Every day, reactive oxygen species, produced during normal oxidative metabolism, generate approximately 30,000 oxidative lesions in the DNA of every nucleated human cell. If not repaired, or if misrepaired, these lesions can be mutagenic or cytotoxic. Nearly all such lesions are repaired in an error-free manner via Base Excision Repair (BER). Our overall goal is to elucidate mechanisms that regulate and promote BER in cells, where access to DNA is limited by its packaging into nucleosomes. In cells, histone chaperones and chromatin remodeling agents help circumvent the nucleosome-associated impediments to DNA replication, transcription, and certain DNA repair pathways. However, chromatin-remodeling agents have not been shown to act at sites of oxidative damage in cells. In fact, we have shown that, in vitro, BER enzymes are able repair oxidative lesions in nucleosomes without the aid of such agents. Nevertheless, it remains to be determined how DNA ligase III?, which must fully encircle its DNA substrate, is able to seal DNA nicks without irreversibly disrupting the host nucleosome. Accordingly, Aim 1 will test the hypothesis that DNA ligase III? discovers DNA nicks in nucleosomes while in a non-specific DNA binding configuration, and then drives nucleosomes into a reversible, ligation-permissive configuration, as it folds into its active configuration. Our studies also suggest that nucleosomes may affect the orderly handoff of BER repair intermediates from one enzyme to the next. This is critical to ensure that potentially cytotoxic or mutagenic BER intermediates remain sequestered. We have shown that, in vitro, the disordered, non-conserved N-terminal tail of the BER glycosylase hNTHL1, helps ensure that hNTHL1 remains bound to its product in nucleosomes until the next enzyme in the BER pathway (APE1) arrives.
Aim 2 will investigate the role of disordered regions in BER glycosylases during substrate handoff in vivo. Finally, we have identified post-translationally modified residues in hNTHL1, and determined some of these modifications (PTMs) are altered by oxidative stress. One of these residues, when replaced with a different residue, and then expressed in human cells, reduces genome stability and increases the risk of transformation. This same hNTHL1-PTM mutant (which is also a germline variant of hNTHL1) is fully active on DNA substrates in vitro.
Aim 3, in collaboration with Project 1, will further investigate the biochemical and cellular properties of this mutant, to determine why it generates the observed cellular defects. We also will identify and characterize additional PTM mutants that are enzymatically normal but generate BER or genome stability defects when expressed in cells. These studies will generate important new information about BER in chromatin and about regulatory mechanisms that ensure that BER helps maintain genome stability.
Defects in the enzymes that repair oxidatively damaged DNA can increase the risk of cancer. Project 3 will investigate molecular mechanisms that regulate these enzymes, and enable them to function properly in cells. These studies will provide further insight into cancer risks linked to oxidative damage in DNA.
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