Evidence for direct contact between the RPA3 subunit of the human replication protein A and single-stranded DNA

Abstract

Replication Protein A is a single-stranded (ss) DNA-binding protein that is highly conserved in eukaryotes and plays essential roles in many aspects of nucleic acid metabolism, including replication, recombination, DNA repair and telomere maintenance. It is a heterotrimeric complex consisting of three subunits: RPA1, RPA2 and RPA3. It possesses four DNA-binding domains (DBD), DBD-A, DBD-B and DBD-C in RPA1 and DBD-D in RPA2, and it binds ssDNA via a multistep pathway. Unlike the RPA1 and RPA2 subunits, no ssDNA-RPA3 interaction has as yet been observed although RPA3 contains a structural motif found in the other DBDs. We show here using 4-thiothymine residues as photoaffinity probe that RPA3 interacts directly with ssDNA on the 3′-side on a 31 nt ssDNA.

Impact of the LH surge on granulosa cell transcript levels as markers of oocyte developmental competence in cattle

“…Single-stranded DNA binding protein 1 (SSCBP1) is essential for the response to DNA damage, and with the help of Integrator3 protein, it controls G(2)/M checkpoint activation (Zhang et al 2009). Replication protein A3 (RPA3), like SSCBP1, is also a single-stranded DNA-binding protein and plays a central role in many aspects of nucleic acid metabolism, including DNA replication, repair, and recombination (Salas et al 2009, Mason et al 2010. Proliferating cells are challenged with maintenance of genome integrity and chromatin organization in order to avoid aberrant gene expression.…”

5′ to 3′ Unfolding Directionality of DNA Secondary Structures by Replication Protein A

“…Second, an inverse relationship between telomeric G4 structural stability and hRPA binding has been established (27,33). Third, the hRPA binding mode (1:1 complexes where one RPA is bound on the DNA in an elongated mode, or 2:1 complexes where two RPA are bound on the DNA in a compact mode) depends on hRPA concentration (18,27). Fourth, cross-linking experiments showed that the RPA1 and RPA2 subunits of the hRPA directly contact the G4 sequence (27).…”

“…RPA carries six DNA-binding domains (DBD), four of them are located in RPA1 (DBD-A, DBD-B, DBD-C and DBD-F), one is located in RPA2 (DBD-D), and one in RPA3 (DBD-E). RPA binds to unstructured, single-stranded DNA (ssDNA) through three different binding modes involving five of the six DBD domains: DBDs A, B, C, D, and E (17)(18)(19). It is now accepted that RPA binds to ssDNA in a sequential pathway with a defined polarity (20,21).…”

“…First, the high-affinity DBD-A and DBD-B domains of RPA1 bind to 8 -10 nucleotides (nt) (22); this initial binding is designated as the "compact" conformation or 8 -10-nt binding mode. This step is followed by weaker interactions of DBD-C of RPA1 with the 3Ј side of the ssDNA, leading to an intermediate named "elongated contracted" conformation or 13-22-nt binding mode (18,23,24). Finally, binding of DBD-D of RPA2 and DBD-E of RPA3 to the 3Ј side of the ssDNA leads to a stable "elongated extended" complex in which RPA covers 30 nt (30-nt binding mode); this is the most stable RPA-ssDNA complex (18).…”

“…This step is followed by weaker interactions of DBD-C of RPA1 with the 3Ј side of the ssDNA, leading to an intermediate named "elongated contracted" conformation or 13-22-nt binding mode (18,23,24). Finally, binding of DBD-D of RPA2 and DBD-E of RPA3 to the 3Ј side of the ssDNA leads to a stable "elongated extended" complex in which RPA covers 30 nt (30-nt binding mode); this is the most stable RPA-ssDNA complex (18). RPA binds ssDNA in a non-sequence-specific manner (15); however, it has a higher affinity for pyrimidine sequences than for other sequence compositions.…”