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Membrane attachment and DNA bending at the origin of the Escherichia coli chromosome
ReS. Microbiol. 1991, 142, 151-154
t~) INSTITUTPASTEUR/EL:~EVlER Paris 1991
Membrane attachment and D N A bending at the origin of the Escherichia coli chromosome M . Schaechter, P. Polaczek and R. Gallegos
Department o f Molecular Biology and Microbiology, Tufts University Medical School, Boston, M A 02111 USA Certain proteins of Escherichia coil that are not required for in vitro initiation of DNA replication are known to interact with the replication origin of the chromosome, oriC. We count among such accessory proteins those involved in the membrane a t t a c h m e n t of oriC and the DNA-hending IHF (integration host factor) protein. Here we discuss the possible role of these proteins attd offer a suggestion for how they may be connected physiologically.
and elsewhere in the chromosome. They found that two sites within oriC as well as a site in the dmtA gene became methylat-
ed after 12-13 miu at 30 °, whereas sites elsewhere in the chromosome required between 0.5 and 7 min.
The Bvent
Replication
I Methylation
Starts
Starts
The attachment of oriC to the membrane In mixing experiment, we have shown that oriC attaches to the m e m b r a n e in a highly specific manner (Hendrickson et al., 1982). We have further demonstrated that this attachmcnt takes place only when oriC is hemimethylated by the Dam methyhransferase, that is, for a period of time after its replication (Ogden et aL, 1988). Our results showed that methylation of newly replicated oriC is delayed when compared with that of other Dam methyiation substrate sites (GATC). In a detailed study, Campbell and Kleckner (1990) compared the methylation kinetics of individual GATC sites in oriC
+++'+I 0
I 1
I 2
I I 3 4 Minutes
, 1 I 5
I 6
I 7
I 8
Fig. L The timing of methylation and membrane attachment of oriC. Replication results in hemimethylated ariC DNA which adheres specifically to the membrane. Methylation of orfC GATC s~quences by the Dam methyhransferase is delayed as compared with GATC sequences elsewhere iA the ch-omosome. After a period of about 6 rain at 30% an event of unknown nature allows the oriC sequences to become methylated and to detach from the membrane. The time for this event is speeuIative. This sequence of events is deduced from in vitro results and its relevance to the in vivo situation is unknown.
‘The mwrpremlion 01 fhese frndin~:.r rc~naim n subject for •,X(.CII~RIIOII.We propose Ihe 61. towing possibilhy: upon rcplicalion. rhr chromosome binds to lhc membrane at newly replicated, hemimclhylalcd oriC. This binding may. by itself. prevent methylation of [his region. After a period ol time (CO 6 min 81 30”). an event happcss Ihal leads 10 rhr delachmenl of oric from rhc membrrme (fig. It. Two minuws or so law. this DNA is Ihe don? ewhytofed by merhyhransfcrase. According to rhir rchemc. E. co/i porrcrres a clock IIW is started by the act of replicalion and is turned off by an unknown event. This may even be racell-cycle-timed modification 01 a membrane binding protein. possibly through the agency of a soluble faclor. Various aher psribiliries have been discussed by Campbell and Kleckncr (1990).
An examinauon of the delay in mcthylation of oriC requires isolating and analyring pulative membrane bindink proteins. We have succeededin showing lhat a membrane protein of E. co/i binds labclled hemimethylaled ONC DNA in the presence of an cxcc~s of untabclted methylated oriC DNA. We inlend 10 determine if Ihe oriC binding ability of this protein becomes modified during the cell cycle.
Binding uf IHF
.-
.
.
..-
t _.
.
.*
prolein lo oriC
II MS found in this laboratory that Ihe inregrarion Host Facmr, the IHF motein. binds at a specific seqience of oriC (Poiaczek. 1990). It war proposed lhal this binding enhancer the aaivhy of Ihe initiator protein DnaA. The followhe mechanism was proposed. 1; oric, [here are four 9 bp sequences 10 which Dnti may
Fig. 2. The effect of IHF on in vitro methyl&on at or;C by Dam methyltransferase. The analysis is based on the fact that methylation of CATC sequences makesthe DNA renairive10 Dpn, and r&tam to MM. Lanes a through f show cleavageby D&M and Mb, al increasingtimes after addition of Da,,, merhyhransferase(a) 1.5 min; b) 3 min; c) 4.5 min; d) 6 min: e) 10 min: 0 lOmint. I” the absenceof IHF, partial digerrs show that all GATC sequences are methylatedby Dam methyltransferase.In rhe presenceof 1°F (3 ag/mt), an IHF consensus sequenceis protectedfrom methyl&m. as are, in part, two GATC sequences beyond rhar region (see fig. 3).
