Publication Date

2015-08-03

Availability

Embargoed

Embargo Period

2017-08-02

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Biochemistry and Molecular Biology (Medicine)

Date of Defense

2015-06-30

First Committee Member

Yanbin Zhang

Second Committee Member

Richard Myers

Third Committee Member

Murray P. Deutscher

Fourth Committee Member

Ramiro E. Verdun

Abstract

Fanconi anemia (FA) is a severe genetic disorder characterized by bone marrow failure, developmental defects, chromosomal instability, and predisposition to cancer. FA cells are hypersensitive to DNA crosslinking compounds including mitomycin C (MMC), cisplatin, and diepoxybutane, which make defective interstrand crosslink (ICL) repair mechanisms a hallmark for the disease. Of the 17 genes associated with FA, mutations in the complementation group A (FANCA) account for ~64% of all patient cases. Studies explored in this dissertation comprise the biochemical details for the functional role of FANCA in DNA ICL repair and the first biochemical evidence for its possible role in double-strand break (DSB) repair. In an oligonucleotide-based assay purified FANCA shows enhancement of MUS81- EME1-mediated ICL incision. On the contrary, FANCA exhibits a two-phase incision regulation when DNA is undamaged or the damage affects only one DNA strand. MUS81-EME1 is a DNA endonuclease involved in replication-coupled repair of ICLs. A prevalent hypothetical role of MUS81-EME1 in ICL repair was to unhook the damage by incising the leading strand on the 3’ side of an ICL lesion. The studies presented in Chapter 3 show that purified MUS81-EME1 incises DNA on the 5’ side of a psoralen ICL residing in fork-like structures. Using truncated FANCA proteins, I determined that both the N- and C-regions of the protein are required for the observed FANCA-dependent MUS81-EME1 incision regulation. Using laser-induced psoralen ICL formation in cells, I found that FANCA colocalizes with and recruits MUS81 to ICL lesions. Due to the specific ICL recognition activity of FANCA in vitro, I hypothesized that it was likely to catalyze strand separation in order to discriminate damage that crosslinks the DNA duplex from damage that only affects one strand. To test my hypothesis, I examined the effect of FANCA on DNA stability. In an oligonucleotide-based assay purified FANCA shows strong helix destabilization, single-strand annealing and strand exchange activities that are completely dependent on protein:oligonucleotide stoichiometric ratios. While low stoichiometric ratios of FANCA to DNA result in helix destabilization, higher ratios catalyze single-strand annealing and strand exchange. Furthermore, FANCA and RAD51 exhibit synergistic DNA strand annealing activity, suggesting their coordinated function might play a role in fork stability. FANCA also promotes the bidirectional annealing of structures that mimic physiologically relevant intermediates in DSB repair. C- and N-terminal truncation mutants reveal that binding to DNA is not sufficient for the helix destabilization or single-strand annealing activities of FANCA. Patient-derived FANCA mutants Q772X, D598N, R1117G, Q1128E, and F1263Δ exhibit deregulated helix destabilization, strand annealing and strand exchange activities. It is conceivable that proteins harboring helix destabilization, single-strand annealing, and strand exchange activities work in concert to protect genome integrity and efficiently process replication and recombination intermediate structures. It’s possible that in the presence of an ICL, FANCA may serve to recognize the damage, help anchor the FA core complex to the DNA, recruit structure-specific endonuclease MUS81/EME1 and enhance its incision activity accordingly. When replication forks are stalled due to sources other than ICLs, FANCA may be recruited to remodel the fork and promote fork- restart through ssDNA annealing of excessively unwound template or fork regression and “chicken foot” formation through single-strand annealing of the nascent DNA. FANCA may also act as a local helix destabilizing protein that promotes fork protection by drawing the nuclease away from the fork, thereby inhibiting it’s incision activity. Collectively, it appears FANCA may have multiple roles in ICL repair, particularly downstream of FA pathway activation during the repair of intermediate DSB structures. It’s possible that FANCA works in concert with RAD51 or RAD52 to promote the repair of DSBs through homology-directed pathways strand invasion and single-strand annealing.

Keywords

Fanconi anemia; single-strand annealing; DSB repair; homology-directed repair; FANCA; interstrand-crosslink repair

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