Dana-Farber Cancer Institute
44 Binney Street
Boston, MA 02115
tel: (617) 632-3499 fax: (617) 632-4326
email: rdkson1@bess.harvard.edu
Genetic recombination is a central aspect of DNA metabolism which
has a number of essential functions. My laboratory is using Escherichia
coli and Saccharomyces cerevisiae as model organisms to study the mechanism
of genetic recombination and related repair events. Our goal is to reconstitute
these reactions in vitro to determine how proteins catalyze them. We are
also working on inherited defects in recombination and repair events and
cancer susceptibility. E. coli is the best understood model system for
studying genetic recombination. Our current work on E. coli is on the RecE
and RecF recombination pathways. We have recently demonstrated that the
RecE and RecT proteins required for the RecE pathway promote homologous
pairing of DNA and constitute a second homologous pairing system that can
substitute for RecA. We have demonstrated that the RecF, RecO and RecR
proteins required for the RecF pathway mediate the assembly of RecA onto
SSB coated ssDNA to form active protein-DNA complexes that promote homologous
pairing. Finally, the RuvC protein, which cleaves Holliday junctions, has
been crystallized in collaboration with Dr. Christin Frederick and structural
studies are ongoing. Our future goals are to identify additional recombination
proteins.
S. cerevisiae is an ideal system for studying eukaryotic recombination and mismatch repair because it is amenable to both genetic and biochemical analysis. Our work on S. cerevisiae recombination and mismatch repair is focused on 8 genes and the proteins they encode. These include: (1) the three genes which encode RPA, an ssDNA binding protein; (2) MSH1, a mitochondrial protein, and the MSH2-MSH3 and MSH2-MSH6 nuclear complexes that are mispaired base recognition proteins/complexes required for mismatch repair in the mitochondria and nucleus, respectively; (3) PMS1 and MLH1 that encode nuclear mismatch repair proteins; and (4) EXO1 and RTH1, which are exonucleases that play a role in different types of DNA repair. Our future goals are to identify other proteins required for recombination and mismatch repair.
Hereditary non-polyposis colon cancer (HNPCC) is a common inherited cancer susceptibility syndrome. We have recently cloned genes encoding the human homologues of the S. cerevisiae mismatch repair genes MSH2 and MLH1 and demonstrated that inherited mutations in these genes are responsible for approximately 90% of HNPCC. A number of other potential human cancer susceptibility genes including MSH3, MSH6 and PMS2 are being analyzed to determine if they play a role in cancer susceptibility. Our future efforts in this area are focused on trying to identify additional cancer susceptibility genes, better defining the diseases that mutations in these genes cause and to develop chemotherapy directed at mismatch repair defective cancer cells.
Selected Publications:
Tishkoff, DX, Filosi, N, Gaida, GM, and Kolodner, R. (1997). A novel mutation suppression mechanism defined in S. cerevisiae rad27 mutants is distinct from DNA mismatch repair. Cell 88:253-263.
Alani, E, Lee, S, Kane, MF, Griffith, JG, and Kolodner, R. (1997). Saccharomyces cerevisiae MSH2, a mispaired base recognition protein, also recognizes Holliday junctions in DNA. J. Mol. Biol. 265:289-301.
Kane, MF, Loda, M, Gaida, GM, Lipman, J, Mishra, R, Goldman, H, Jessup, JM, and Kolodner, R. (1997). Methylation of the hMLH1 promoter correlates with lack of expression of hMLH1 in sporadic colon tumors and mismatch repair defective human tumor cell lines. Cancer Res. 57:808-811.