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DNA replication. DNA repair. DNA helicase. genetic analysis. genome stability. protein purification. yeastOur lab uses the budding yeast S. cerevisiae as a model system to study DNA replication and genome stability in eukaryotic cells. Our approach is to apply genetics. biochemistry and molecular biology to analyze highly-conserved genes involved in this process. The main project focuses on a DNA helicase-topoisomerase complex that is known to control genome stability in humans. The yeast gene SGS1 is the homolog of the genes responsible for both Bloom's Syndrome and Werner's Syndrome in humans. We have shown that Sgs1 is a 3' - 5' DNA helicase that interacts with Top3 and is composed of two functional domains. In the absence of either domain the yeast genome undergoes hyper-recombination and rearrangement. A second project involves the use of genetic screens to identify and characterize new genes required for genome stability. Here we are focused on determining the function of six conserved but poorly characterized "SLX" genes that were identified based on their genetic interaction with SGS1. Enzymatically. we have shown that four of these these genes encode two heterodimeric structure-specific endonucleases while a third pair appears to encode a novel protein-modifying activity. The long-term goal of this project is to determine the mechanism by which these enzymes maintain genome stability. A third project involves Replication Protein A (RPA) which is a ssDNA binding protein required for DNA replication. repair. and recombination. RPA is a three-subunit ssDNA binding protein that is found in all eukaryotes from yeast to humans. Using a combination of biochemistry and molecular biology we have shown that RPA contains at least four non-identical ssDNA binding domains. Genetically. we have determined which of these domains are required for cell viability. and have identified other essential regions of the protein that interact directly with the DNA replication machinery. Our current focus is to determine the role of RPA in the DNA damage response. Selected PublicationsYang L, Mullen JR, Brill SJ. (2006) Purification of the yeast Slx5-Slx8 protein complex and characterization of its DNA-binding activity. Nucleic Acids Res. 34(19):5541-51. Roberts TM. Kobor MS. Bastin-Shanower SA. Ii M. Horte SA. Gin JW. Emili A. Rine J. Brill SJ. Brown GW. (2006) Slx4 regulates DNA damage checkpoint-dependent phosphorylation of the BRCT domain protein Rtt107/Esc4. Mol Biol Cell. 17(1):539-48. Fricke. W. M.. Bastin-Shanower. S. A.. Mullen. J. R.. and Brill. S. J. (2005). Substrate specificity of the Mus81-Mms4 endonuclease from S. cerevisiae. DNA Repair 4:243-251. Mullen. J.R.. Nallaseth. F. S.. Lan. Y. Q.. Slagle. C. E.. and Brill. S. J. (2005). Yeast Rmi1/Nce4 controls genome stability as a subunit of the Sgs1-Top3 complex. Mol. Cell. Biol. 25:4476-4487. Hollingsworth. N. M. and Brill. S. J. (2004). The Mus81 solution to resolution: generating meiotic crossovers without Holliday junctions. Genes Dev. In Press. Kim. H.-S.. and Brill. S. J. (2003). MEC1-dependent phosphorylation of yeast RPA1 in-vitro. DNA Repair. In Press. Fricke. W. M. and Brill. S. J. (2003). Slx1-Slx4 is a second structure-specific endonuclease that functionally overlaps with Sgs1-Top3. Genes Dev. 17:1768-1778. Bastin-Shanower. S. A.. Fricke. W. M. Mullen. J. R. and Brill. S. J. (2003). The mechanism of Mus81-Mms4 cleavage-site selection distinguishes it from the homologous endonuclease Rad1-Rad10. Mol. Cell. Biol. 23:3487-3496. Bae. K. H.. Kim. H. S.. Bae. S. H.. Kang. H. Y.. Brill. S.. and Seo. Y. S. (2003). Bimodal interaction between replication-protein A and Dna2 is critical for Dna2 function both in vivo and in vitro. Nucleic Acids Res 31:006-3015. Kaliraman. V. and Brill. S. J. (2002). Role of SGS1 and SLX4 in rDNA replication in Saccharomyces cerevisiae. Current Genetics 41: 389-400. Bastin-Shanower. S.A. and Brill. S. J. (2001) Functional analysis of the four DNA binding domains of Replication Protein A: the role of RPA2 in ssDNA binding. J. Biol. Chem. 276:36446-36453. Kaliraman. V.. Mullen. J. R.. Fricke. W. M.. and Brill. S.J. (2001). Functional overlap between Sgs1-Top3 and the Mms4-Mus81 endonuclease: Genes Dev. 15. 2730-27. Mullen. J. R.. Kaliraman. V.. and Brill. S. J. (2001). Requirement for three novel protein complexes in the absence of the Sgs1 DNA helicase. Genetics 157:103-118. Fricke. W. M.. Kaliraman. V.. and Brill. S. J. (2001). Mapping of the DNA topoisomerase III binding domain of the Sgs1 DNA helicase J. Biol. Chem. 276:8848 - 8855. Kim. H.S.. and Brill. S.J. (2001). Rfc4 interacts with Rpa1 and is required for both DNA replication and DNA damage checkpoints in Saccharomyces cerevisiae. Mol. Cell Biol. 21. 3725-3737. Mullen. J. R.. Kaliraman. V.. and Brill. S. J. (2000). Bipartite structure of the SGS1 DNA helicase of S. cerevisiae. Genetics 154:1101-1114. |
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