eriC, ,~4IEMBRANEA TFACHMENT AND DNA BENDING bind (DnaA boxes). However, DnaA appears to function in initiation by interacting with three nearby sites identified as repeats of a 13-bp sequence (13-mers; Bramhill and Kernberg, 1988). How can DnaA protein recognize these relatively distant sequences, which have no homology to the primary binding sites.'? One possibility is that the transfer of DnaA is facilitated by bending of DNA in this region. IHF does indeed cause bending between the DnaA boxes and the 13-mers (Polaczek, 1990), allowing DnaA boxes to face the 13-met region. It was shown by in rive footprinting that thrc. of the boxes appear to be occupied at all times, whereas one, R3, is not (Samitt et aL, 1989). This suggests that R3 may be directly involved in regulation by DnaA. Bending of e r i c DNA by IHF places three D n a A boxes in register with the 13-mers, with R3 in the best position to "'face'" the 13-mers. TM
Is I H F indispensable for initiating replication'?. Apparently not. IHF stimulates the unwinding o f the 13-mer region, but so does H U (Bramhill and Kornberg, 1988). In addition, IHF deficient mutants are viable and so are HU-deficient mutants (Ogawa et uL, 1989; Kano and lmamoto, 1990). However, double mutants in these two proteins are difficult to grow and maintain (Kano and lmamoto, 1990). Thus, IHF is not essential and its function may be substituted by H U , possibly by a different mechanism of action. In an attempt to make a connection between the bending of e r i c and membrane binding, we have determined the influence of [HF on methylation. The IHF binding site in e r i c overlaps a recognition sequence for dam methyltransferase. This site is protected from in vitro methylation by Dam methyltransferase.
153
106-122
i
gain HI (92) Region grotectecl agslnst Dnese I
I
I
Regton erotectad against Darn methylgsa
Fig. 3. Schematic diagram of the in vitro protection studies with IHF. Binding of IHF to eriC results in protection from Dnase 1 to the left of an [HF consensus sequence located between bp 106 and 122 (Polaczek, 1990). [HF protects from methylation by the Dam methyhransferase the GATC sites within or overlapping the consensus sequence and also, in part, sites to the right of this region. A GATC site to the left of the IHF consensus sequence is protected from DnaseI but not flora methylation by the Dam methyltransferase.
To our surprise, we found that GATC sites not protected by IHF in a Dnase 1 footprint were protected from Dam methylation (figs 2 and 3). Lacking a better explanation, we attribute this resistance to methylarion to changes in tile conformation of the DNA.
Acknowledgements
If IHF-binding influences mefhylation, which in turn affects the binding of eriC to the membrane, might this protein play a role in the timing of initiation and segregation? IHF may be an element in a regulatory circuit consisting of a series of protein-DNA interactions that lead to precisely timed initiation at eriC. Ceils may overcome the lack of 1HF by adjusting the level of other proteins, thus functionally substituting IHF and achieving well regulated initiations.
References
Key-words:
DNA, oriC, E. coil; Chromosome, Bending, Attachment, Membrane.
This w o r k wa> ~rried ~ut with sup-
port from a U.S. Puhiic Heagh Service (National Institutes of Health)grant No. ROI 09465.
nramhil[, D. & Kornberg, A. (1988), Duplex opening by DnaA protein at novel sequences in initiation of replication at the origin of the E. coli chromosome. Cell, 52, 743-755. Campbt:ll. J.L. & Kleckncr, N. (1990). E. colt eric and the dnaA gene promoter are sequestered from dam mcthyltransferase following the passage of the chromosoma; replication fork. Cell, 62. 967-979. Hendrickson, W.G.. Kusano, T., Yamaki. H,. Balakrishnan. R., King, M.. Murchie. J. & Schaechter, M. (1982). Binding of the origin of replication of Escherichia colt to the outermembrane. Cell. 30, 915-923.
154
Kann, Y. & hnamoto, F (1990), Rcquirenlefll for integration bo~t factor (IHF) for growth of Eschcrichia coil deficicnl in HU prolein, Gene, 89, 133-137, Ogawa, T., Wada, M., Kano, Y., [ff ]lllOIO, F. & Okazaki, T, (1989). DNA replication in Escherichia coil mutants Ihal
M. SCHAECHTER E T AL. lack protein HU, J. Bac,, 171, 5672-5679. Ogden, O., Pratt, M.J. & Sehaechter, M. (1988), The replicative origin or the Escherichla coil chromosome binds to ceil membranes only when hemimelhylated. Cell, 54, 127-135. Polaczck, P. (1990), Bending of the
origin of replication of E. coil by binding of [HF at a specific site. New Biol., 2, 265-271. Samitt, C.E., Hansen. F.G.. Miller, J,F. & Scha¢chter, M. (1989), In vivo studies of DnaA binding to the origin of replication of Escherichia coll. EMBO J.. 8, 989-993.
