The Werner Syndrome Helicase/Exonuclease Processes Mobile D-Loops through Branch Migration and Degradation

Patricia L Opresko,Hong Wang,Gregory Sowd,Mikhail V Blagosklonny

doi:10.1371/journal.pone.0004825

Patricia L Opresko, Hong Wang + Show 2 more

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https://doi.org/10.1371/journal.pone.0004825

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Journal: PloS one	Publication Date: Mar 13, 2009
Citations: 105	License type: CC BY 4.0

Affiliation: University of Pittsburgh, UPMC Hillman Cancer Center

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.

Highlights

Werner syndrome (WS) is an autosomal recessive disorder marked by the premature onset of numerous features associated with aging and a predisposition to mesenchymal cancers [1]
These data demonstrate that the mobile displacement loop (D-loop) are stable and contain an invading strand that is completely base paired with the complementary plasmid strand
Increasing evidence supports an important role for Werner syndrome protein (WRN) protein in mediating the proper dissociation of joint DNA molecules that arise in genomic and telomeric regions during homologous recombination and repair of breaks at collapsed replication forks

Summary

Introduction

Werner syndrome (WS) is an autosomal recessive disorder marked by the premature onset of numerous features associated with aging and a predisposition to mesenchymal cancers [1]. WS is caused by loss of the DNA repair protein WRN which is a member of the RecQ family of DNA helicases [2]. E. coli and S. cerevisiase each have a single family member, whereas five members exist in humans: RECQ1, RECQ4, RECQ5, BLM and WRN [3,4]. Mutations in the human RecQ helicases BLM and RECQ4 give rise to the cancer predisposition disorders Bloom syndrome (BS) and Rothmund-Thomson syndrome (RTS), respectively [5]. Despite the common feature of genomic instability, these disorders are clinically distinct from WS. While there are no known human disorders caused by defects in RECQ1 or RECQ5, cellular and transgenic mouse studies indicate that loss of these proteins leads to genomic instability [5]. RecQ helicases have been classified as ‘‘caretakers’’ of the genome [3]

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