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Replisome structure and conformational dynamics underlie fork progression past obstacles.

ABSTRACT: Replisomes are multiprotein complexes that unzip the parental helix and duplicate the separated strands during genome replication. The antiparallel structure of DNA poses unique geometric constraints to the process, and the replisome has evolved unique dynamic features that solve this problem. Interestingly, the solution to duplex DNA replication has been co-opted to solve many other important problems that replisomes must contend with during the duplication of long chromosomes. For example, along its path the replisome will encounter lesions and DNA-bound proteins. Recent studies show that the replisome can circumvent lesions on either strand, using the strategy normally applied to the lagging strand synthesis. Circumventing lesions can also be assisted by other proteins that transiently become a part of the replisome. The replisome must also contend with DNA-binding proteins and recent studies reveal a fascinating process that enables it to bypass RNA polymerase without stopping.

SUBMITTER: Yao NY

PROVIDER: S-EPMC3732650 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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Replisome structure and conformational dynamics underlie fork progression past obstacles.

Yao Nina Y NY O'Donnell Mike M

Current opinion in cell biology 20090416 3

Replisomes are multiprotein complexes that unzip the parental helix and duplicate the separated strands during genome replication. The antiparallel structure of DNA poses unique geometric constraints to the process, and the replisome has evolved unique dynamic features that solve this problem. Interestingly, the solution to duplex DNA replication has been co-opted to solve many other important problems that replisomes must contend with during the duplication of long chromosomes. For example, alo ...[more]

PMID: 19375905

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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.

Dataset Information

Replisome structure and conformational dynamics underlie fork progression past obstacles.

Publications

Replisome structure and conformational dynamics underlie fork progression past obstacles.

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