t~) INSTITUTPASTEUR/EL:~EVlER Paris 1991
Membrane attachment and D N A bending at the origin of the Escherichia coli chromosome M . Schaechter, P. Polaczek and R. Gallegos
Department o f Molecular Biology and Microbiology, Tufts University Medical School, Boston, M A 02111 USA Certain proteins of Escherichia coil that are not required for in vitro initiation of DNA replication are known to interact with the replication origin of the chromosome, oriC. We count among such accessory proteins those involved in the membrane a t t a c h m e n t of oriC and the DNA-hending IHF (integration host factor) protein. Here we discuss the possible role of these proteins attd offer a suggestion for how they may be connected physiologically.
and elsewhere in the chromosome. They found that two sites within oriC as well as a site in the dmtA gene became methylat-
ed after 12-13 miu at 30 °, whereas sites elsewhere in the chromosome required between 0.5 and 7 min.
The Bvent
Replication
I Methylation
Starts
Starts
The attachment of oriC to the membrane In mixing experiment, we have shown that oriC attaches to the m e m b r a n e in a highly specific manner (Hendrickson et al., 1982). We have further demonstrated that this attachmcnt takes place only when oriC is hemimethylated by the Dam methyhransferase, that is, for a period of time after its replication (Ogden et aL, 1988). Our results showed that methylation of newly replicated oriC is delayed when compared with that of other Dam methyiation substrate sites (GATC). In a detailed study, Campbell and Kleckner (1990) compared the methylation kinetics of individual GATC sites in oriC
+++'+I 0
I 1
I 2
I I 3 4 Minutes
, 1 I 5
I 6
I 7
I 8
Fig. L The timing of methylation and membrane attachment of oriC. Replication results in hemimethylated ariC DNA which adheres specifically to the membrane. Methylation of orfC GATC s~quences by the Dam methyhransferase is delayed as compared with GATC sequences elsewhere iA the ch-omosome. After a period of about 6 rain at 30% an event of unknown nature allows the oriC sequences to become methylated and to detach from the membrane. The time for this event is speeuIative. This sequence of events is deduced from in vitro results and its relevance to the in vivo situation is unknown.
‘The mwrpremlion 01 fhese frndin~:.r rc~naim n subject for •,X(.CII~RIIOII.We propose Ihe 61. towing possibilhy: upon rcplicalion. rhr chromosome binds to lhc membrane at newly replicated, hemimclhylalcd oriC. This binding may. by itself. prevent methylation of [his region. After a period ol time (CO 6 min 81 30”). an event happcss Ihal leads 10 rhr delachmenl of oric from rhc membrrme (fig. It. Two minuws or so law. this DNA is Ihe don? ewhytofed by merhyhransfcrase. According to rhir rchemc. E. co/i porrcrres a clock IIW is started by the act of replicalion and is turned off by an unknown event. This may even be racell-cycle-timed modification 01 a membrane binding protein. possibly through the agency of a soluble faclor. Various aher psribiliries have been discussed by Campbell and Kleckncr (1990).
An examinauon of the delay in mcthylation of oriC requires isolating and analyring pulative membrane bindink proteins. We have succeededin showing lhat a membrane protein of E. co/i binds labclled hemimethylaled ONC DNA in the presence of an cxcc~s of untabclted methylated oriC DNA. We inlend 10 determine if Ihe oriC binding ability of this protein becomes modified during the cell cycle.
Binding uf IHF
.-
.
.
..-
t _.
.
.*
prolein lo oriC
II MS found in this laboratory that Ihe inregrarion Host Facmr, the IHF motein. binds at a specific seqience of oriC (Poiaczek. 1990). It war proposed lhal this binding enhancer the aaivhy of Ihe initiator protein DnaA. The followhe mechanism was proposed. 1; oric, [here are four 9 bp sequences 10 which Dnti may
Fig. 2. The effect of IHF on in vitro methyl&on at or;C by Dam methyltransferase. The analysis is based on the fact that methylation of CATC sequences makesthe DNA renairive10 Dpn, and r&tam to MM. Lanes a through f show cleavageby D&M and Mb, al increasingtimes after addition of Da,,, merhyhransferase(a) 1.5 min; b) 3 min; c) 4.5 min; d) 6 min: e) 10 min: 0 lOmint. I” the absenceof IHF, partial digerrs show that all GATC sequences are methylatedby Dam methyltransferase.In rhe presenceof 1°F (3 ag/mt), an IHF consensus sequenceis protectedfrom methyl&m. as are, in part, two GATC sequences beyond rhar region (see fig. 3).
