)

o/0 Minus strand genotype 27+1 28+4 41, 37 22+7

The mismatch-repair proficient E. coli host employed was ICR540 x ICR36. t The 1st base in each base-pair was in the minus strand and the 2nd was in the plus strand. The mutant base is underlined and represents a C to T or A to G transition in the minus strand for the T .G heteroduplex and the inverted T.G (G.T) heteroduplex, respectively. For the C. A mismatch-containing heteroduplex, a G to A transition in the plus strand, which was obtained after oligonucleotide-directed mutagenesis and transfection, provided the mutant base. $ The values reported are mean percentageskstandard deviation, except in the case of the G.T heteroduplex, for which only 2 determinations were obtained and are separately reported, each from a distinct but identically prepared heteroduplex sample. The total number of determinations from duplicate transfections for the other samples is reported in parentheses. 8 These values are those for transfection of the mut+ host shown in Table 3.

synthesis in vitro of the minus strand (Baas et al., 1980; Schlomai & Kornberg, 1978). When the host for the transfection with this T *G mismatchcontaining heteroduplex was mutt rather than mut’ E. coli, there was a change in the ratio of Hin? to Hint’ progeny phage as well as an increase in the percentage of mixed burst plaques (Table 3). With E. coli ES553 (mutL25), ES652 (mutS3) (Siegel & Kamel, 1974), or ES914 (mutH34) hosts plaques (Vaccaro & Siegel, 1977), mixed-burst constituted 53, 50 and 33% of the total plaques, respectively, as compared to 3 to 4% in mut’ hosts. Similarly, E. coli that had a TnlO insertion mutation in the mutL or mutS genes (ES1484 and 1481; Siegel et al., 1982) or a deletion of the mutH gene (RH721; Hoess & Fan, 1975) gave about 50, 56 and 80% pure bursts, respectively, in the infective center assay. Only in mut+ E. coli was preferential repair of the G residue from the minus strand of the T. G mismatch always seen (Tables 2 and 3). This bias in repair of Ml3 T *G mismatches (Tables 2 and 3) might have been due to incorporation of a rare atypical deoxynucleoside triphosphate, e.g. dITP (Baas et al., 1980; Karran & Lindahl, 1980), present as a contaminant in the triphosphate mixture used for synthesis of the heteroduplex. Such an atypical base, which would be found only on the minus strand, might be a repairable DNA lesion and trigger co-repair of the mismatched residue on the minus strand in some of the heteroduplex molecules. However, a heteroduplex CCC DNA prepared by a denaturation-reannealing method without DNA synthesis in vitro and containing a C. A mismatch (Fig. 1(b)) was repaired preferentially on the minus strand by mut+ E. coli to approximately the same extent as were in-vitrosynthesized, T. G mismatch-containing heteroduplex DNAs (Table 4).

(c) Influence of dcm methylation and strand orientation on mismatch repair Because of the absence of detectable dam methylation in the Dam- heteroduplex DNA (see above), the preferential repair in mut’ hosts of the minus strand in T. G mismatch-containing heteroduplex DNA (Tables 2 and 3) should not be due to dam methylation-directed repair (Kramer et al., 1984; Radman & Wagner, 1984; Dohet et al., 1985). However, the Dam- template, M13mp18 DNA derived from E. coli GM1675, should be methylated at the internal cytosine residue of its seven 5’-CC(A/T)GG-3’ (dcm) sequences (Marinus, 1973; Yanisch-Perron et al., 1985). This was confirmed by the resistance of in-vivo-derived M13mp18 RF propagated on this strain to digestion by EcoRII at 5’-CC(A/T)GG-3’ sequences (Roberts, 1985). Heteroduplex RF DNA constructed by synthesis in vitro of the minus strand using a Dcm+ template would be hemimethylated at dcm sites, which might influence DNA mismatch repair (Lieb, 1983), although the nearest dcm site in this heteroduplex DNA is 64 base-pairs from the mismatch and this is less than the repair tract size of the very-shortpatch repair system (Lieb, 1983). To provide the first direct test of the influence of dcm hemimethylation on repair of a neighboring mismatch, a heteroduplex was synthesized with Dam- DcmM13mp18 viral strand DNA (from E. coli GM1674; 1973; M. G. Marinus, Marinus & Morris, and the oligonucleotide unpublished results) containing the C to T transition in the HincII site. The Dcm- Dam- state of this RF DNA was confirmed by its sensitivity to EcoRII and to MhoI. The ratio of wild-type to mutant phage in progeny produced upon transfection of mut’ E. coli with T. G mismatch-containing heteroduplex this symmetrically unmethylated at its dcm sites was

