The Intriguing Mystery of RPA Phosphorylation in DNA Double-Strand Break Repair.

Human Replication Protein A (RPA) was historically discovered as one of the six components needed to reconstitute simian virus 40 DNA replication from purified components. RPA is now known to be involved in all DNA metabolism pathways that involve single-stranded DNA (ssDNA). Heterotrimeric RPA comprises several domains connected by flexible linkers and is heavily regulated by post-translational modifications (PTMs). The structure of RPA has been challenging to obtain. Various structural methods have been applied, but a complete understanding of RPA's flexible structure, its function, and how it is regulated by PTMs has yet to be obtained. This review will summarize recent literature concerning how RPA is phosphorylated in the cell cycle, the structural analysis of RPA, DNA and protein interactions involving RPA, and how PTMs regulate RPA activity and complex formation in double-strand break repair. There are many holes in our understanding of this research area. We will conclude with perspectives for future research on how RPA PTMs control double-strand break repair in the cell cycle.

Combined inhibition of aurora kinases and Bcl-xL induces apoptosis through select BH3-only proteins.

Aurora kinases (AURKs) are mitotic kinases important for regulating cell cycle progression. Small-molecule inhibitors of AURK have shown promising antitumor effects in multiple cancers; however, the utility of these inhibitors as inducers of cancer cell death has thus far been limited. Here, we examined the role of the Bcl-2 family proteins in AURK inhibition-induced apoptosis in colon cancer cells. We found that alisertib and danusertib, two small-molecule inhibitors of AURK, are inefficient inducers of apoptosis in HCT116 and DLD-1 colon cancer cells, the survival of which requires at least one of the two antiapoptotic Bcl-2 family proteins, Bcl-xL and Mcl-1. We further identified Bcl-xL as a major suppressor of alisertib- or danusertib-induced apoptosis in HCT116 cells. We demonstrate that combination of a Bcl-2 homology (BH)3-mimetic inhibitor (ABT-737), a selective inhibitor of Bcl-xL, Bcl-2, and Bcl-w, with alisertib or danusertib potently induces apoptosis through the Bcl-2 family effector protein Bax. In addition, we identified Bid, Puma, and Noxa, three BH3-only proteins of the Bcl-2 family, as mediators of alisertib-ABT-737-induced apoptosis. We show while Noxa promotes apoptosis by constitutively sequestering Mcl-1, Puma becomes associated with Mcl-1 upon alisertib treatment. On the other hand, we found that alisertib treatment causes activation of caspase-2, which promotes apoptosis by cleaving Bid into truncated Bid, a suppressor of both Bcl-xL and Mcl-1. Together, these results define the Bcl-2 protein network critically involved in AURK inhibitor-induced apoptosis and suggest that BH3-mimetics targeting Bcl-xL may help overcome resistance to AURK inhibitors in cancer cells.