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

Abstract

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:

Leptospira genomes are modified at 5'-GTAC

“…One exception to the rule that sequence-specific modification systems are present in some, but not all, strains in any species is the dam methylation system (5'-Gm6ATC), which occurs in all strains of several species in the family Enterobacteriaceae and related genera (3). Rather than being part of a restriction modification system, dam methylation is part of a daughter strand repair system (1,14) and also regulates replication (25,26). It is presumably because of a role in maintaining all cells that the dam modification is found throughout those species in which it occurs.…”