Sunday, November 24, 2024
11/24/2024
ABSTRACT Efficient repair of DNA base lesions, such as UV-induced cyclobutane pyrimidine dimers (CPDs) or DNA alkylation damage, is critical to maintain genome stability and prevent mutations that can lead to cancer. Repair of these abundant classes of DNA lesions is the responsibility of the nucleotide excision repair (NER) and base excision repair (BER) pathways. The importance of efficient excision repair is highlighted by the severe phenotypes of patients with Xeroderma pigmentosum (XP), which have inherited defects in NER genes, and the cancer predisposition of individuals with variants in key BER genes. While the basic mechanisms by which the NER and BER pathways repair DNA base lesions are well understood, how these excision repair pathways efficiently recognize and repair DNA lesions resident in eukaryotic chromatin is unclear. To address this question, we have developed a new high-throughput sequencing method known as MNase-CPD-seq, which provides an unprecedented snapshot of the dynamics of damaged nucleosomes in UV-irradiated cells. Our preliminary MNase-CPD-seq data indicate that damaged nucleosomes rapidly alter their rotational setting to move CPDs to more accessible Minor-Out rotational settings, and at later repair time points, alter translational positioning to expand linker regions between nucleosomes. These preliminary data form the basis of Aim I, where we will use MNase-CPD-seq to characterize the role of damage recognition factors, such as XPC or UV-DDB, and histone modifications in promoting repositioning of damaged nucleosomes in yeast and human cells and in vitro. In parallel, we also plan to develop related methods to map nucleosome dynamics associated with other classes of DNA lesions (i.e., UV-induced 6-4 photoproducts and N-methylpurine (NMP) lesions). In Aim II, we will test the hypothesis that ACR complexes in yeast and human cells are required for efficient repair of UV damage in chromatin by repositioning damaged nucleosomes during repair. We will also investigate how ACR mutants affect UV mutagenesis in UV-exposed yeast cells and human skin cancers. Finally, in the previous award we identified novel roles for histone PTMs in regulating distinct NER and BER pathways in chromatin. Aim III will build on the studies by characterizing the mechanisms by which histone acetylation regulates NER and BER in yeast chromatin, and the role of histone H3 K36 methylation by Set2 in yeast and SETD2 in human cells in regulating canonical and cryptic transcription coupled-nucleotide excision repair (TC-NER). Since ACR subunits (e.g., ARID2) and SETD2 are frequently mutated in human cancers, including melanoma, these studies have important implications to mechanisms of carcinogenesis and chemotherapeutic resistance in cancer.
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