Biochemistry of eukaryotic homologous recombination

Abstract

The biochemistry of eukaryotic homologous recombination caught fire with the discovery that Rad51 is the eukaryotic homolog of the bacterial RecA and T4 UvsX proteins; and this field is still hot. The core reaction of homologous recombination, homology search and DNA strand invasion, along with the proteins catalyzing it, are conserved throughout evolution in principle. However, the increased complexity of eukaryotic genomes and the diversity of eukaryotic cell biology pose additional challenges to the recombination machinery. It is not surprising that this increase in complexity coincided with the evolution of new recombination proteins and novel support pathways, as well as changes in the properties of those eukaryotic recombination proteins that are evidently conserved in evolution. In humans, defects in homologous recombination lead to increased cancer predisposition, underlining the importance of this pathway for genomic stability and tumor suppression. This review will focus on the mechanisms of homologous recombination in eukaryotes as elucidated by the biochemical analysis of yeast and human proteins.

Fig. 1

(overleaf). Pathways of homologous recombination. A. Repair of a frank DSB by double-strand break repair (DSBR) and synthesis-dependent strand annealing (SDSA). After Rad51-mediated D-loop formation, the DSBR and SDSA pathways split. In DSBR, a double Holliday junction (dHJ) is generated that can be resolved by a HJ-specific endonuclease into crossover and non-crossover products or dissolved by the action of BLM-TopoIIIα leading to non-crossover products only. In SDSA, the invading strand reanneals after DNA repair synthesis with the second DSB end without generating a dHJ intermediate, leading to non-crossover products only. B. Repair of a one-sided DSB. Cleavage of a stalled replication fork yields a one-sided DSB, a situation that is similar to break-induced replication (BIR) and recombination at chromosome ends (telomeres). Rad51-mediated DNA strand invasion (D-loop) can establish a replication fork with a single Holliday junction. C. Bypass of DNA damage blocking the lagging strand of a replication fork. Rad51-mediated DNA strand invasion using the blocked 3′ end leads to D-loop formation and may proceed either by a DSBR-type pathway (involving a dHJ) or an SDSA-type pathway (without dHJ). This pathway requires a 5′-3′ DNA helicase that peels the blocked strand off the template. Alternatively, Rad51 assembles a filament on the template strand to form a paranemic joint (no free end available), allowing the blocked end to use the new sister strand as a template, giving rise to a nicked dHJ. After DNA synthesis either a DSBR-type pathway (involving a dHJ) or an SDSA-type pathway (without dHJ) may ensue. Paranemic joint formation likely requires an additional factor(s) to stabilize the joint.

Fig. 2

Model reactions to study homologous recombination in vitro. A. DNA annealing. Annealing of protein-free DNA is a relatively non-specific reaction that can occur when proteins aggregate DNA non-specifically. Annealing of RPA-coated ssDNA instead is a highly specific reaction, catalyzed by the UvsY, RecO, and Rad52 proteins. B. D-loop reaction with either linear ssDNA (shown) or linear tailed DNA (not shown) and a supercoiled dsDNA substrate. Note that the RPA requirement for this reaction depends on the length and secondary-structure potential of the ssDNA. Low level, non-specific apparent D-loop formation can occur in particular when the supercoiled dsDNA substrate has been prepared by procedures involving DNA denaturation. C. Three strand DNA strand exchange between circular ssDNA and linear dsDNA. A well-known potential artifact with this assay is exonucleoytic resection followed by DNA strand annealing that will lead to the formation of intermediates and products that resemble DNA strand exchange intermediates. D-loop and DNA strand exchange reactions typically follow strict order of addition protocols: ssDNA + Rad51 → +RPA → +dsDNA. Inhibition of in vitro recombination by early RPA addition is overcome by the mediator proteins. Inhibition by early addition of dsDNA can be overcome by the Rad54 motor protein. Release of the product DNA is achieved in all reactions by treatment with detergent and proteinase, thus side-stepping a requirement for turnover by the proteins.

Fig. 3

Mechanistic stages of homologous recombination. HR can be conceptually divided into three stages. First, during presynapsis the ends are processed and the Rad51 filament is assembled. The potential functions of cofactors in Rad51 filament assembly are indicated as anchoring the non-growing end, binding to the growing end or binding the filament laterally. In addition, cofactors may control the Rad51 protomer pool. Second, in synapsis the Rad51 filament undergoes homology search and DNA strand invasion, likely in conjunction with Rad54 protein. Third, postsynapsis comprises all ensuing steps including branch migration, Rad51 turnover, and DNA synthesis, common to the DSBR and SDSA pathways, as well as the subpathway-specific functions of dHJ resolution by a putative resolvase (Resolvase A) and dHJ dissolution by BLM-TOPOIIIα. The SDSA sub-pathway requires a protein, likely a DNA helicase, to dissolve the D-loop and likely employs Rad52 in reannealing the broken ends. The representation of the proteins is for illustration purposes only and does not imply specific stoichiometries or a specific oligomeric assembly status.