Mutations in theE. coliRep helicase increase the amount of DNA per cell

Abstract

The mbrA4 mutation confers camphor resistance, severe growth defects and up to a two-fold increase in the amount of chromosomal DNA per cell. The extra DNA is replicated from oriC in a synchronous fashion. Cells containing mbrA4 are more resistant to X-rays, indicating that the extra DNA represents complete or nearly complete chromosomes. I report here that mbrA4 is an unusual allele of the leading strand DNA helicase, Rep. Eight independently isolated alleles of rep(mbrA) contain the same three changes in the rep gene: a G to A at position 344 from the start of the mRNA (+1); an opal stop at codon 142; and a glycine to serine at codon 414 (G414S). My data indicate that rep(mbrA4) is not a null mutation and that the third mutation, G414S, is necessary for camphor resistance, the phenotype associated with increased DNA content per cell. I also show that increase in DNA content does not lead to independently segregating chromosomes. ß

Habitat Visualization and Genomic Analysis of “Candidatus Pantoea carbekii,” the Primary Symbiont of the Brown Marmorated Stink Bug

“…Rep helicase is required for replication in E. coli , preventing double-stranded breaks and acting in a replication fork restart pathway ( Michel et al 1997 ; Sandler 2000 ). The absence of rep in E. coli results in severe growth problems, namely the accumulation of DNA within a single cell ( Trun 2003 ). We detected the 8-oxo-dGTP diphosphatase ( mutT ) gene on pBMSBPS2 and it encodes half of the base excision repair (BER) system whereas the formamidopyrimidine DNA glycosylase, encoded by mutM was absent; mutM deletions in E. coli have been shown to result in an increase in GC→AT transversions ( Cox 1976 ; Cabrera et al 1988 ).…”

“…The observed cell morphology may reflect cells undergoing cell division but the loss of the Rep helicase ( rep ) and presence of a truncated FtsK and pseudogenized FtsN gene may impair cytokinesis. Mutations in rep in E. coli resulted in severe growth defects and a near-doubling of the amount of chromosomal DNA present within each cell ( Trun 2003 ). FtsK directs DNA translocation and chromosome segregation during cytokinesis in E. coli and the P. carbekii ortholog (932 amino acids) is approximately 80% of the length of FtsK in P. ananatis (1,112 amino acids) and the P. stali symbiont (1,143 amino acids).…”

Unwinding of the Nascent Lagging Strand by Rep and PriA Enables the Direct Restart of Stalled Replication Forks

“…The overall replication rate is reduced in the absence of Rep (29), manifested by an increase in the total amount of DNA per cell (30,44) and an increase in the average number of growing forks in exponentially growing cells (45). One potential explanation of these observations is that the actual speed of the replication fork is reduced, but a more likely reason is that the replication fork frequently pauses or stalls.…”