The long term goal of our laboratory is to elucidate the molecular mechanisms of DNA replication, repair and recombination by studying the DNA joining step that is common to these different DNA transactions. There are three genes encoding DNA ligases in human cells. The participation of these enzymes in different cellular functions is directed by specific protein-protein interactions with different partner proteins. In this proposal we are focused on human DNA ligase I (hLigI) which plays a key role in DNA replication and DNA repair. We have shown that hLigI interacts with PCNA, a DNA sliding clamp, and RFC, the loader that loads PCNA onto DNA and, using cell-based assays, we have demonstrated that these interactions are biologically relevant. In the first Specific Aim, we will elucidate the functional consequences of the interactions among RFC, PCNA and hLigI during replication and repair with a particular emphasis on the interaction between hLig1 and RFC. hLigI also interacts with the checkpoint clamp and its loader, hRad17-RFC. Our in vitro studies have shown that the physical and functional interaction between hLigI and the clamp loaders are sensitive to the phosphorylation status of hLigI. In the second Specific Aim, we will elucidate the role in hLigI phosphorylation in regulating its cellular functions using a combination of quantitative mass spectrometry and biochemical and cell-based assays. Small molecule inhibitors of hLigI and the other human DNA ligases have been identified by a structure-based approach. In the third Specific Aim, we will characterize the in vitro and in vivo activities of the small molecule inhibitors of hLigI. These inhibitors will not only serve as novel probes of the ligation reaction but also provide a complimentary approach to delineating the cellular functions of hLigI and the other human DNA ligases. Furthermore, the cytostatic and cytotoxic activities of the inhibitors suggest that they may have clinical applications as a novel class of DNA repair inhibitors that can be used in combination with DNA damaging agents used to treat cancer.
It is well established that genomic instability drives the progression from a normal cell into a cancer cell. Human cells have a complex network of pathways that act together to maintain genome stability. A mechanistic understanding of these pathways will provide fundamental insights into tumor suppression. In addition, genomic instability is a characteristic of tumor cells, indicating that there are differences in the pathways that normally maintain stability. These differences between normal and cancer cells offer an opportunity to develop therapeutic strategies that selectively target cancer cells.
|Howes, Timothy R L; Sallmyr, Annahita; Brooks, Rhys et al. (2018) Erratum to ""Structure-activity relationships among DNA ligase inhibitors; characterization of a selective uncompetitive DNA ligase I inhibitor"" [DNA Repair 60C (2017) 29-39]. DNA Repair (Amst) 61:99|
|Sallmyr, Annahita; Tomkinson, Alan E (2018) Repair of DNA double-strand breaks by mammalian alternative end-joining pathways. J Biol Chem 293:10536-10546|
|Howes, Timothy R L; Sallmyr, Annahita; Brooks, Rhys et al. (2017) Structure-activity relationships among DNA ligase inhibitors: Characterization of a selective uncompetitive DNA ligase I inhibitor. DNA Repair (Amst) 60:29-39|
|Wiest, Nathaniel E; Tomkinson, Alan E (2017) Optimization of Native and Formaldehyde iPOND Techniques for Use in Suspension Cells. Methods Enzymol 591:1-32|
|Greco, George E; Matsumoto, Yoshihiro; Brooks, Rhys C et al. (2016) SCR7 is neither a selective nor a potent inhibitor of human DNA ligase IV. DNA Repair (Amst) 43:18-23|
|Slean, Meghan M; Panigrahi, Gagan B; Castel, Arturo López et al. (2016) Absence of MutS? leads to the formation of slipped-DNA for CTG/CAG contractions at primate replication forks. DNA Repair (Amst) 42:107-18|
|Peng, Zhimin; Liao, Zhongping; Matsumoto, Yoshihiro et al. (2016) Human DNA Ligase I Interacts with and Is Targeted for Degradation by the DCAF7 Specificity Factor of the Cul4-DDB1 Ubiquitin Ligase Complex. J Biol Chem 291:21893-21902|
|Hegde, Pavana M; Dutta, Arijit; Sengupta, Shiladitya et al. (2015) The C-terminal Domain (CTD) of Human DNA Glycosylase NEIL1 Is Required for Forming BERosome Repair Complex with DNA Replication Proteins at the Replicating Genome: DOMINANT NEGATIVE FUNCTION OF THE CTD. J Biol Chem 290:20919-33|
|Shanmugam, Ilanchezhian; Abbas, Mohammad; Ayoub, Farhan et al. (2014) Ubiquitin-specific peptidase 20 regulates Rad17 stability, checkpoint kinase 1 phosphorylation and DNA repair by homologous recombination. J Biol Chem 289:22739-48|
|Hegde, Muralidhar L; Hegde, Pavana M; Bellot, Larry J et al. (2013) Prereplicative repair of oxidized bases in the human genome is mediated by NEIL1 DNA glycosylase together with replication proteins. Proc Natl Acad Sci U S A 110:E3090-9|
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