Abstract
Replicative helicases unwind double-stranded DNA in front of the polymerase and ensure the processivity of DNA synthesis. In Escherichia coli, the helicase loader DnaC as well as factors involved in the formation of the open complex during the initiation of replication and primosomal proteins during the reactivation of arrested replication forks are required to recruit and deposit the replicative helicase onto single-stranded DNA prior to the formation of the replisome. dnaC2 is a thermosensitive allele of the gene specifying the helicase loader; at non-permissive temperature replication cannot initiate, but most ongoing rounds of replication continues through to completion (18% of dnaC2 cells fail to complete replication at non-permissive temperature). An assumption, which may be drawn from this observation, is that only a few replication forks are arrested under normal growth conditions. This assumption, however, is at odds with the severe and deleterious phenotypes associated with a null mutant of priA, the gene encoding a helicase implicated in the reactivation of arrested replication forks. We developed an assay that involves an abrupt inactivation of rounds of synchronized replication in a large population of cells, in order to evaluate the ability of dnaC2 cells to reactivate arrested replication forks at non-permissive temperature. We compared the rate at which arrested replication forks accumulated in dnaC2 priA+ and dnaC2 priA2 cells and observed that this rate was lower in dnaC2 priA+ cells. We conclude that while replication cannot initiate in a dnaC2 mutant at non-permissive temperature, a class of arrested replication forks (PriA-dependent and DnaC-independent) are reactivated within these cells.
Highlights
The processivity of DNA replication requires a 59R39 replicative helicase - DnaB in Escherichia coli - to unwind doublestranded DNA in front of, and in interaction with, the replisome [1]
Assessing the fraction of cells with active replication forks (RF) The appreciation of the ability of dnaC2 cells to reactivate arrested RF requires a precise evaluation of the proportion of cells with active RF in a given population, which is made possible by flow cytometry
Inactivating RF with Novobiocin We developed a procedure to generate a large quantity of arrested RF in cultures of synchronized cells to assess the capacity of dnaC2 cells to reactivate RF at non-permissive temperature
Summary
The processivity of DNA replication requires a 59R39 replicative helicase - DnaB in Escherichia coli - to unwind doublestranded DNA in front of, and in interaction with, the replisome [1]. The most prominent pathway involves PriA and the primosomal proteins PriB and DnaT [8] In this case, the 39R59 helicase activity specified by PriA is required to make available a sufficient length of single-stranded DNA to allow the assembly of the primosomal proteins and the subsequent loading of DnaB onto the lagging strand [9]. The 39R59 helicase activity specified by PriA is required to make available a sufficient length of single-stranded DNA to allow the assembly of the primosomal proteins and the subsequent loading of DnaB onto the lagging strand [9] Other proteins, such as Rep, which specifies a 39R59 helicase activity, and PriC may have important functions during the reactivation of arrested RF [8,9,10]
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