Single-Stranded-DNA Binding Alters Human Replication Protein A Structure and Facilitates Interaction with DNA-Dependent Protein Kinase

Abstract

Human replication protein A (hRPA) is an essential single-stranded-DNA-binding protein that stimulates the activities of multiple DNA replication and repair proteins through physical interaction. To understand DNA binding and its role in hRPA heterologous interaction, we examined the physical structure of hRPA complexes with single-stranded DNA (ssDNA) by scanning transmission electron microscopy. Recent biochemical studies have shown that hRPA combines with ssDNA in at least two binding modes: by interacting with 8 to 10 nucleotides (hRPA 8nt ) and with 30 nucleotides (hRPA 30nt ). We find the relatively unstable hRPA 8nt complex to be notably compact with many contacts between hRPA molecules. In contrast, on similar lengths of ssDNA, hRPA 30nt complexes align along the DNA and make few intermolecular contacts. Surprisingly, the elongated hRPA 30nt complex exists in either a contracted or an extended form that depends on ssDNA length. Therefore, homologous-protein interaction and available ssDNA length both contribute to the physical changes that occur in hRPA when it binds ssDNA. We used activated DNA-dependent protein kinase as a biochemical probe to detect alterations in conformation and demonstrated that formation of the extended hRPA 30nt complex correlates with increased phosphorylation of the hRPA 29-kDa subunit. Our results indicate that hRPA binds ssDNA in a multistep pathway, inducing new hRPA alignments and conformations that can modulate the functional interaction of other factors with hRPA.

In vitro analysis of the zinc-finger motif in human replication protein A

“…It is likely that a mutation at the zincfinger domain inhibits the conformational change in p70 that is necessary for the switch from an unstable and weak RPA-ssDNA interaction to a stable and strong RPA-ssDNA interaction. This is also supported by recent biochemical studies [29], which indicate that human RPA combines with ssDNA in at least two binding modes : one is a relatively unstable and compact complex and the other is an elongated and stable complex.…”

“…Upon DNA binding, the structural change in p70 allows p34 (and its phosphorylation sites) access to DNA-PK for phosphorylation. In keeping with this notion, others have shown that formation of the extended hRPA30nt complex, the stable form of RPA-DNA interaction, correlates with increased phosphorylation of RPA p34 [29]. In this way, the phosphorylation of p34 by DNA-PK is controlled by the p70 subunit in a zinc-fingerdomain dependent manner.…”

“…ZFM1 with a mutation at Cys-487 (mutated to alanine) showed ssDNA-binding activity similar to that of wt-RPA ( Figure 6). The two distict bands seen in Figure 6 represent RPA-DNA complexes with different ratios of RPA : DNA [25,29]. In contrast, ZFM2 and ZFM4 showed 2-3-fold lower ssDNA-binding activity in the presence of 0.1 µg of RPA (lanes 2 and 5 compared with lanes 8 and 11 in Figure 6).…”

“…Previous studies on RPA-ssDNA interaction and limited proteolysis strongly suggest that RPA undergoes a structural change when it interacts with DNA [29,30]. p70 has multiple functional domains, including a pol α stimulation domain, an ssDNA-binding domain, and a conserved zinc-finger domain of the 4-cysteine type [17,31,32].…”

Chromatin Association of Replication Protein A

“…Two binding modes have been identified: an intermediate and relatively weak binding mode, characterized by an occluded binding site of 8 -10 nucleotides (8), and a more stable binding mode in which RPA covers ϳ30 nucleotides (7,9,10). Single-stranded DNA covered by RPA remains in an extended conformation under low ionic strength conditions, but more physiological salt concentrations induce a severe compaction of RPA⅐DNA complexes (11), which may reflect yet another DNA-binding mode possibly including 90 nucleotides of bound DNA, as first suggested by work with yeast RPA (12).…”