The DnaM Protein Plays Roles In Maintaining The Escherichia Coli Genome Integrity

The genome stability ensures that each daughter cell receives a complete genome without mutation after cell division during the cell cycle.Functions of the dnaM(ybfEis renamed as dnaM regarding its functions found here)gene and its product in Escherichia coli are unknown.The study showed that the dnaM gene was not essential since deletion of dnaM was not lethal for cell survival.Deletion or overexpression of the dnaM gene led to abnormal replications including asynchronous replication initiation and incomplete replication.Further,deletion of the dnaM gene also increased the rate of mutagenesis,expression of uvrB(the SOS gene)and of dnaM itself,leading to that the cell was sensitive to the UV irradiation.The results indicate that DnaM is likely involved in maintaining the genome stability.Fluorescent microscopy analsysis showed that DnaM-GFP co-localizeed with nucleoids in exponentially growing cells,and the colocalization was only observed during the replication of chromosomes(C-period).It was found that purified DnaM-His digested linear double-stranded DNA(dsDNA),single-stranded DNA(ssDNA),and also relaxed supercoiled plasmid DNA(scDNA),indicating that DnaM possesses exonuclease and topoisomerase activities.Interestingly,digestion of the 3'-biotin labeled DNA with DnaM showed that DnaM cut linear DNA molecule from its 3' end,having activity of the 3'?5' exonuclease.Further,DnaM nicked on one strand of closed dsDNA plasmid so that it relaxed the supercoiled DNA molecule.To find out the active site of DnaM,19 mutants carrying a single amino acid change were generated.Functions of the DnaMK38I,DnaMK51E,DnaMR52G,DnaMK56E and DnaMR76G mutant proteins were changed.Very interestingly,DnaMR92G but not DnaMK51E still colocalized with nucleoids,and DnaM functioned while DnaMK51Enot,suggesting that DnaM functions through colocalizing with nucleoids.Further,K51E and R92G mutations decreased the exonuclease activities but strengthened the topoisomerase activities of DnaM,indicating that the two amino acids are important in function of DnaM.To understand how DnaM colocalizes with nucleiod,the interaction of DnaM with DnaB,DnaN or DnaX was detected by the bacterial two-hybrid.The result showed that DnaM interacted physically with DnaN to colocalize with nucleoids only during the chromosome replication period of the cell cycle.Taken all data described together,we propose that DnaM is a back-up process for maintaining the E.coli genome integrity,where DnaM colocalizes with chromosome during replication to correct errors and relax supercoils in front of replication fork,possessing 3'?5' exonuclease and topoisomerase activities.During chromosome replication,DNA helicase unwinds duplex DNA and introduces the supercoiled dsDNA which is relaxed by topoisomerase in front of replication fork.Sliding clamp,the DnaN dimer,anchors DNA polemerase III on the template strand,ensuring the processiveness of chromosome replication.After release of the DNA polemerase,the DnaN dimer leaves the template DNA.Therefore,during the chromosome replication,DnaM localizes at the replication fork through interacting with DnaN and removes wrong nucleotide newly-incooperated with its 3'?5 activity,making sure that replication is free-error;DnaM also assists DNA gyrase to relax the supercoils of replicating chromosomes for guaranteeing smooth process of replication due to its topoisomerase activity.