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Robert M. Brosh, Jr., Ph.D., Senior Investigator Section on DNA Helicases Laboratory of Molecular Gerontology Phone: 410-558-8578 Fax: 410-558-8157 E-mail: broshr@grc.nia.nih.gov |
| Biography: Dr. Robert Brosh received his Ph.D. in Biology from the University of North Carolina at Chapel Hill in 1996, his M.S. in Biochemistry from Texas A&M University in 1988, and his B.S. in Chemistry from Bethany College in 1985. He conducted postdoctoral training at the Laboratory of Molecular Genetics (NIA, NIH) and served as an adjunct faculty member at Towson University before assuming his position at NIA in 2000. He became a tenured Senior Investigator in 2006. Dr. Brosh serves as a Mentor for the NIH Summer Student Intramural Research Training Award Program and the NIH Undergraduate Scholarship Program. |
| Roles of DNA Helicases in Genomic Stability: 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 are still under investigation. 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. |
| RecQ Helicases as Caretakers of the Genome: 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 functions in DNA metabolism to preserve genome integrity. To understand the DNA structures and cellular pathways that WRN impacts, we have examined the mechanism of DNA unwinding by WRN helicase 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. Real-time kinetic evidence demonstrate that WRN unwinds duplex DNA substrate as a monomer. The physical interaction between WRN and the single-stranded DNA binding protein RPA plays a critical role in the mechanism for RPA stimulation of WRN helicase activity on long DNA duplexes. |
| 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. |
| Unique and Important Consequences of RECQ1 Deficiency in Mammalian Cells: 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. RECQ1 helicase is the most highly expressed of the human RecQ helicases, suggesting an important role in cellular DNA metabolism. Recent advances from our lab have elucidated a unique role of RECQ1 to suppress genomic instability. Embryonic fibroblasts from RECQ1-deficient mice displayed aneuploidy, chromosomal instability, and increased load of DNA damage. Acute depletion of human RECQ1 renders cells sensitive to DNA damage and results in spontaneous gamma-H2AX foci and elevated sister chromatid exchanges, indicating aberrant repair of DNA breaks. Consistent with a role in DNA repair, RECQ1 relocalizes to irradiation-induced nuclear foci and associates with chromatin. RECQ1 catalytic activities and interactions with DNA repair proteins are likely to be important for its molecular functions in genome homeostasis. Collectively, these studies provide the first evidence for an important role of RECQ1 to confer chromosomal stability that is unique from that of other RecQ helicases and suggest its potential involvement in tumorigenesis. |
| Unraveling the Linkage of FANCJ Helicase to DNA Repair and Replicational Stress Fanconi anemia (FA) is an autosomal recessive disorder characterized by multiple congenital anomalies, progressive bone marrow failure, and high cancer risk. Cells from FA patients exhibit spontaneous chromosomal instability and hypersensitivity to DNA interstrand cross-linking (ICL) agents. Although the precise mechanistic details of the FA/BRCA pathway of ICL-repair are not well understood, progress has been made in the identification of the FA proteins that are required for the pathway. Among the 13 FA complementation groups from which all the FA genes have been cloned, only a few of the FA proteins are predicted to have direct roles in DNA metabolism. One of the more recently identified FA proteins, shown to be responsible for complementation of the FA complementation group J, is the BRCA1 Associated C-terminal Helicase (BACH1, designated FANCJ), originally identified as a protein associated with breast cancer. FANCJ has been proposed to function downstream of FANCD2 monoubiquitination, a critical event in the FA pathway. Evidence supports a role for FANCJ in a homologous recombination (HR) pathway of double strand break (DSB) repair. Our current studies have examined FANCJ functions through its enzymatic activities and protein interactions. This work has begun to elucidate the molecular roles of FANCJ in DNA repair and replication fork stabilization, and demonstrate that the cellular defects associated with FANCJ mutation extend beyond the reduced ability to repair ICLs and involve other types of DNA structural roadblocks to replication such as G-quadruplexes. |
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| Section on DNA Helicases (left to right). Row 1: Monika Aggarwal, Suhasini Avvaru; row 2: Yuliang Wu, Robert Brosh, Josh Sommers. |
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