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

Abstract

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.

Starting DNA Synthesis: Initiation Processes during the Replication of Chromosomal DNA in Humans

“…These activated helicase complexes start to untwist and unwind dsDNA establishing replication bubbles in origins and move in opposite directions forming two RFs at the active origins [ 3 , 51 ]. To stabilise the ssDNA and protect the ssDNA against nucleases, CMG complexes load the eukaryotic ssDNA-binding protein, replication protein A (RPA), a heterotrimer consisting of RPA70, RPA32, and RPA14, onto the unwound ssDNA strands [ 59 , 60 , 61 , 62 ]. In the following step, DNA polymerase α-primase (Pol α) associates with the CMG complex via the AND-1/CTF4/WDGD homotrimer, and in turn the primase function of Pol α forms RNA primers on ssDNA templates at an origin, the start of the elongation step in eukaryotic DNA replication [ 1 , 9 , 32 , 46 , 61 , 63 ].…”

“…Here, it is important to note that RNA primers produced by primase are sufficient for checkpoint initiation [ 117 , 142 ]. The 9-1-1 complex and Pol α on the lagging strand template then recruit TOPBP1 to the stalled RF, whereas RPA, an ATR substrate, on the leading strand binds to the ATR-binding protein ATRIP ( Figure 3 B, [ 62 , 119 , 122 , 143 ]). In addition, the stalled Pol ε may interact with TOPBP1 and stabilise its interaction with the stalled RF.…”