In all cells, the process of genetic recombination repairs DNA damage that might otherwise lead to mutations or cell death. The loss of various DNA repair mechanisms in human cells has, in some cases, been correlated with a predisposition to cancer. Studies of genetic recombination in the bacterium Escherichia coli has defined in detail many of the proteins that mediate steps of genetic exchange. Our previous work has focused on understanding the role that DNA exonucleases play in genetic recombination in E. coli. We propose to characterize the biochemical properties of the RecJ exonuclease that is involved in several pathways for DNA repair. We will determine the properties of the RecJ exonuclease in coupled in vitro reactions with the RecA protein of E. coli. RecA plays a central role in genetic recombination and can promote strand exchange between homologous DNA molecules. We will investigate the reactions that mimic the types of biochemical steps that are thought to constitute recombinational DNA repair. These experiments will provide a biochemical framework to understand the role of RecJ in the bacterial cell. We also propose to define those regions of the RecJ protein that are important for the genetic and biochemical functions of RecJ. We will isolate and characterize mutant forms of the protein by genetic and biochemical means. We will isolate, sequence and compare the predicted amino acid sequence or other bacterial RecJ proteins to determine those residues of the protein that have been conserved throughout evolution. Our goal from this analysis will be to develop a motif shared among RecJ and other 5' exonucleases. Although 5' exonucleases are found in many genetic recombination systems, there is no great similarity among these proteins in primary sequence. It is therefore not possible to identify putative 5' DNA exonucleases based on sequence information alone. Extensive mutational analysis of exonuclease proteins has not been performed.From our proposed analysis, we will be able to identify the regions of the protein likely to be involved in catalysis; these regions are most likely to share similar structure among exonucleases. We will use the structural information about RecJ to seek RecJ-homologs from the eukaryotic organism Saccharomyces cerevisiae. Molecular genetics in the yeast S. cerevisiae is well-developed and many genes involved in genetic recombinationand DNA repair have been identified. Recently, homologs of several genes which mediate genetic recombination and DNA repair in bacteria have been found in yeast. The yeast homologs have been useful, in turn, in finding human counterparts to these genes. The conservation of DNA repair pathways from bacteria to yeast suggests that RecJ-like proteins should be found in eukaryotes. Our proposed experiments seek to identify these counterparts in yeast and to begin to investigate their genetic and biochemical properties in this eukaryotic organism.
Showing the most recent 10 out of 24 publications
