Publication Date

2015

Document Type

Dissertation

Committee Members

Steven Berberich (Committee Member), Thomas Brown (Committee Member), Michael Leffak (Advisor), Courtney Sulentic (Committee Member), Yongjie Xu (Committee Member)

Degree Name

Doctor of Philosophy (PhD)

Abstract

Noncanonical DNA structure-forming sequences, such as hairpin structures, stall replication forks in vivo, expand or contract during DNA replication, and colocalize with chromosome fragile sites. Since the frequency of hairpin formation is higher than the frequency of expansion or contraction, the cell may possess mechanisms to resolve hairpin structures prior to replication fork stalling. One possible activity for hairpin resolution is the FANCJ DNA helicase, known to unwind noncanoncial DNA structures in vitro. Indeed, mutations or loss of any of the nineteen FANC genes, including FANCJ, leads to genome instability and the cancer-prone syndrome Fanconi anemia (FA). To test whether (CTG)n·(CAG)n trinucleotide repeat (TNR) stability is protected by FANCJ in human cells, HeLa derived cell lines were created by integration of cassettes containing the c-myc replication origin and (CTG)n · (CAG)n TNRs. Small pool PCR was used to assess genome instability after siRNA depletion of FANCJ helicase in the presence of a replication stress. The results of these experiments indicate FANCJ helicase is essential for the maintenance of (CTG)n·(CAG)n TNR stability independent of replication polarity, and DNA double strand breaks (DSBs) occur near (CTG)n·(CAG)n microsatellites in the absence of FANCJ helicase. These results were confirmed in FA patients cells null for FANCJ. Remarkably, several other types of microsatellite sequences capable of forming noncanoncial DNA structures (hairpins, triplexes, G-quadruplexes, unwound DNA) also suffered DSBs in FANCJ depleted cells, indicating a novel role for FANCJ in microsatellite stabilization across the genome. In contrast, HeLa cells depleted for other FANC proteins or patient cells null for other FA proteins did not show a similar phenotype suggesting FANCJ stabilizes microsatellite sequences apart from activation of the FA repair pathway. Genome instability present at microsatellite sequences results in loss of small pool PCR signal leading us to believe double strand break formation has occurred. We suspect loss of PCR signal results from unrepaired DSBs or DSBs repaired by a translocation event to another site along the genome. To understand the mechanism of double strand break repair at the ectopic site (CTG)·(CAG) trinucleotide repeats, determined by small pool PCR signal loss, inverse PCR coupled with next generation sequencing was employed. The results of next generation sequencing revealed that DSBs are occurring at or near the (CTG)·(CAG) repeats of the ectopic site in cells depleted of FANCJ and treated with aphidicolin lead to translocations with other sites along the genome. Next generation sequencing identified chromosomal positions of translocation sites. These chromosomal positions correspond to cytogenetic positions which have previously been identified as being associated with chromosome breaks in tumors and cells from patients with several developmental disorders. These results suggest a role for FANCJ helicase in cancer and other developmental diseases as a protector of microsatellite stability.

Page Count

195

Department or Program

Biomedical Sciences


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