Putting together and taking apart: assembly and disassembly of the Rad51 nucleoprotein filament in DNA repair and genome stability

Abstract

Homologous recombination is a key mechanism providing both genome stability and genetic diversity in all living organisms. Recombinases play a central role in this pathway: multiple protein subunits of Rad51 or its orthologues bind single-stranded DNA to form a nucleoprotein filament which is essential for initiating recombination events. Multiple factors are involved in the regulation of this step, both positively and negatively. In this review, we discuss Rad51 nucleoprotein assembly and disassembly, how it is regulated and what functional significance it has in genome maintenance.

Keywords: DNA repair; Rad51 filament; Rad51 regulation; double-stranded DNA break; homologous recombination.

Figure 1. FIGURE 1: A general overview of DSB repair mechanisms.

A DNA molecule with a DSB (blue lines) can either be repaired by NHEJ (A) or resected, thereby committing to HR. Sometimes, resected breaks can be healed by telomerase (DNTA, B) rather than repaired by HR but these events are rare. Resected DNA can be repaired by SSA (C) when there are direct repeats flanking the break (pink lines). Alternatively, resected ssDNA might invade a homologous donor molecule (red lines) enabling repair by BIR (D) or GC (E). During BIR, the invading strand is extended and the newly synthesised DNA is displaced from the donor to act as a template for the second strand. GC can proceed via SDSA or DSBR pathways. During SDSA, the invading ssDNA is extended, displaced from the donor and annealed to the other end of the break. DSBR involves the extension of the invading strand, capture of the second end of the break and the resolution of the dHJ intermediate formed. Arrow heads indicate 3' ends of the DNA. Dotted lines show newly-synthesised DNA and their colours correspond to the DNA molecules that have been used as templates. The black zigzag represents the telomere. Abbreviations: NHEJ - non-homologous end joining; DNTA - de novo telomere addition; SSA - single-strand annealing; BIR - break-induced replication; GC - gene conversion; SDSA - synthesis-dependent strand annealing; DSBR - double-strand break repair.

Figure 2. FIGURE 2: Rad51-catalysed strand exchange reaction.

Circular ssDNA is pre-incubated with Rad51 to allow Rad51 binding to DNA without competition. RPA is then added to the reaction to remove secondary DNA structures. Nucleated Rad51 can replace RPA and form a functional continuous nucleofilament. When linear dsDNA is added Rad51 can catalyse the strand exchange between the double-stranded donor and the circular ssDNA.

Figure 3. FIGURE 3: A model of Rad51 nucleofilament formation in budding yeast.

ssDNA generated as a result of resection or strand separation is rapidly covered by RPA (yellow spheres). Rad52 (purple spheres) binds RPA-coated ssDNA, recruits Rad51 (green spheres) and facilitates RPA-Rad51 exchange in the vicinity, thereby promoting Rad51-ssDNA filament formation. Rad55-Rad57 dimers (dark-green spheres) are also incorporated into the Rad51 nucleofilament and stabilise it by providing additional protein-protein interactions as well as antagonising Rad51 removal by Srs2 (teal ring). Only the main regulators of S. cerevisiae Rad51 are shown.

Figure 4. FIGURE 4: A hypothetical model for the complementing roles of Rad54 and Srs2 in Rad51 removal at dsDNA-ssDNA junctions.

In wild-type cells, Srs2 (teal ring) and Rad54 (orange ring) remove Rad51 from ssDNA and dsDNA respectively to allow RFC-PCNA (pink ellipsoid and light-brown ring) to access the dsDNA-ssDNA junction and recruit DNA polymerase (light-purple sphere). In the absence of Srs2, Rad54 removes Rad51 from dsDNA and either directly or indirectly promotes Rad51 dissociation from ssDNA at the junction.