Catalysis of Strand Annealing by Replication Protein A Derives from Its Strand Melting Properties

Abstract

Eukaryotic DNA-binding protein replication protein A (RPA) has a strand melting property that assists polymerases and helicases in resolving DNA secondary structures. Curiously, previous results suggested that human RPA (hRPA) promotes undesirable recombination by facilitating annealing of flaps produced transiently during DNA replication; however, the mechanism was not understood. We designed a series of substrates, representing displaced DNA flaps generated during maturation of Okazaki fragments, to investigate the strand annealing properties of RPA. Until cleaved by FEN1 (flap endonuclease 1), such flaps can initiate homologous recombination. hRPA inhibited annealing of strands lacking secondary structure but promoted annealing of structured strands. Apparently, both processes primarily derive from the strand melting properties of hRPA. These properties slowed the spontaneous annealing of unstructured single strands, which occurred efficiently without hRPA. However, structured strands without hRPA displayed very slow spontaneous annealing because of stable intramolecular hydrogen bonding. hRPA appeared to transiently melt the single strands so that they could bind to form double strands. In this way, melting ironically promoted annealing. Time course measurements in the presence of hRPA suggest that structured single strands achieve an equilibrium with double strands, a consequence of RPA driving both annealing and melting. Promotion of annealing reached a maximum at a specific hRPA concentration, presumably when all structured single-stranded DNA was melted. Results suggest that displaced flaps with secondary structure formed during Okazaki fragment maturation can be melted by hRPA and subsequently annealed to a complementary ectopic DNA site, forming recombination intermediates that can lead to genomic instability.

Mechanistic Studies of Hairpin to Duplex Conversion for Trinucleotide Repeat Sequences

“…A similar destabilization has been reported for replication protein A. For single strands of DNA capable of adopting a folded conformation, replication protein A altered the equilibrium between melting and formation of duplex (38). Furthermore, our fluorescence measurements established that the FL-(CAG) 10 -DCL molecular beacon hairpin melts at 38.8°C; optical analysis reveals that duplex formation occurs at 37.3°C.…”

Reconstitution of recombination-associated DNA synthesis with human proteins

“…This explains why we observe a reduction in D-loops levels in reactions lacking RPA in the absence of DNA synthesis (Figure 2B). On canonical templates, RPA binds both the primer-template junction of DNA, facilitating initiation of DNA synthesis, and to the template strand downstream of the 3′-primer terminus, facilitating synthesis by removing secondary structures (41,49,50). We hypothesized that during recombination-associated DNA synthesis from the invading strand of the D-loop, RPA would also bind the template strand in addition to the demonstrated binding of the displaced D-loop strand.…”

Yeast Pif1 Accelerates Annealing of Complementary DNA Strands

“…An example of such complex behavior is exhibited by the RPA protein. Both human 52 and yeast 53 RPA can stimulate or inhibit annealing in vitro depending on the type of substrate and conditions. It is feasible that Pif1 and other positively charged enzymes simply act as chemical attractants of negatively charged DNA.…”