Date of Award

2017

Document Type

Open Access Dissertation

Department

Biological Sciences

Sub-Department

College of Arts and Sciences

First Advisor

Alan Waldman

Abstract

Homologous recombination (HR) serves critical roles in DNA repair to maintain genome stability, and the malfunction of HR contributes to carcinogenesis and cancer development. Current research focuses on the regulation against deleterious recombination between imperfectly matching sequences, which has been documented in certain myeloid leukemias, hereditary nonpolyposis colorectal cancers and other genetic diseases. Homology dependency of recombination was examined in cultured thymidine kinase-deficient mouse fibroblasts. Cells have chromosomal integration of DNA constructs harboring a herpes tk gene (the “recipient”) and a closely linked truncated “donor” tk sequence. The recipient was rendered non-functional by insertion of the recognition site for endonuclease I-SceI, and the donor sequence could restore the function of the recipient through spontaneous gene conversion or via recombinational repair provoked by a double-strand break (DSB) at the I-SceI site. Recombination events were recoverable by HAT selection for tk-positive clones. Three different donor sequences contained 16, 25, or 33 mismatches relative to the recipient, and these mismatches were clustered within these “homeologous” sequences surrounded by region of high homology. Previous work indicated that mammalian cells fastidiously avoid recombination between homeologous sequences, while our results revealed that when homeologous sequences are surrounded by high homology, mismatches are frequently included in gene conversion events. Knock-down of DNA mismatch repair provided evidence that incorporation of mismatches into gene conversion tracts involved repair of mismatched heteroduplex intermediates. Our results demonstrate that mismatch repair of multiple mispaired bases does not function to impede exchange between homeologous sequences. Moreover, gene conversion tracts from spontaneous recombination showed that either all or none of the mismatches were transferred from donor to recipient, suggesting that recombination must begin and end in high homology. But this requirement was somewhat relaxed in DSB-induced events with recombination ending in homeology. Further experiments with a rearranged construct were attempted to investigate the relaxed homology requirement during DSB repair. In addition to the study on homology requirement of recombination, research works were also carried out to characterize the roles of RecQ4 helicases in DSB repair. It is the first demonstration that RecQ4 deficiency reduces the fraction of crossover events in DSB-induced recombination. Moreover, BLM deficiency failed to boost crossover events in RecQ4 deficient cells. It is postulated that these two helicases act agonistically to determine the generation of crossover events.

Rights

© 2017, Shen Li

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