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Localization of an accessory helicase at the replisome is critical in sustaining efficient genome duplication.

ABSTRACT: Genome duplication requires accessory helicases to displace proteins ahead of advancing replication forks. Escherichia coli contains three helicases, Rep, UvrD and DinG, that might promote replication of protein-bound DNA. One of these helicases, Rep, also interacts with the replicative helicase DnaB. We demonstrate that Rep is the only putative accessory helicase whose absence results in an increased chromosome duplication time. We show also that the interaction between Rep and DnaB is required for Rep to maintain rapid genome duplication. Furthermore, this Rep-DnaB interaction is critical in minimizing the need for both recombinational processing of blocked replication forks and replisome reassembly, indicating that colocalization of Rep and DnaB minimizes stalling and subsequent inactivation of replication forks. These data indicate that E. coli contains only one helicase that acts as an accessory motor at the fork in wild-type cells, that such an activity is critical for the maintenance of rapid genome duplication and that colocalization with the replisome is crucial for this function. Given that the only other characterized accessory motor, Saccharomyces cerevisiae Rrm3p, associates physically with the replisome, our demonstration of the functional importance of such an association indicates that colocalization may be a conserved feature of accessory replicative motors.

SUBMITTER: Atkinson J

PROVIDER: S-EPMC3035471 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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Localization of an accessory helicase at the replisome is critical in sustaining efficient genome duplication.

Atkinson John J Gupta Milind K MK Rudolph Christian J CJ Bell Hazel H Lloyd Robert G RG McGlynn Peter P

Nucleic acids research 20101004 3

Genome duplication requires accessory helicases to displace proteins ahead of advancing replication forks. Escherichia coli contains three helicases, Rep, UvrD and DinG, that might promote replication of protein-bound DNA. One of these helicases, Rep, also interacts with the replicative helicase DnaB. We demonstrate that Rep is the only putative accessory helicase whose absence results in an increased chromosome duplication time. We show also that the interaction between Rep and DnaB is required ...[more]

PMID: 20923786

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Project description:Replicative DNA helicases expose the two strands of the double helix to the replication apparatus, but accessory helicases are often needed to help forks move past naturally occurring hard-to-replicate sites, such as tightly bound proteins, RNA/DNA hybrids, and DNA secondary structures. Although the Schizosaccharomyces pombe 5'-to-3' DNA helicase Pfh1 is known to promote fork progression, its genomic targets, dynamics, and mechanisms of action are largely unknown. Here we address these questions by integrating genome-wide identification of Pfh1 binding sites, comprehensive analysis of the effects of Pfh1 depletion on replication and DNA damage, and proteomic analysis of Pfh1 interaction partners by immunoaffinity purification mass spectrometry. Of the 621 high confidence Pfh1-binding sites in wild type cells, about 40% were sites of fork slowing (as marked by high DNA polymerase occupancy) and/or DNA damage (as marked by high levels of phosphorylated H2A). The replication and integrity of tRNA and 5S rRNA genes, highly transcribed RNA polymerase II genes, and nucleosome depleted regions were particularly Pfh1-dependent. The association of Pfh1 with genomic integrity at highly transcribed genes was S phase dependent, and thus unlikely to be an artifact of high transcription rates. Although Pfh1 affected replication and suppressed DNA damage at discrete sites throughout the genome, Pfh1 and the replicative DNA polymerase bound to similar extents to both Pfh1-dependent and independent sites, suggesting that Pfh1 is proximal to the replication machinery during S phase. Consistent with this interpretation, Pfh1 co-purified with many key replisome components, including the hexameric MCM helicase, replicative DNA polymerases, RPA, and the processivity clamp PCNA in an S phase dependent manner. Thus, we conclude that Pfh1 is an accessory DNA helicase that interacts with the replisome and promotes replication and suppresses DNA damage at hard-to-replicate sites. These data provide insight into mechanisms by which this evolutionarily conserved helicase helps preserve genome integrity.

Project description:BackgroundThe health policy and systems research literature increasingly observes that knowledge translation (KT) practices are difficult to sustain. An important issue is that it remains unclear what sustainability of KT practices means and how it can be improved. The aim of this study was thus to identify and explain those processes, activities, and efforts in the literature that facilitate the sustaining of KT practices in health policy-making processes.MethodsWe used a critical interpretive synthesis (CIS) to review the health policy and systems research and Science and Technology Studies (STS) literature. The STS literature was included as to enrich the review with constructivist social scientific perspectives on sustainability and KT. The CIS methodology allowed for creating new theory by critically combining both literatures. We searched the literature by using PubMed, Google Scholar, Web of Science, and qualitative sampling. Searches were guided by pre-set eligibility criteria and all entries were iteratively analysed using thematic synthesis.ResultsEighty documents were included. Our synthesis suggests a shift from sustainability as an end-goal towards sustaining as actors' relatively mundane work aimed at making and keeping KT practices productive. This 'sustaining work' is an interplay of three processes: (i) translating, (ii) contexting, and (iii) institutionalising. Translating refers to activities aimed at constructing and extending networks. Contexting emphasises the activities needed to create contexts that support KT practices. Institutionalising addresses how actors create, maintain, and disrupt institutions with the aim of sustaining KT practices.ConclusionThe 'sustaining work' perspective of our CIS emphasises KT actors' ongoing work directed at sustaining KT practices. We suggest that this perspective can guide empirical study of sustaining work and that these empirical insights, combined with this CIS, can inform training programmes for KT actors, and thereby improve the sustainability of KT practices.

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Localization of an accessory helicase at the replisome is critical in sustaining efficient genome duplication.

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Localization of an accessory helicase at the replisome is critical in sustaining efficient genome duplication.

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