Importance of state of methylation of oriC GATC sites in initiation of DNA replication in Escherichia coli.

Smith, Douglas W.; Garland, Adella M.; Herman, Gail E.; Enns, Robert E.; Baker, Tania A.; Zyskind, Judith W.

doi:10.1002/j.1460-2075.1985.tb03779.x

Cited by 136 publications

(76 citation statements)

References 37 publications

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“…Previous experiments demonstrated that dam bacterial strains are poorly transformed by fully methylated and hemimethylated plasmids dependent on either the chromosomal replication origin oriC or the pMBl plasmid origin (28,38,39). This is due to the lack of replication of hemimethylated molecules, which accumulate in dam strains transformed by methylated plasmids (38).…”

Section: Resultsmentioning

confidence: 99%

“…The dam sites in oriC lie in a complex of repeats which may adopt several different secondary structures (29,39), suggesting similarities to the inverted repeat in the pMB1 primer promoter. The smaller effects of methylation on pMB1 replication in mutH strains and the regulation of oriC by methylation which is independent of MutH may represent similar phenomena.…”

Section: Resultsmentioning

confidence: 99%

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The mutH gene regulates the replication and methylation of the pMB1 origin

Russell

Horiuchi

1991

J Bacteriol

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DNA methylation is known to regulate several prokaryotic replication origins. In particular, the Escherichia coli chromosomal origin oriC and the pMB1 plasmid origin (which is homologous to the ColEl origin) replicate poorly when hemimethylated at dam (GATC) sites. Because the mismatch repair protein MutH is known to recognize hemimethylated dam sites, its role in the replication of these origins was investigated. The results presented here show that the mutH gene product is partially responsible for the poor replication of the pMBl origin when hemimethylated but has no effect on the replication of oriC. Methylation levels at individual dam sites suggest that the MutH protein binds to an inverted repeat in the pMBl replication primer promoter. These findings suggest a mechanism for the coordinated control of DNA repair and replication.Both DNA replication and repair require the differentiation of newly replicated molecules from unreplicated molecules. In Escherichia coli, this distinction is made by recognizing hemimethylated dam sites (5'-GATC-3' sequences) produced during semiconservative DNA replication of fully methylated parent molecules. This mechanism was first suggested by Wagner and Meselson (42), who proposed that the absence of methylated adenine residues in newly synthesized DNA strands enabled their identification and preferential correction during mismatch repair. This hypothesis was subsequently verified both in vivo (34) and in vitro (24). The repair system requires the mutH, mutL, mutS, and uvrD gene products, exonuclease I, DNA polymerase III, singlestranded DNA-binding protein (21,24), and the presence of dam sites (20,22). The Dam methylase of E. coli methylates the N6 position of adenines in both strands at dam sites (11,13,19). The MutH protein recognizes and shows weak endonucleolytic activity towards hemimethylated and unmethylated dam sites (43).Newly replicated molecules must also be distinguished during DNA replication, in order to prevent immediate reinitiation and origin amplification. In E. coli, the inhibition of replication produced by hemimethylation prevents reinitiation of newly replicated, hemimethylated daughter molecules. Both oriC and the pMB1 plasmid origin (which is similar to the ColEl origin) replicate effectively when fully methylated or unmethylated but not when hemimethylated (38). In addition, replication of the plasmid prophage of bacteriophage P1 is controlled by methylation (1). The exact molecular mechanisms of these regulations remain unknown at present. However, the fact that recognition of hemimethylated DNA plays a role in both mismatch repair and replication suggests that common processes may be involved.In this report we show that the MutH protein, known to recognize hemimethylated and unmethylated dam sites dur-* Corresponding author. t Present address:

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

The mutH gene regulates the replication and methylation of the pMB1 origin

Russell

Horiuchi

1991

J Bacteriol

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“…Methylation at ori-C also influences the local DNA structure and stability (62, 66 -71) and its interaction with replication proteins (72). Hemimethylated ori-C is refractory to further initiation events (73)(74)(75) due to the inability of DnaA protein to access ori-C (76), thus preventing premature reinitiation when free DnaA levels are still high (64,65,76). OriC persists in this hemimethylated/sequestered state for ϳ8 min, or about 10 times longer than for most other sites on the E. coli chromosome (64,65).…”