eriC, ,~4IEMBRANEA TFACHMENT AND DNA BENDING bind (DnaA boxes). However, DnaA appears to function in initiation by interacting with three nearby sites identified as repeats of a 13-bp sequence (13-mers; Bramhill and Kernberg, 1988). How can DnaA protein recognize these relatively distant sequences, which have no homology to the primary binding sites.'? One possibility is that the transfer of DnaA is facilitated by bending of DNA in this region. IHF does indeed cause bending between the DnaA boxes and the 13-mers (Polaczek, 1990), allowing DnaA boxes to face the 13-met region. It was shown by in rive footprinting that thrc. of the boxes appear to be occupied at all times, whereas one, R3, is not (Samitt et aL, 1989). This suggests that R3 may be directly involved in regulation by DnaA. Bending of e r i c DNA by IHF places three D n a A boxes in register with the 13-mers, with R3 in the best position to "'face'" the 13-mers. TM
Is I H F indispensable for initiating replication'?. Apparently not. IHF stimulates the unwinding o f the 13-mer region, but so does H U (Bramhill and Kornberg, 1988). In addition, IHF deficient mutants are viable and so are HU-deficient mutants (Ogawa et uL, 1989; Kano and lmamoto, 1990). However, double mutants in these two proteins are difficult to grow and maintain (Kano and lmamoto, 1990). Thus, IHF is not essential and its function may be substituted by H U , possibly by a different mechanism of action. In an attempt to make a connection between the bending of e r i c and membrane binding, we have determined the influence of [HF on methylation. The IHF binding site in e r i c overlaps a recognition sequence for dam methyltransferase. This site is protected from in vitro methylation by Dam methyltransferase.
153
106-122
i
gain HI (92) Region grotectecl agslnst Dnese I
I
I
Regton erotectad against Darn methylgsa
Fig. 3. Schematic diagram of the in vitro protection studies with IHF. Binding of IHF to eriC results in protection from Dnase 1 to the left of an [HF consensus sequence located between bp 106 and 122 (Polaczek, 1990). [HF protects from methylation by the Dam methyhransferase the GATC sites within or overlapping the consensus sequence and also, in part, sites to the right of this region. A GATC site to the left of the IHF consensus sequence is protected from DnaseI but not flora methylation by the Dam methyltransferase.
To our surprise, we found that GATC sites not protected by IHF in a Dnase 1 footprint were protected from Dam methylation (figs 2 and 3). Lacking a better explanation, we attribute this resistance to methylarion to changes in tile conformation of the DNA.
Acknowledgements
If IHF-binding influences mefhylation, which in turn affects the binding of eriC to the membrane, might this protein play a role in the timing of initiation and segregation? IHF may be an element in a regulatory circuit consisting of a series of protein-DNA interactions that lead to precisely timed initiation at eriC. Ceils may overcome the lack of 1HF by adjusting the level of other proteins, thus functionally substituting IHF and achieving well regulated initiations.
References
Key-words:
DNA, oriC, E. coil; Chromosome, Bending, Attachment, Membrane.
This w o r k wa> ~rried ~ut with sup-
port from a U.S. Puhiic Heagh Service (National Institutes of Health)grant No. ROI 09465.
nramhil[, D. & Kornberg, A. (1988), Duplex opening by DnaA protein at novel sequences in initiation of replication at the origin of the E. coli chromosome. Cell, 52, 743-755. Campbt:ll. J.L. & Kleckncr, N. (1990). E. colt eric and the dnaA gene promoter are sequestered from dam mcthyltransferase following the passage of the chromosoma; replication fork. Cell, 62. 967-979. Hendrickson, W.G.. Kusano, T., Yamaki. H,. Balakrishnan. R., King, M.. Murchie. J. & Schaechter, M. (1982). Binding of the origin of replication of Escherichia colt to the outermembrane. Cell. 30, 915-923.
154
Kann, Y. & hnamoto, F (1990), Rcquirenlefll for integration bo~t factor (IHF) for growth of Eschcrichia coil deficicnl in HU prolein, Gene, 89, 133-137, Ogawa, T., Wada, M., Kano, Y., [ff ]lllOIO, F. & Okazaki, T, (1989). DNA replication in Escherichia coil mutants Ihal
M. SCHAECHTER E T AL. lack protein HU, J. Bac,, 171, 5672-5679. Ogden, O., Pratt, M.J. & Sehaechter, M. (1988), The replicative origin or the Escherichla coil chromosome binds to ceil membranes only when hemimelhylated. Cell, 54, 127-135. Polaczck, P. (1990), Bending of the
origin of replication of E. coil by binding of [HF at a specific site. New Biol., 2, 265-271. Samitt, C.E., Hansen. F.G.. Miller, J,F. & Scha¢chter, M. (1989), In vivo studies of DnaA binding to the origin of replication of Escherichia coll. EMBO J.. 8, 989-993.