My long term research goal is to define at the molecular level, the linked pathways of DNA replication, DNA repair and genetic recombination in eukaryotes. Since alterations in these functions have been described in cancer-prone human diseases and in cancer cells, this information will be used to investigate the mechanisms of carcinogenesis. This proposal will focus on DNA joining, which is an essential common step in the replication, repair and recombination of DNA molecules. Mammalian cells contain three distinct DNA ligase activities. Genetic and biochemical studies on one of these enzymes, DNA ligase I, have implicated this enzyme in the joining of Okazaki fragments at the replication fork and in DNA repair. the cellular functions of mammalian DNA ligases II and III cannot be predicted from their biochemical properties. Therefore I am proposing to search for similar enzymes in a eukaryotic organism, S. cerevisiae, that is amenable to genetic analysis. Prior to initiating these studies, the product of the CDc9 gene, which is functionally homologous to mammalian DNA ligase I and is quantitatively the major DNA ligase activity in extracts of vegetatively growing S. cerevisiae cells, has been purified and characterized. In preliminary experiments, a second DNA ligase activity has been partially purified and characterized from these extracts. This activity, which is antigenically distinct from Cdc9 DNA ligase and has similar biochemical properties to mammalian DNA ligase II, will be further purified and characterized. It is possible that the relative distribution of the yeast DNA ligases will be significantly different in extracts prepared from cells undergoing meiosis or these extracts may contain a meiosis- specific DNA ligase or meiotic extracts are enriched for activity, which is present at low levels in vegetatively growing cells, then the DNA ligase activity will be purified and characterized from meiotic extracts. The goal of these studies is to purify sufficient quantities of these enzymes for amino acid sequencing studies and for raising polyclonal antiserum. The specific molecular probes generated by these studies will be used to isolate the gene from a yeast genomic library and the phenotypic consequence of gene disruption will be investigated. As an alternative approach to identifying S. cerevisiae DNA ligase genes, I will purify bovine DNA ligase II. Using the same strategy as outlined above, I will isolate mammalian DNA ligase II cDNA sequences. These sequences will be used as a probe to screen for the functionally homologous enzyme in S. cerevisiae. Having identified the yeast gene, the phenotypic consequences of gene disruption will be investigated. by this integrated biochemical and genetic approach, the cellular function(s) of DNA ligase(s) in eukaryotic DNA metabolism will be determined.
| Ghezraoui, Hind; Piganeau, Marion; Renouf, Benjamin et al. (2014) Chromosomal translocations in human cells are generated by canonical nonhomologous end-joining. Mol Cell 55:829-842 |
| Grob, Patricia; Zhang, Teri T; Hannah, Ryan et al. (2012) Electron microscopy visualization of DNA-protein complexes formed by Ku and DNA ligase IV. DNA Repair (Amst) 11:74-81 |
| Chen, Xi; Tomkinson, Alan E (2011) Yeast Nej1 is a key participant in the initial end binding and final ligation steps of nonhomologous end joining. J Biol Chem 286:4931-40 |
| Song, Wei; Pascal, John M; Ellenberger, Tom et al. (2009) The DNA binding domain of human DNA ligase I interacts with both nicked DNA and the DNA sliding clamps, PCNA and hRad9-hRad1-hHus1. DNA Repair (Amst) 8:912-9 |
