Article

The Disposition of Nascent Strands at Stalled Replication Forks Dictates the Pathway of Replisome Loading during Restart

Program in Molecular Biology, Weill Graduate School of Medical Sciences, Cornell University, New York, New York 10021, USA.
Molecular Cell (Impact Factor: 14.02). 04/2005; 17(5):733-43. DOI: 10.1016/j.molcel.2005.01.019
Source: PubMed

ABSTRACT

Rescue of arrested and collapsed replication forks is essential for maintenance of genomic integrity. One system for origin of replication-independent loading of the DnaB replicative helicase and subsequent replisome reassembly requires the structure-specific recognition factor PriA and the assembly factors PriB and DnaT. Here, we provide biochemical evidence for an alternate system for DnaB loading that requires only PriC. Furthermore, the choice of which system is utilized during restart is dictated by the nature of the structure of the stalled replication fork. PriA-dependent reactions are most robust on fork structures with no gaps in the leading strand, such as is found at the junction of a D loop, while the PriC-dependent system preferentially utilizes fork structures with large gaps in the leading strand. These observations suggest that the type of initial damage on the DNA template and how the inactivated fork is processed ultimately influence the choice of enzymatic restart pathway.

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    • "Alternatively, DnaB reloading can be catalysed by PriC. PriC has complementary forked DNA binding specificity to that of PriA, targeting forks lacking a 3' OH group of a nascent strand close to the fork branch point, but the outcome again is replicative helicase reloading onto the lagging strand template[25,27]. If a single stranded DNA region is absent on the lagging strand A C C E P T E D M A N U S C R I P T "
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    ABSTRACT: Replicative helicases play central roles in chromosome duplication and their assembly onto DNA is regulated via initiators and helicase loader proteins. The E. coli replicative helicase DnaB and the helicase loader DnaC form a DnaB6-DnaC6 complex that is required for loading DnaB onto ssDNA. Overexpression of dnaC inhibits replication by promoting continual rebinding of DnaC to DnaB and consequent prevention of helicase translocation. Here we show that overexpression of dnaB also inhibits growth and chromosome duplication. This inhibition is countered by co-overexpression of wild type DnaC but not of a DnaC mutant that cannot interact with DnaB, indicating that a reduction in DnaB6-DnaC6 concentration is responsible for the phenotypes associated with elevated DnaB concentration. Partial defects in the oriC-specific initiator DnaA and in PriA-specific initiation away from oriC during replication repair sensitise cells to dnaB overexpression. Absence of the accessory replicative helicase Rep, resulting in increased replication blockage and thus increased re-initiation away from oriC, also exacerbates DnaB-induced defects. These findings indicate that elevated levels of helicase perturb replication initiation not only at origins of replication but also during fork repair at other sites on the chromosome. Thus imbalances in levels of the replicative helicase and helicase loader can inhibit replication both via inhibition of DnaB6-DnaC6 complex formation with excess DnaB, as shown here, and promotion of formation of DnaB6-DnaC6 complexes with excess DnaC [1, 2]. Thus there are two mechanisms by which an imbalance in the replicative helicase and its associated loader protein can inhibit genome duplication.
    Full-text · Article · Jan 2016 · Journal of Molecular Biology
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    • "The importance of this replisome reassembly is underlined by the inviability of cells lacking PriA and a second structure-specific initiator PriC (Sandler 2000). PriA and PriC have complementary DNA substrate specificities, binding preferentially to forked DNA with and without a leading strand present at the branch point, respectively (Heller and Marians 2005b). The inviability of cells lacking both enzymes provides direct evidence for the low probability of replisomes assembled at oriC being able to complete chromosome duplication. "
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    ABSTRACT: The links between recombination and replication have been appreciated for decades and it is now generally accepted that these two fundamental aspects of DNA metabolism are inseparable: Homologous recombination is essential for completion of DNA replication and vice versa. This review focuses on the roles that recombination enzymes play in underpinning genome duplication, aiding replication fork movement in the face of the many replisome barriers that challenge genome stability. These links have many conserved features across all domains of life, reflecting the conserved nature of the substrate for these reactions, DNA.
    Full-text · Article · Oct 2014 · Cold Spring Harbor perspectives in biology
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    • "PriA interacts with SSB [38], PriB [15, 39, 40], and DnaT [15]. PriA can unwind the nascent lagging strand DNA to create a suitable binding site to help PriC load the DnaB helicase onto stalled replication forks where a gap exists in the nascent leading strand [41, 42]. The crystal structures of the N-terminal 105 amino acid residue segment of E. coli PriA (EcPriA) in complex with different deoxydinucleotides show a feasible interaction model for the base-non-selective recognition of the 3′-terminus of DNA between the nucleobase and the DNA-binding sites of EcPriA [43]. "
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    ABSTRACT: Replication restart primosome is a complex dynamic system that is essential for bacterial survival. This system uses various proteins to reinitiate chromosomal DNA replication to maintain genetic integrity after DNA damage. The replication restart primosome in Escherichia coli is composed of PriA helicase, PriB, PriC, DnaT, DnaC, DnaB helicase, and DnaG primase. The assembly of the protein complexes within the forked DNA responsible for reloading the replicative DnaB helicase anywhere on the chromosome for genome duplication requires the coordination of transient biomolecular interactions. Over the last decade, investigations on the structure and mechanism of these nucleoproteins have provided considerable insight into primosome assembly. In this review, we summarize and discuss our current knowledge and recent advances on the DNA-binding mode of the primosomal proteins PriA, PriB, and DnaT.
    Full-text · Article · Jul 2014 · BioMed Research International
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