In vitro and in vivo characterization of RAD51AP1 in homologous recombination DNA repair
Date
2020
Authors
Pires, Elena, author
Wiese, Claudia, advisor
Argueso, Lucas, committee member
Thamm, Douglas, committee member
Yao, Tingting, committee member
Journal Title
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Volume Title
Abstract
Cancer embodies a large group of diseases that is responsible for illness and deaths in millions of people annually around the world. Many tumors arise due to accumulated, unrepaired damage and alterations to genes, from endogenous or exogenous sources of DNA damage. Among the DNA lesions associated with cancer, DNA double-strand breaks (DSBs) are considered the most dangerous and require coordinated and conserved machinery to prevent unfavorable consequences, such as apoptosis and cancer-causing mutations. One crucial DNA repair pathway for mending DSBs and maintaining genome integrity is homologous recombination (HR) DNA repair. This relatively error-free mechanism employs the RAD51 recombinase and involves the joining of homologous DNA strands to restore lost DNA sequence information at the damage site. RAD51-Associated Protein 1 (RAD51AP1) is a key protein that interacts with RAD51 and stimulates its activities during HR. Nonetheless, there are knowledge gaps in understanding how this HR player functions mechanistically and in vivo for protection against DNA damage. To test our overarching hypothesis that disrupted RAD51AP1 inhibits cellular and organismal protection against spontaneous or induced DNA damage, we assessed the biochemical and biological functions of RAD51AP1 through three main avenues of study: its role in the context of chromatin, the effects of its post-translational modifications in cells, and the penalties of its loss in an animal system. This dissertation describes findings from these pursuits that have not been previously characterized and offers new insights into RAD51AP1's functions in vitro and in vivo. At the start of this dissertation, our first objective was to define key attributes of RAD51AP1 in the HR reaction by further characterizing the DNA binding properties of recombinant human RAD51AP1. Using the electrophoretic mobility shift assay, we found that RAD51AP1 avidly associates with both naked and chromatinized double-stranded (ds)DNA. Deletional and mutational analyses were used to further define the chromatin-binding region in RAD51AP1, which occurs within its C-terminal DNA binding domain. Two post-translational modification (PTM) sites, which undergo phosphorylation at S277 and S282 (in isoform 2) and lie within its C-terminal DNA binding region, were also evaluated and showed decreased affinity to chromatinized dsDNA. These results unveil a novel RAD51AP1 interaction with chromatin DNA. Next, we further assessed these PTMs in regard to their impacts on RAD51AP1 function and HR capability in cells facing spontaneous or induced DNA damage. Using RAD51AP1 KO cells expressing phosphorylation mimic (S2D) or non-phosphorylatable (S2A) mutants, we found that S2D expressing cells behaved similarly to wild-type expressing cells. Notably, S2A expressing cells were significantly compromised in their growth, cell survival, cell cycle progression, and HR kinetics. The results of these studies provide an important role for PTMs that affect RAD51AP1's functions during HR. To examine the role of RAD51AP1 in providing protection against DNA damage in an animal system, we utilized a recently available mouse knockout model to evaluate the impacts of Rad51ap1 deletion from spontaneous DNA damage. Given the role of RAD51AP1 in meiotic HR and its high expression in murine testes, we specifically monitored fertility ratios, spermatogenesis in testes cross sections, and meiosis via synaptonemal complex formation. We found that Rad51ap1 heterozygous mice do not breed in a Mendelian pattern. Furthermore, while synaptonemal complex formation was not impaired in Rad51ap1 KO mice, advanced stages of spermatogenesis were impacted, suggestive of a biological role for RAD51AP1 in maintaining the fidelity of this process. Collectively, the results of these studies characterizing the in vitro and in vivo roles of RAD51AP1 provide new insights into this important HR player. For the first time, we reveal a new association between RAD51AP1 and chromatinized dsDNA and propose a model integrating this interaction within the HR reaction, when homology search and hetero-duplex formation after presynaptic filament formation occurs. Additionally, previously uncharacterized PTMs were assessed functionally in cells, and we unveil that the lack of these PTMs negatively impacts cells against spontaneous and induced DNA damage. Lastly, our studies on the biological effects of Rad51ap1 loss in a recently available Rad51ap1 KO mouse describe a novel role of Rad51ap1/RAD51AP1 during late spermatogenesis in an animal system for the first time. Ultimately, by understanding the mechanisms and biology of this important HR protein, this knowledge can guide the optimization of treatments for cancers that exploit DNA repair factors as well as help us comprehend how this factor protects against DNA damage in mammals.
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Subject
DNA repair
RAD51AP1
homologous recombination
DNA damage