the same as that observed using tht, atlalogous heteroduplex dcm rnethylat.ed on one strand (Table 4). Another possible sour(*e of the bias observed for the repair of the minus strand was a preference for the repair of the pyrimidine residue in T. G and a heteroduplex Therefore. c . c: mismatches. M13mpl8 DNA was synthesized with a T.G mismatch (G ‘I’) of opposite polarity to the ‘I’. (: mismatch in the previously described hrteroduplex. Tn t,he latter ((’ to T) heteroduplex. the ‘I’ residue of t’he mismatch was in the minus strand and was the mutant base conferring restriction resistance (Fig. l(a)). Tn the former (A to G) het,eroduplex, the T residue of the mismatch was in the plus &and and was the wild-type base associat’ed with KpnI sensitivity. The mixed progeny phage population obtained after transfection of mut+ E. coli with this G . T heteroduplex contained about 60 “/:, KIms (plus strand genotype) phage and about 409zb Kpn’ strand genotype) phage. Therefore, (minus inversion of the strand orientation of the T. G mismatch in M13mp18 DNA did not rause an inversion in bias for the strand repaired in E. coli. It is unclear whether the difference between approximately 73% plus strand genotype phage in the experiments with the T. G mismatch at the HincII site and approximately 60% plus strand genotype wit)h the G. T mismatch at the KpnT site is significant,. However, it might reflect a minor base preference of the mismatch repair system or the influence of surrounding sequences (Claverys & Lacks, 1986: Fazakerley et (II., 1986) on mismatch repair.

4. Discussion This paper presents the first comparison of the efficiency of repair in viva of I!. G and T. G mismatches in DNA. As expected, repair at the U .G mismatch in CCC M13mp18 RF DNA transfected into repair-proficient E. coli was highly efficient with about 97% of the transfecting heteroduplex molecules having the U residue excised (Table 2). In contrast, in the same strain, about 73% of the analogous T. G mismatchcontaining heteroduplexes had the T residue excised (Table 2). The very high degree of directionality of repair of U. G mismatches in repair-proficient E. coli was due to uracil-excision repair, because it was not observed in an ung mutant host (Table 2). Although the ung- E. ~011;host did repair U. G mismatches, giving a very small percentage of mixed bursts, repair occurred with a much lower frequency of removal specifically of the U residue at the mismatch (Table 2). The frequency of elimination of U from a I:. G mispair in ung- cells, monitored by the ratio of Hint” to Hint’ progeny phage or pure bursts, was essentially the same as the frequency of loss of T from a T. G mispair in ung- or ung+ cells, but not in mutcells (Tables 2 and 3). This suggests that, in the absence of uracilDNA glycosylase, U. G mispairs are repaired as well

