WRN Helicase and FEN-1 Form a Complex upon Replication Arrest and Together Process Branchmigrating DNA Structures Associated with the Replication Fork

Abstract

Werner Syndrome is a premature aging disorder characterized by genomic instability, elevated recombination, and replication defects. It has been hypothesized that defective processing of certain replication fork structures by WRN may contribute to genomic instability. Fluorescence resonance energy transfer (FRET) analyses show that WRN and Flap Endonuclease-1 (FEN-1) form a complex in vivo that colocalizes in foci associated with arrested replication forks. WRN effectively stimulates FEN-1 cleavage of branch-migrating double-flap structures that are the physiological substrates of FEN-1 during replication. Biochemical analyses demonstrate that WRN helicase unwinds the chicken-foot HJ intermediate associated with a regressed replication fork and stimulates FEN-1 to cleave the unwound product in a structuredependent manner. These results provide evidence for an interaction between WRN and FEN-1 in vivo and suggest that these proteins function together to process DNA structures associated with the replication fork. INTRODUCTIONWerner Syndrome (WS) is a premature aging disorder characterized by genomic instability and increased cancer risk (Martin, 1978). The WRN gene product defective in WS belongs to the RecQ family of DNA helicases (Yu et al., 1996). In humans, mutations in RecQ family members BLM and RECQ4 are responsible for two other disorders associated with elevated chromosomal instability and cancer, Bloom and Rothmund-Thomson syndromes, respectively (Ellis et al., 1995;Kitao et al., 1998Kitao et al., , 1999. RecQ helicase mutants display defects in DNA replication, recombination, and DNA repair, suggesting a role for RecQ helicases in maintaining genomic integrity (Wu and Hickson, 2002;Cobb et al., 2002).A DNA processing defect during replication or recombination has been suggested to contribute to the molecular pathology of WS. WS cells have a prolonged S phase (Poot et al., 1992), slower rate of repair associated with DNA damage induced in S-phase, reduced induction of RAD51 foci, and higher level of DNA strand breaks (Pichierri et al., 2001).More recently, it was demonstrated that WS cells initiate recombination at a normal rate but fail to resolve recombination intermediates in a RAD51-dependent pathway (Prince et al., 2001;Saintigny et al., 2002). A DNA substrate for WRN was suggested by the demonstration that RusA, a bacterial enzyme that cleaves four-way junctions, rescued cell survival and restored the ability to generate viable recombinants following exposure of WRNϪ/Ϫ cells to DNA damaging agents (Saintigny et al., 2002).A stalled replication fork can be converted to a four-way junction resembling a Holliday Junction (HJ) by branch migration and reannealing of nascent DNA strands (McGlynn et al., 2001;Postow et al., 2001). WRN has been shown to unwind the HJ and catalyze branch fork migration on ␣-structures (Constantinou et al., 2000), suggesting a potential role in processing the recombination intermediate to prevent aberrant recombination events at sites of stalled replication forks. Evidence ...

Interaction of the Human DNA Glycosylase NEIL1 with Proliferating Cell Nuclear Antigen

“…Repair of the damage occurs in the reannealed duplex DNA, and replication resumes after a helicase, e.g. Werner or Bloom protein, resolves the collapsed fork (50). In support of this scenario, we have recently shown that NEIL1 stably interacts with and is activated by the Werner protein (33).…”

Biochemical and Kinetic Characterization of the DNA Helicase and Exonuclease Activities of Werner Syndrome Protein

“…Inhibition of a mutation avoidance system (mismatch repair) in yeast cells or human cell extracts by an environmentally relevant concentration of a metal ion, as opposed to direct DNA damage by the mutagen, was recently suggested to be the primary mechanism for genomic destabilization (46); however, the cellular protein targets remain to be identified in that system. Evidence suggests that WRN helicase activity is important in the metabolism of DNA structures associated with a blocked replication fork (40) or homologous recombination (7). The proposed role of WRN in DNA repair-related processes that might be affected by metal ions led us to a second line of inquiry concerning metal effects on WRN helicase activity.…”

“…Proteins-Recombinant hexahistidine-tagged full-length WRN proteins (wild-type WRN or the exonuclease point mutant WRN-E84A) were overexpressed using a baculovirus/Sf9 insect system and purified as described previously (40). A recombinant hexahistidine-tagged truncated form of WRN (amino-terminal 368 amino acids, designated N-WRN) was overexpressed using the baculovirus/Sf9 insect system and purified as described previously (41).…”

FEN1 contributes to telomere stability in ALT-positive tumor cells

“…Indeed, this has been observed with the Werner protein (Crabbe et al, 2004) Role of FEN1 in ALT tumor cells A Saharia and SA Stewart as well as FEN1 (this study). Flap endonuclease 1 functions with the Werner protein to process branchmigrating structures that resemble stalled replication forks (Sharma et al, 2004). Therefore, given that unresolved stalled replication forks lead to DNA double-strand breaks (Branzei and Foiani, 2005), loss of Werner or FEN1 activity would be expected to result in telomere loss and subsequent end-to-end fusions.…”