Maintenance of genomic integrity is fundamental to life. The genome is under constant assault by endogenous metabolites and environmental sources that damage the DNA molecule, giving rise to heritable changes in the sequence of genes. DNA repair pathways are 'molecular guardians'of genomic integrity and are one of the first lines of defense against a wide range of human diseases, including spontaneous and environmentally induced cancers. The broad objective of this proposal is to understand the molecular details of DNA packaging in both DNA damage and its repair, and the response of DNA packaging to these events. The target of DNA damaging agents and the 'landscape'of DNA repair enzymes in eukaryotes is the highly compact and dynamic structure of chromatin. The mechanisms by which the cell gains access to the occluded DNA are not clearly defined, but include combinations of posttranslational modifications (PTMs) of histone proteins and recruitment of chromatin remodeling (CR) complexes. Understanding the regulation of these chromatin modulating activities is essential for understanding the management of DNA repair. We will use UV radiation and a DNA methylating chemical as prototype environmental agents for studies on nucleotide excision repair (NER) and base excision repair (BER), respectively, in yeast and human cells. As we found that CR complexes are recruited to NER sites in yeast chromatin, and physically interact with a DNA damage recognition complex, we will examine the efficiency of both NER and BER in yeast cells deficient in each of the four different classes of CR complexes (Aim I). We will also examine BER in human cells with variable CR activity (Aim I). Secondly, we have identified a conserved sequence cassette in yeast histone H2B (named the HBR cassette) that plays a critical role in gene repression, sensitivity to DNA damaging agents, and efficiency of ER.
In Aim II, we will investigate the mechanisms by which the HBR cassette regulates ER, using nuclease digestion and DNA topology assays to follow nucleosome stability and loading, and chromatin immunoprecipitation (ChIP) to follow recruitment of chromatin modifying enzymes. Thirdly, as we found that the DNA polymerase ss step in BER is blocked by oligo-nucleosome (Oligo-N) formation in vitro, we will analyze NER of UV damage in Oligo-Ns, including histone PTMs during NER and their effect on NER efficiency (Aim III). Finally, we will examine the role of histone PTMs in regulating ER in chromatin in yeast and human cells (Aim IV). We will characterize the functions of both well-established and potentially novel histone PTMs in ER, including examination of a potentially novel role for histone methylation-ubiquitylation in the ER pathway. This proposal is an ongoing investigation of the effects of DNA packaging in chromatin on the two major ER pathways (NER and BER) found in cells. As all eukaryotes, including humans, must deal with this 'packaging paradox'for surveillance of the genome, results from these studies are relevant to the broad spectrum of cancer etiology, prevention and treatment.
DNA damage is detrimental to all organisms and repair of these lesions in mammalian cells is a 'frontline defense'against mutations and cancer. We expose cells to prototype DNA-damaging agents to elicit DNA repair in regions of DNA associated with different states of packaging to determine how cells cope with repair of 'chromatin-buried'lesions. Thus, the project results have implications for the broad spectrum of cancer etiology, prevention and treatment. These studies will also help provide a rational basis for regulatory policies associated with environmental exposure to DNA damaging agents.
Showing the most recent 10 out of 34 publications
