The growing number of DNA helicases implicated in human disease and processes to maintain genome stability suggests that these enzymes have important roles in cellular DNA metabolism. RecQ and DNA repair helicases are of particular interest because several human genomic instability disorders that display premature aging and/or cancer arise from mutations in the helicase genes. Werner syndrome (WS) is a hereditary premature aging disorder characterized by chromosomal instability. The WRN gene product is a helicase/exonuclease that presumably functions in DNA metabolism to preserve genome integrity. To understand the DNA structures and cellular pathways that WRN impacts, we have systematically examined the DNA substrate preferences of WRN helicase for unwinding and its interactions with human nuclear proteins. Our biochemical studies indicate that WRN preferentially unwinds DNA replication structures in a defined orientation and utilizes specific DNA structures for recognition. We have initiated a kinetic analysis of WRN helicase activity to define the mechanism of DNA unwinding. To further understand its molecular functions, we have characterized the functional interaction of WRN protein with human Flap Endonuclease 1 (FEN-1), a structure-specific nuclease implicated in DNA repair, replication, and recombination. WRN and FEN-1 form a complex in vivo that co-localizes with arrested replication forks. To better understand the mechanism of action by the WRN-FEN-1 complex, we have investigated their organized function to process branch-migrating DNA structures associated with the replication fork. These studies reveal helicase-dependent and helicase-independent mechanisms for coordinate nucleolytic processing that is DNA structure-specific. It has been proposed that the helicase-endonuclease Dna2 and Rad27 (FEN-1) act sequentially to remove long 5? flaps during Okazaki fragment maturation in S. cerevisiae. Yeast genetic complementation analysis of dna2 mutants was used to further examine the biological significance of the WRN-FEN-1 interaction. Our results indicate that ectopic expression of human WRN in a dna2-1 mutant background rescues the replication and repair phenotypes. Genetic complementation of dna2-1 does not require WRN catalytic activity since expression of a WRN protein fragment devoid of enzymatic activity complements the mutant. We suggest that RecQ helicases modulate Rad2 nucleolytic processing of key DNA replication and repair intermediates to preserve genomic integrity. Although the biochemical properties and protein interactions of the WRN and BLM helicases have been extensively investigated, less information is available concerning the functions of the other human RecQ helicases. We have begun to focus our attention on human RECQ1. RECQ1 was found to stably bind a variety of DNA structures, enabling it to unwind a diverse set of DNA substrates. RECQ1 was shown to catalyze efficient strand annealing between complementary single-stranded DNA molecules. To acquire a better understanding of RECQ1 cellular functions, we have investigated its protein interactions. Our results suggest a role of RECQ1 in regulation of genetic recombination by its interaction with mismatch repair factors. Recent work has focused on the roles of helicases in the DNA damage response. Mutations in the BRCA1-associated helicase BACH1/BRIP1 have been associated with early-onset breast cancer and cellular data suggest a role of the helicase in double strand break repair and checkpoint control. To better understand the molecular functions and biological substrates that BACH1 helicase acts upon, we have systematically evaluated the ability of purified recombinant BACH1 to unwind a panel of related DNA substrates with distinct tail variations including single-stranded versus double-stranded character, tail length, or backbone continuity. In addition, we have assessed the ability of BACH1 to catalytically unwind DNA structures proposed to be key intermediates of cellular DNA metabolism. The results from these unwinding studies provide a platform to investigate the molecular interactions of the BACH1/BRIP1 helicase with its protein partners in double strand break repair by homologous recombination. Molecular and genetic studies such as these will help to elucidate the functions of human DNA helicases and better understand the molecular deficiencies of helicase disorders.

National Institute of Health (NIH)
National Institute on Aging (NIA)
Intramural Research (Z01)
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Wu, Yuliang; Rawtani, Nina; Thazhathveetil, Arun Kalliat et al. (2008) Human replication protein A melts a DNA triple helix structure in a potent and specific manner. Biochemistry 47:5068-77
Gupta, Rigu; Brosh Jr, Robert M (2007) DNA repair helicases as targets for anti-cancer therapy. Curr Med Chem 14:503-17
Brosh Jr, Robert M; Bohr, Vilhelm A (2007) Human premature aging, DNA repair and RecQ helicases. Nucleic Acids Res 35:7527-44
Sharma, Sudha; Stumpo, Deborah J; Balajee, Adayabalam S et al. (2007) RECQL, a member of the RecQ family of DNA helicases, suppresses chromosomal instability. Mol Cell Biol 27:1784-94
Sharma, Sudha; Brosh Jr, Robert M (2007) Human RECQ1 is a DNA damage responsive protein required for genotoxic stress resistance and suppression of sister chromatid exchanges. PLoS ONE 2:e1297
Muftuoglu, Meltem; Sharma, Sudha; Thorslund, Tina et al. (2006) Cockayne syndrome group B protein has novel strand annealing and exchange activities. Nucleic Acids Res 34:295-304
Brosh Jr, Robert M; Opresko, Patricia L; Bohr, Vilhelm A (2006) Enzymatic mechanism of the WRN helicase/nuclease. Methods Enzymol 409:52-85
Brosh Jr, Robert M; Sharma, Sudha (2006) Biochemical assays for the characterization of DNA helicases. Methods Mol Biol 314:397-415
Choudhary, Saba; Doherty, Kevin M; Handy, Christopher J et al. (2006) Inhibition of Werner syndrome helicase activity by benzo[a]pyrene diol epoxide adducts can be overcome by replication protein A. J Biol Chem 281:6000-9
Gupta, Rigu; Sharma, Sudha; Doherty, Kevin M et al. (2006) Inhibition of BACH1 (FANCJ) helicase by backbone discontinuity is overcome by increased motor ATPase or length of loading strand. Nucleic Acids Res 34:6673-83

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