In order to dissect the biochemical steps involved in genetic recombination we have chosen to focus on a key early step: strand exchange between homologous parental DNAs. The product of this strand exchange reaction,is a joint molecule composed of a single-strand circle joined to one end of a linear duplex. Three proteins responsible for this step have been purified: uvsx from phage T4; RecA from E. coli; and recl from U. maydis. Over the last few years we have reported the partial purification and Characterization of similar strand-exchange proteins or recombinases from nuclear extracts of human cells and tissues and embryos of D. melanogaster have shown that the structure of the protein-free intermediate in strand-- exchange is most likely that of a three-stranded DNA. Recently, we have built a model of the precise hydrogen bonding interactions between the third strand and, the duplex. Critical tests of the model are now possible; e.g., chemical probing (footprinting) of reactive groups in the third strand. In addition, we have searched for eukaryotic proteins other than recombinases that might either form or bind to three-stranded or triplex DNA. We have purified and characterized a protein from HeLa cells that binds to the TAT triplex. We used a gel shift assay to detect the protein. The protein is greatly enriched by passage through a triplex DNA-affinity column. Finally, in order to assess the role that homologous recombination might play in immunoglobulin heavy chain class switching, we have sequenced several human switch recombination sites. From an examination of these sequences, we have argued that they probably are not substrates for the homologous recombination machinery.

Project Start
Project End
Budget Start
Budget End
Support Year
11
Fiscal Year
1990
Total Cost
Indirect Cost
City
State
Country
United States
Zip Code
Cotroneo, M S; Haag, J D; Zan, Y et al. (2007) Characterizing a rat Brca2 knockout model. Oncogene 26:1626-35
Pezza, Roberto J; Petukhova, Galina V; Ghirlando, Rodolfo et al. (2006) Molecular activities of meiosis-specific proteins Hop2, Mnd1, and the Hop2-Mnd1 complex. J Biol Chem 281:18426-34
Sokolov, M V; Smirnova, N A; Camerini-Otero, R D et al. (2006) Microarray analysis of differentially expressed genes after exposure of normal human fibroblasts to ionizing radiation from an external source and from DNA-incorporated iodine-125 radionuclide. Gene 382:47-56
Smirnova, Natalya A; Romanienko, Peter J; Khil, Pavel P et al. (2006) Gene expression profiles of Spo11-/- mouse testes with spermatocytes arrested in meiotic prophase I. Reproduction 132:67-77
Petukhova, Galina V; Pezza, Roberto J; Vanevski, Filip et al. (2005) The Hop2 and Mnd1 proteins act in concert with Rad51 and Dmc1 in meiotic recombination. Nat Struct Mol Biol 12:449-53
Bellani, Marina A; Romanienko, Peter J; Cairatti, Damian A et al. (2005) SPO11 is required for sex-body formation, and Spo11 heterozygosity rescues the prophase arrest of Atm-/- spermatocytes. J Cell Sci 118:3233-45
Khil, Pavel P; Oliver, Brian; Camerini-Otero, R Daniel (2005) X for intersection: retrotransposition both on and off the X chromosome is more frequent. Trends Genet 21:3-7
Difilippantonio, Simone; Celeste, Arkady; Fernandez-Capetillo, Oscar et al. (2005) Role of Nbs1 in the activation of the Atm kinase revealed in humanized mouse models. Nat Cell Biol 7:675-85
Volodin, Alexander A; Voloshin, Oleg N; Camerini-Otero, R Daniel (2005) Homologous recombination and RecA protein: towards a new generation of tools for genome manipulations. Trends Biotechnol 23:97-102
Khil, Pavel P; Camerini-Otero, R Daniel (2005) Molecular features and functional constraints in the evolution of the mammalian X chromosome. Crit Rev Biochem Mol Biol 40:313-30

Showing the most recent 10 out of 30 publications