In vivo function of the conserved non-catalytic domain of Werner syndrome helicase in DNA replication.
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.
Laboratory of Molecular Gerontology, National Institute on Aging, NIH, 5600 Nathan Shock Drive, Baltimore, Maryland 21224, USA.
Sudha
S
Sharma
Joshua A
JA
Sommers
Robert M
RM
Brosh
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D002453:Cell Cycle; D002478:Cells, Cultured; D017124:Conserved Sequence; D004265:DNA Helicases / Q000235:genetics* / Q000378:metabolism; D004261:DNA Replication; D024202:Electrophoretic Mobility Shift Assay; D005090:Exodeoxyribonucleases; D045585:Flap Endonucleases / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D047468:Immunoprecipitation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D053484:RecQ Helicases; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D014898:Werner Syndrome / Q000201:enzymology*; D000071657:Werner Syndrome Helicase
D029701:Saccharomyces cerevisiae Proteins; D005090:Exodeoxyribonucleases; D045585:Flap Endonucleases; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; D053484:RecQ Helicases; C098785:WRN protein, human; D000071657:Werner Syndrome Helicase; D006918:Hydroxyurea