In vivo function of the conserved non-catalytic domain of Werner syndrome helicase in DNA replication

Laboratory of Molecular Gerontology, National Institute on Aging, NIH, 5600 Nathan Shock Drive, Baltimore, Maryland 21224, USA.
Human Molecular Genetics (Impact Factor: 6.39). 11/2004; 13(19):2247-61. DOI: 10.1093/hmg/ddh234
Source: PubMed


Werner syndrome is a genetic disorder characterized by genomic instability, elevated recombination and replication defects. The WRN gene encodes a RecQ helicase whose function(s) in cellular DNA metabolism is not well understood. To investigate the role of WRN in replication, we examined its ability to rescue cellular phenotypes of a yeast dna2 mutant defective in a helicase-endonuclease that participates with flap endonuclease 1 (FEN-1) in Okazaki fragment processing. Genetic complementation studies indicate that human WRN rescues dna2-1 mutant phenotypes of growth, cell cycle arrest and sensitivity to the replication inhibitor hydroxyurea or DNA damaging agent methylmethane sulfonate. A conserved non-catalytic C-terminal domain of WRN was sufficient for genetic rescue of dna2-1 mutant phenotypes. WRN and yeast FEN-1 were reciprocally co-immunoprecipitated from extracts of transformed dna2-1 cells. A physical interaction between yeast FEN-1 and WRN is demonstrated by yeast FEN-1 affinity pull-down experiments using transformed dna2-1 cells extracts and by ELISA assays with purified recombinant proteins. Biochemical analyses demonstrate that the C-terminal domain of WRN or BLM stimulates FEN-1 cleavage of its proposed physiological substrates during replication. Collectively, the results suggest that the WRN-FEN-1 interaction is biologically important in DNA metabolism and are consistent with a role of the conserved non-catalytic domain of a human RecQ helicase in DNA replication intermediate processing.

3 Reads
    • "We show that both RECQL5 and WRN physically interact with each other and that their association is likely enhanced during S-phase under replicative stress. WRN plays a critical role in response to replicative stress and significantly contributes to the recovery of stalled replication forks (53,63,64). WRN re-localizes from the nucleolus to the nucleus after replicative stress and co-localizes with the MRN complex at proliferating cell nuclear antigen (PCNA) sites during S-phase, and it was further shown that this re-localization of WRN requires MRE11 (42,65). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Humans have five RecQ helicases, whereas simpler organisms have only one. Little is known about whether and how these RecQ helicases co-operate and/or complement each other in response to cellular stress. Here we show that RECQL5 associates longer at laser-induced DNA double-strand breaks in the absence of Werner syndrome (WRN) protein, and that it interacts physically and functionally with WRN both in vivo and in vitro. RECQL5 co-operates with WRN on synthetic stalled replication fork-like structures and stimulates its helicase activity on DNA fork duplexes. Both RECQL5 and WRN re-localize from the nucleolus into the nucleus after replicative stress and significantly associate with each other during S-phase. Further, we show that RECQL5 is essential for cell survival in the absence of WRN. Loss of both RECQL5 and WRN severely compromises DNA replication, accumulates genomic instability and ultimately leads to cell death. Collectively, our results indicate that RECQL5 plays both co-operative and complementary roles with WRN. This is an early demonstration of a significant functional interplay and a novel synthetic lethal interaction among the human RecQ helicases.
    Nucleic Acids Research 11/2012; 41(2). DOI:10.1093/nar/gks1134 · 9.11 Impact Factor
  • Source
    • "However, stimulation of FEN-1 is mediated by direct protein-protein interaction but does not require WRN catalytic activity [108, 112]. In fact, expression of a conserved noncatalytic C-terminal domain of WRN necessary and sufficient for the physical and functional interaction with FEN-1 is sufficient to rescue the yeast dna2-1 mutant phenotypes [113]. The conserved C-terminal in BLM was subsequently also found to mediate a physical and functional interaction with FEN-1 [109], and the phenotypes of yeast dna2 mutants can be rescued by expression of BLM [114]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: In addition to the canonical B-form structure first described by Watson and Crick, DNA can adopt a number of alternative structures. These non-B-form DNA secondary structures form spontaneously on tracts of repeat sequences that are abundant in genomes. In addition, structured forms of DNA with intrastrand pairing may arise on single-stranded DNA produced transiently during various cellular processes. Such secondary structures have a range of biological functions but also induce genetic instability. Increasing evidence suggests that genomic instabilities induced by non-B DNA secondary structures result in predisposition to diseases. Secondary DNA structures also represent a new class of molecular targets for DNA-interactive compounds that might be useful for targeting telomeres and transcriptional control. The equilibrium between the duplex DNA and formation of multistranded non-B-form structures is partly dependent upon the helicases that unwind (resolve) these alternate DNA structures. With special focus on tetraplex, triplex, and cruciform, this paper summarizes the incidence of non-B DNA structures and their association with genomic instability and emphasizes the roles of RecQ-like DNA helicases in genome maintenance by resolution of DNA secondary structures. In future, RecQ helicases are anticipated to be additional molecular targets for cancer chemotherapeutics.
    Journal of nucleic acids 10/2011; 2011(8):724215. DOI:10.4061/2011/724215
  • Source
    • "Thus, the helicase activity of BLM is required to resolve secondary structure in the flaps, thereby facilitating Fen1- catalyzed cleavage. This activity was not determined with WRN (Sharma et al., 2004a; Wang and Bambara, 2005). BLM could resolve the secondary structure in the 5′ flap or remove a blocking primer annealed to the 5′ flap by translocating from the base of the flap in the 3′ to 5′ direction. "
    [Show abstract] [Hide abstract]
    ABSTRACT: DNA replication is a primary mechanism for maintaining genome integrity, but it serves this purpose best by cooperating with other proteins involved in DNA repair and recombination. Unlike leading strand synthesis, lagging strand synthesis has a greater risk of faulty replication for several reasons: First, a significant part of DNA is synthesized by polymerase alpha, which lacks a proofreading function. Second, a great number of Okazaki fragments are synthesized, processed and ligated per cell division. Third, the principal mechanism of Okazaki fragment processing is via generation of flaps, which have the potential to form a variety of structures in their sequence context. Finally, many proteins for the lagging strand interact with factors involved in repair and recombination. Thus, lagging strand DNA synthesis could be the best example of a converging place of both replication and repair proteins. To achieve the risky task with extraordinary fidelity, Okazaki fragment processing may depend on multiple layers of redundant, but connected pathways. An essential Dna2 endonuclease/helicase plays a pivotal role in processing common structural intermediates that occur during diverse DNA metabolisms (e.g. lagging strand synthesis and telomere maintenance). Many roles of Dna2 suggest that the preemptive removal of long or structured flaps ultimately contributes to genome maintenance in eukaryotes. In this review, we describe the function of Dna2 in Okazaki fragment processing, and discuss its role in the maintenance of genome integrity with an emphasis on its functional interactions with other factors required for genome maintenance.
    Critical Reviews in Biochemistry and Molecular Biology 04/2010; 45(2):71-96. DOI:10.3109/10409230903578593 · 7.71 Impact Factor
Show more