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The Werner syndrome helicase/exonuclease processes mobile D-loops through branch migration and degradation.

ABSTRACT: RecQ DNA helicases are critical for preserving genome integrity. Of the five RecQ family members identified in humans, only the Werner syndrome protein (WRN) possesses exonuclease activity. Loss of WRN causes the progeroid disorder Werner syndrome which is marked by cancer predisposition. Cellular evidence indicates that WRN disrupts potentially deleterious intermediates in homologous recombination (HR) that arise in genomic and telomeric regions during DNA replication and repair. Precisely how the WRN biochemical activities process these structures is unknown, especially since the DNA unwinding activity is poorly processive. We generated biologically relevant mobile D-loops which mimic the initial DNA strand invasion step in HR to investigate whether WRN biochemical activities can disrupt this joint molecule. We show that WRN helicase alone can promote branch migration through an 84 base pair duplex region to completely displace the invading strand from the D-loop. However, substrate processing is altered in the presence of the WRN exonuclease activity which degrades the invading strand both prior to and after release from the D-loop. Furthermore, telomeric D-loops are more refractory to disruption by WRN, which has implications for tighter regulation of D-loop processing at telomeres. Finally, we show that WRN can recognize and initiate branch migration from both the 5' and 3' ends of the invading strand in the D-loops. These findings led us to propose a novel model for WRN D-loop disruption. Our biochemical results offer an explanation for the cellular studies that indicate both WRN activities function in processing HR intermediates.

SUBMITTER: Opresko PL

PROVIDER: S-EPMC2653227 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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The Werner syndrome helicase/exonuclease processes mobile D-loops through branch migration and degradation.

Opresko Patricia L PL Sowd Gregory G Wang Hong H

PloS one 20090313 3

RecQ DNA helicases are critical for preserving genome integrity. Of the five RecQ family members identified in humans, only the Werner syndrome protein (WRN) possesses exonuclease activity. Loss of WRN causes the progeroid disorder Werner syndrome which is marked by cancer predisposition. Cellular evidence indicates that WRN disrupts potentially deleterious intermediates in homologous recombination (HR) that arise in genomic and telomeric regions during DNA replication and repair. Precisely how ...[more]

PMID: 19283071

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Project description:BackgroundThe cancer-prone and accelerated aging disease Werner syndrome is caused by loss of function of the WRN gene product that possesses ATPase, 3' to 5' helicase and 3' to 5' exonuclease activities. Although WRN has been most prominently suggested to function in telomere maintenance, resolution of replication blockage and/or recombinational repair, its exact role in DNA metabolism remains unclear. WRN is the only human RecQ family member to possess both helicase and exonuclease activity, but the mechanistic relationship between these activities is unknown. In this study, model single-stranded and 3' overhang DNA substrates of varying length and structure were used to examine potential coordination between the ATPase/helicase and exonuclease activities of WRN.ResultOur results show that WRN can not only bind to but also catalyze the 3' to 5' degradation of single-stranded and 3' overhang DNA substrates, structures that were previously thought to be refractory to WRN exonuclease activity. The length of the single-stranded regions in these structures is a critical parameter in determining both the binding affinity and the level of exonuclease activity of WRN. Most importantly, specific nucleotide cofactors dramatically stimulate WRN exonuclease activity on these substrates, with conditions that permit ATP hydrolysis not only resulting in enhanced exonuclease activity but also altering its length dependence on these structures. Parallel experiments show that a deletion mutant containing only the WRN exonuclease domain lacks both this DNA length and nucleotide cofactor dependence, demonstrating that the interaction of the ATPase/helicase domain of WRN with the DNA substrate has a profound influence on exonuclease activity.ConclusionOur results indicate that, under conditions that permit ATP hydrolysis, there is a dynamic and cooperative relationship between the distinct ATPase/helicase and exonuclease domains of WRN with regard to their orientation on DNA. Based on these results, models are proposed for the coordinated, unidirectional 3' to 5' movement of the helicase and exonuclease domains of WRN on DNA that should be informative for elucidating its function in genome maintenance.

Dataset Information

The Werner syndrome helicase/exonuclease processes mobile D-loops through branch migration and degradation.

Publications

The Werner syndrome helicase/exonuclease processes mobile D-loops through branch migration and degradation.

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