Degree

Doctor of Philosophy (PhD)

Department

Comparative Biomedical Sciences, Vet School

Document Type

Dissertation

Abstract

In eukaryotes, DNA repair mechanisms detect and repair damaged DNA. DNA damage is primarily caused by a variety of exogenous and endogenous sources. Several types of damage to DNA are repaired by different kinds of DNA repair pathways. This dissertation focused on repair of N-methylpurines (NMPs) and double-strand breaks (DSBs) in human fibroblasts.

NMPs, including N7-methylguanine (7MeG) and N3-methyladenine (3MeA), can be induced by environmental methylating agents (e.g. the soil fumigant methyl bromide), chemotherapeutics (e.g. nitrogen mustards), and natural cellular methyl donors like S-adenosylmethionine. In human cells, NMPs are repaired by the multi-step base excision repair pathway initiated by human alkyladenine glycosylase (hAAG). Repair of NMPs has been shown to be affected by DNA sequence contexts. However, the nature of the sequence contexts has been poorly understood. We developed a sensitive method, LAF-Seq (Lesion-Adjoining Fragment Sequencing), which allows nucleotide-resolution digital mapping of DNA damage and repair in multiple genomic fragments of interest in human cells. We also developed a strategy that allows accurate measurement of the excision kinetics of NMP bases in vitro. We demonstrate that 3MeAs are induced to a much lower level by the SN2 methylating agent dimethyl sulfate (DMS) and repaired much faster than 7MeGs in human fibroblasts. Induction of 7MeGs by DMS is affected by nearest-neighbor nucleotides, being enhanced at sites neighbored by a G or T on the 3’ side, but impaired at sites neighbored by a G on the 5’ side. Repair of 7MeGs is also affected by nearest-neighbor nucleotides, being slow if the lesions are between purines, especially Gs, and fast if the lesions are between pyrimidines, especially Ts. Excision of 7MeG bases from the DNA backbone by hAAG in vitro is similarly affected by nearest-neighbor nucleotides, suggesting that the effect of nearest-neighbor nucleotides on repair of 7MeGs in the cells is primarily achieved by modulating the initial step of the base excision repair process.

DSBs can be induced by hydrogen peroxide (H2O2), endonuclease I-PpoI and ionizing radiation. Senataxin is a putative RNA/DNA helicase. We demonstrate that senataxin facilitates repair of DSBs and modulates the activation of DNA damage response pathway of ATM-Chk2 and ATR-Chk1 upon DSB damage.

Date

12-19-2017

Committee Chair

Li, Shisheng

DOI

10.31390/gradschool_dissertations.4186

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