Section: Figmentioning

confidence: 99%

DNA Methylation at Mammalian Replication Origins

Rein

Kobayashi²,

Malott³

et al. 1999

Journal of Biological Chemistry

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In Escherichia coli, DNA methylation regulates both origin usage and the time required to reassemble prereplication complexes at replication origins. In mammals, at least three replication origins are associated with a high density cluster of methylated CpG dinucleotides, and others whose methylation status has not yet been characterized have the potential to exhibit a similar DNA methylation pattern. One of these origins is found within the ϳ2-kilobase pair region upstream of the human c-myc gene that contains 86 CpGs. Application of the bisulfite method for detecting 5-methylcytosines at specific DNA sequences revealed that this region was not methylated in either total genomic DNA or newly synthesized DNA. Therefore, DNA methylation is not a universal component of mammalian replication origins. To determine whether or not DNA methylation plays a role in regulating the activity of origins that are methylated, the rate of remethylation and the effect of hypomethylation were determined at origin ␤ (ori-␤), downstream of the hamster DHFR gene. Remethylation at ori-␤ did not begin until ϳ500 base pairs of DNA was synthesized, but it was then completed by the time that 4 kilobase pairs of DNA was synthesized (<3 min after release into S phase). Thus, DNA methylation cannot play a significant role in regulating reassembly of prereplication complexes in mammalian cells, as it does in E. coli. To determine whether or not DNA methylation plays any role in origin activity, hypomethylated hamster cells were examined for ori-␤ activity. Cells that were >50% reduced in methylation at ori-␤ no longer selectively activated ori-␤. Therefore, at some loci, DNA methylation either directly or indirectly determines where replication begins.In early embryos undergoing rapid cell cleavage (e.g. frogs, flies, sea urchin, fish), initiation of DNA replication appears neither to require specific DNA sequences nor to occur at specific DNA sites. However, as development progresses past the blastula stage, initiation of DNA replication begins to occur at specific sites (1, 2). Thus, it is not surprising that initiation sites for DNA replication in cultured mammalian cells occur at specific genomic loci (3). For example, a 200-kb 1 region at the human ␤-globin gene (4, 5) and a 500-kb region at the mouse IgH gene (6) are both replicated from a single initiation locus. Moreover, what previously had been viewed as random initiation events distributed throughout "initiation zones" in Schizosaccharomyces pombe and hamster cells more likely reflects the presence of several strongly preferred initiation sites (7,8).Nevertheless, the precise size and composition of these initiation loci, as well as the parameters that define them remain the subject of intense investigation.The fact that specific sites for initiation of DNA replication can be developmentally acquired makes it clear that initiation sites in the metazoa are determined at least in part by epigenetic parameters. These parameters include nuclear structure, chromatin structure, and even...

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“…The first type of evidence is the effect of dam mutations on replication of plasmids dependent on the oriC origin. E. coli dam mutants are transformed inefficiently by fully methylated oriC plasmids (20,26). Finding that unmethylated oriC plasmids replicate efficiently in dam mutants, that hemimethylated daughter molecules accumulate in dam mutant strains after transformation with fully methylated plasmids, and that hemimethylated plasmids transform dam mutant bacteria poorly, Russell and Zinder (25) concluded that hemimethylated oriC replicons cannot undergo initiation.…”

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confidence: 99%

“…2A. The strain of E. coli used in these studies was DS1310 F-dam-3 rpsL metBI trpR55 lacYl galK2 galT22 supE44 supF58 hsdR514 (26). The plasmid pAB20 (Ptac dam' bla lacIq) was constructed by ligating the 1.3-kb PvuII fragment of pPJ39 (14) into the EcoRI-HindIII fragment of pJF118EH (10).…”

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confidence: 99%

Escherichia coli cells lacking methylation-blocking factor (leucine-responsive regulatory protein) have precise timing of initiation of DNA replication in the cell cycle

et al. 1992

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A protein that is required for specific methylation inhibition of two GATC sites in the papBA pilin promoter region, known as methylation-blocking factor (Mbf) and recently shown to be identical to the leucineresponsive regulatory protein (Lrp), is not responsible for the delayed methylation at oniC implicated in an eclipse period following initiation of DNA replication. CeUs containing a transposon mutation within the mbf (bp) gene initiate DNA replication at the correct time during the cel cycle, whereas ceUs with increased amounts of the Dam methyltransferase initiate DNA replication randomly throughout the cell cycle.

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Importance of state of methylation of oriC GATC sites in initiation of DNA replication in Escherichia coli.

Cited by 136 publications

References 37 publications

The mutH gene regulates the replication and methylation of the pMB1 origin

The mutH gene regulates the replication and methylation of the pMB1 origin

DNA Methylation at Mammalian Replication Origins

Escherichia coli cells lacking methylation-blocking factor (leucine-responsive regulatory protein) have precise timing of initiation of DNA replication in the cell cycle

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