as T.(i mispairs b>, the rout mismatch repair svst,em. That the rn& mismat,c*h repair pathwa\- in i. roli can repair both (’ 9A and T . G transition mismatches very efficiently. and even A . A mispairs with intermediate efficiency (Kramer et al.. 1984). is consistent with the mrrt system repairing I’.(: mismatches. Our data also indicate that only the uracil-excision repair pathway. which is dependent upon wag func+on. confers a strong directionalit!, upon I’ G repair. Similarly. higher spontaneous tnutation rates were found in ung- E. coli than in urLg+ E. coli (Duncaan & Weiss, 1978: 1)uncan Pt (Il.. 1978: Duncan & Miller. 1980) and completr restoration of the viability of uracil-substituted phage T4 DXA was seen in an ung- E. coli host but not, in an ung+ host (Warner & Duncan. 1978). Repair of T.G (T on the minus strand). ittverted T. G (T on the plus strand). and (‘. A mismatches in repair-proficient hosts occurred on the minus strand in 60 to 800;, of the transfecting ~113mplX RF hetrroduplexes (Tables 2 to 4). This bias, which was not seen in the study by Kramer uf nl. (1984). D’WS not due to darri methylat,ion or rlcvrr methglation nor to art,ifacts associated wit)h synthesis in vitro of the minus strand (Table 4). The preferential removal of the mismatched nucleotide on the minus strand of the &Ml.3 RF DNA might reflect’ some unequal interaction of its strands with t,he mismatch repair svstem. Alternatively, it might be due to a &incidental effect of neighboring sequences influencing the directionality of DNA repair in the absence of dnm methylat,ion ((‘laverys & l,acks. 1986: Fazakerley et 01.. 1986). The mismatches used in this studv are in t,he region replicated early during RF 1)hA synthesis (Baas, 1985). Only extretnel$ low levels of mixed bursts were observed in repair-proficient hosts: therefore. mismatch repair and uracil-excision repair were probably completed soon after entry of the heteroduplex int,o the host cell. Because less than 502, of the progeny phage were mutant upon transfection of ung+ rnuf+ B. coli with tjhe I’. (:containing RF DNA (Table 2). uracil-excision repair probably occurs more rapidly than does mismatch repalr: ot,herwise, more mutant progen)’ would have been observed due t,o the excision of the wild-type G residue of the mismat)ch. Also. it can be concluded from this result that t’here was no significant loss of t,he plus strand and, therefore. presumably none of the minus strand during M 13 DNA uptake. When Ml3mp18 RF DNA possessing a ‘r .Q mismatch was t,ransfected int,o hosts containing point mut,ations, delet,ions. or transposon insertions in mutl,, mu,tS or mutfir genes, from 50 to SOY, of total burstas were pure bursts compared to more t,han 96O/, from isogenic mtlt+ E. coli hosts (Tables 2 and 3). Tf strand loss was responsible for the purr bursts in ma- cells. then it had to take place. by some undefined mechanism, after DNA upt.ake (and the t*ime of uracil-DNA excision) but’ before the second round of replication. Also. the minus strand would have to be lost about 50 to 60 Ocjof the time in ~!utL and ,mutS hosts and

DNA Mismatch Repair and Uracil-Excision only 30% of the time in an isogenic mutH host. Furthermore, if there were strand loss after DNA uptake, the parental plus strand might be the more likely to be lost in all mut hosts, given the delay in its conversion to the double-stranded form during RF DNA replication (Baas, 1985; Messing, 1983). Therefore, our analysis of infective centers after transfection suggests that considerable mismatch repair can occur on Ml 3 heteroduplexes in the absence of the m&L or mutS products, although this was seen only with mutH hosts in the experiments reported by Kramer et al. (1984). In that study, the genotype of Ml3mpl phage was determined by plaque color, which could not be interpreted unambiguously. In our study of mutH hosts, not only were even more pure bursts seen than from m&L and mutS hosts, but also a very different ratio of plus strand genotype infective centers to minus strand genotype infective centers was obtained (Table 3). This suggests that, in the absence of the mutH product, much mismatch repair (or abortive repair; Doutriaux et al., 1986) can still occur but generally with a different directionality than in its presence. As proposed by Claverys & Lacks (1986), these data further suggest that, in the absence of the mutH product (a putative endonuclease; Modrich et al., 1986; LgngleRouault et al., 1987), the nick that begins mismatch repair might be the nick that is specifically made in the plus strand by the Ml3 gene II product (Baas, 1985). That nick, which begins Ml3 RF DNA replication and plus strand synthesis (FidaniLn & Ray, 1972; Messing, 1983), must not often be used for initiating mismatch repair in mutH+ cells or else, in these cells too, the mismatched base would have been excised preferentially from the plus strand. We hypothesize that mismatch repair on Ml3 RF DNA molecules in the presence of the mutH product occurs before RF DNA replication is initiated, whereas it is often delayed in a mutHhost until the initiation of DNA replication. We thank Kim Daigle for technical assistance. Martin Eli Siegel, Joachim Messing and Barbara Ma&us, Bachman kindly supplied the E. coli strains. We are very grateful to Martin Marinus and Xian-Yang Zhang for critical reading of the manuscript. This research was supported in part by PU’SF grant PCM 821 900.

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Edited by M. Gottesmnn