Helicases are molecular motor proteins that couple the hydrolysis of nucleoside triphosphate to nucleic acid unwinding. Enzymes of this class function coordinately with other proteins as a complex machine and play essential roles in pathways of DNA metabolism that include replication, DNA repair, recombination, transcription, and chromosome segregation. Despite considerable efforts to understand biochemical, structural, and genetic aspects of helicase function, the precise mechanisms by which helicases catalyze strand separation and perform their biological roles remain to be fully understood. The growing number of DNA helicases implicated in human disease suggests that these enzymes have vital specialized roles in cellular pathways important for the maintenance of genome stability. Recent evidence indicates that mutations in genes of the RecQ family of DNA helicases result in chromosomal instability diseases of premature aging and/or cancer predisposition. Currently known RecQ helicase-deficient disorders include Werner, Bloom, and Rothmund-Thomson syndromes. The WRN gene product, defective in Werner syndrome, 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 structural elements for recognition. A real-time kinetic analysis of WRN helicase activity was used by our group to characterize the mechanism of DNA unwinding by WRN. Biochemical studies were performed to investigate the mechanism for stimulation of WRN helicase activity by its auxiliary factor RPA. Our results indicate that the physical interaction between RPA and WRN plays a critical role in the functional interaction. To further understand the molecular functions of WRN protein, we have characterized the functional interaction of WRN with human Flap Endonuclease 1 (FEN-1), a structure-specific nuclease implicated in DNA repair, replication, and recombination. Our results indicate that WRN stimulates FEN-1 cleavage of important DNA intermediates by a unique mechanism whereby the efficiency of FEN-1 cleavage is dramatically enhanced. Our most recent work has elucidated a role for WRN in resolving stalled replication forks and recombination intermediates. Our hypothesis is that the aberrant mitotic recombination and genomic instability arises from inappropriate processing of replication/recombination intermediates in Werner syndrome cells. In vivo evidence for a role of WRN in cellular DNA replication was attained using a model genetic system for WRN structure-function studies. 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 focused our attention on human RECQ1, a DNA helicase whose cellular functions remain largely uncharacterized. 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. Currently, we are utilizing model systems to determine the biological functions of RECQ1.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIAAG000741-13
Application #
8736605
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
13
Fiscal Year
2013
Total Cost
$579,461
Indirect Cost
Name
National Institute on Aging
Department
Type
DUNS #
City
State
Country
Zip Code
Bharti, Sanjay Kumar; Brosh Jr, Robert M (2016) Fine-tuning DNA repair by protein acetylation. Cell Cycle 15:1952-3
Khan, Irfan; Crouch, Jack D; Bharti, Sanjay Kumar et al. (2016) Biochemical Characterization of the Human Mitochondrial Replicative Twinkle Helicase: SUBSTRATE SPECIFICITY, DNA BRANCH MIGRATION, AND ABILITY TO OVERCOME BLOCKADES TO DNA UNWINDING. J Biol Chem 291:14324-39
Bharti, Sanjay Kumar; Awate, Sanket; Banerjee, Taraswi et al. (2016) Getting Ready for the Dance: FANCJ Irons Out DNA Wrinkles. Genes (Basel) 7:
Mendoza, Oscar; Bourdoncle, Anne; Boulé, Jean-Baptiste et al. (2016) G-quadruplexes and helicases. Nucleic Acids Res 44:1989-2006
Calì, Federica; Bharti, Sanjay Kumar; Di Perna, Roberta et al. (2016) Tim/Timeless, a member of the replication fork protection complex, operates with the Warsaw breakage syndrome DNA helicase DDX11 in the same fork recovery pathway. Nucleic Acids Res 44:705-17
Marchese, Francesco P; Grossi, Elena; Marín-Béjar, Oskar et al. (2016) A Long Noncoding RNA Regulates Sister Chromatid Cohesion. Mol Cell 63:397-407
Banerjee, Taraswi; Aggarwal, Monika; Sommers, Joshua A et al. (2016) Biochemical and cell biological assays to identify and characterize DNA helicase inhibitors. Methods 108:130-41
Crouch, Jack D; Brosh Jr, Robert M (2016) Mechanistic and Biological Considerations of Oxidatively Damaged DNA for Helicase-dependent Pathways of Nucleic Acid Metabolism. Free Radic Biol Med :
Khan, Irfan; Sommers, Joshua A; Brosh Jr, Robert M (2015) Close encounters for the first time: Helicase interactions with DNA damage. DNA Repair (Amst) 33:43-59
Banerjee, Taraswi; Brosh Jr, Robert M (2015) RECQL: a new breast cancer susceptibility gene. Cell Cycle :0

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