A dynamic model for replication protein A (RPA) function in DNA processing pathways

Abstract

Processing of DNA in replication, repair and recombination pathways in cells of all organisms requires the participation of at least one major single-stranded DNA (ssDNA)-binding protein. This protein protects ssDNA from nucleolytic damage, prevents hairpin formation and blocks DNA reannealing until the processing pathway is successfully completed. Many ssDNA-binding proteins interact physically and functionally with a variety of other DNA processing proteins. These interactions are thought to temporally order and guide the parade of proteins that 'trade places' on the ssDNA, a model known as 'hand-off', as the processing pathway progresses. How this hand-off mechanism works remains poorly understood. Recent studies of the conserved eukaryotic ssDNA-binding protein replication protein A (RPA) suggest a novel mechanism by which proteins may trade places on ssDNA by binding to RPA and mediating conformation changes that alter the ssDNA-binding properties of RPA. This article reviews the structure and function of RPA, summarizes recent studies of RPA in DNA replication and other DNA processing pathways, and proposes a general model for the role of RPA in protein-mediated hand-off.

Figure 1

The modular structure of RPA. (a) Schematic diagram: arrows indicate intersubunit associations; protein-binding domains are denoted by red bars; ssDNA binding domains A–D by hatching; OB-folds by blue boxes; linkers by yellow boxes; winged helix by a green box; phosphoamino acid cluster by a circled P [adapted from (29) with permission]. (b) Structural models of RPA domains. [Reprinted from (20,24,25,55) with permission.]

Figure 2

(a) Covalent linkage of RPA70 ssDNA-binding domains A and B enhances their affinity to ssDNA. Binding constants of A or B with d(CTTCA) and AB with d(CTTCA CTTCA) were determined (29). (b) Sequential 5′→3′ binding of RPA to ssDNA. Positioning of RPA70N and RPA14 relative to other domains is speculative (24,103). Dashed lines depict a potential pathway for RPA displacement from ssDNA. (c) Schematic diagram of the primer–template junction-binding mode of the RPA trimerization core (70C-32D-14) (38).

Figure 3

(a–d) A model for protein-mediated RPA displacement from ssDNA in concert with loading of the next protein in the pathway [Reprinted from (48), with permission, Nature Publication Group]. For discussion see text.

Figure 4

Phosphorylation of RPA32N and its potential functional roles. (a) Diagram of the phosphoamino acid cluster in RPA32N. Boldface, sites phosphorylated by CDK; underlined, likely phosphorylated by PIKKs; asterisks, phosphorylated by unknown kinases. (b) Hyperphosphorylation of RPA32N is proposed to shift the equilibrium distribution of RPA conformation states/binding modes to favor the high-affinity extended mode.