Eylem Seç
Molecular Characterization of a RAD51-independent Conservative Homologous Recombination Mechanism Driven by Human RAD52 in the Model Organism Saccharomyces cerevisiae
Başlık:
Molecular Characterization of a RAD51-independent Conservative Homologous Recombination Mechanism Driven by Human RAD52 in the Model Organism Saccharomyces cerevisiae
Yazar:
Clear, Alissa Denise, author.
ISBN:
9780438058910
Yazar Ek Girişi:
Fiziksel Tanımlama:
1 electronic resource (142 pages)
Genel Not:
Source: Dissertation Abstracts International, Volume: 79-10(E), Section: B.
Advisors: Adam M. Bailis; Jeremy M. Stark Committee members: Mark P. Boldin; Susan L. Neuhausen; Jefferson P. Perry.
Özet:
In the absence of canonical RAD51-dependent HR repair pathway proteins BRCA1 or BRCA2, human RAD52 (HsRAD52) supports conservative homologous recombination (HR) repair of DNA double strand breaks (DSBs), thus implicating HsRAD52 in an alternative DNA repair mechanism. The HsRAD52 mechanism is critical for canonical HR-deficient cancer cell survival, as well as for cancer cells under high replication stress, indicating that this alternative pathway has biological significance. This synergistic genetic relationship also identifies HsRAD52 as a potential target for cancer therapy.
The role of HsRAD52 in conservative HR remains unclear, perhaps due to the difficulty of studying the protein in a mammalian system. RAD52 was discovered and extensively characterized in the model organism Saccharomyces cerevisiae, where yeast Rad52 (ScRad52) plays a role in ScRad51-independent non-conservative HR, as well as supporting ScRad51-dependent HR. While HsRAD52 has retained the non-conservative repair role, it is unable to support a critical in vitro HsRAD51-dependent activity required for canonical conservative HR. This indicates that HsRAD52 drives a conservative HR mechanism which is separate and distinct from the canonical HsRAD51-dependent pathway.
We have expressed HsRAD52 in budding yeast and have demonstrated that it is both stable and capable of robust self-association, indicating that the protein folds properly in our model system. Genetic assays of cellular HR demonstrated that HsRAD52 can completely suppress the effect of the loss of endogenous ScRad52 in DSB repair by non-conservative HR. Similarly, expression of HsRAD52 can substantially suppress the loss of ScRad52 in DSB repair by conservative HR. However, the function of HsRAD52 in conservative HR is distinct from the function of ScRad52 as HsRAD52 works independently from ScRAD51 while ScRad52 does not. This is supported by the failure to observe an interaction between HsRAD52 and ScRad51 by co-immunoprecipitation (co-IP), which is integral to the function of ScRad52 in conservative HR. Consistent with this, ChIP-qPCR experiments showed that HsRAD52 associates with both HR substrates during DSB repair by conservative HR, but that the level and kinetics were consistent with a mechanism that is less efficient and slower than that supported by ScRad52.
We have characterized two variant HsRAD52 alleles expressed stably in yeast cells; HsRad52-G59R and HsRad52-S346X . Our analysis indicates that both are separation-of-function alleles, as they completely suppress the loss of DSB repair by non-conservative HR in cells without ScRad52, but do not suppress the inability to repair a DSB by conservative HR. Importantly, HsRad52-S346X has been preliminarily epidemiologically linked to suppression of breast cancer in pathogenic BRCA2 mutation carriers, further implicating the HsRAD52-driven conservative HR mechanism as the critical function underlying its role in tumor suppression. Accordingly, both mutant proteins display the ability to associate with the single-stranded DNA at DSBs, but are unable to associate with, or support repair synthesis from the donor substrate during conservative HR. We speculate that this block is due to a loss of dsDNA binding activity by these mutant proteins. Importantly, this identifies the glycine at position 59, the 72 C-terminal amino acids, and the dsDNA-binding domain as regions of HsRAD52 that are essential to its function in conservative HR in yeast, and may provide targets for development of drugs that kill BRCA-deficient tumors in people.
Notlar:
School code: 0517
Mevcut:*
Yer Numarası | Demirbaş Numarası | Shelf Location | Lokasyon / Statüsü / İade Tarihi |
---|---|---|---|
XX(682833.1) | 682833-1001 | Proquest E-Tez Koleksiyonu | Arıyor... |
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