Biochemical Mechanisms of Genetic Recombination and DNA Repair

Abstract

Genetic recombination involves the exchange of genetic material between homologous sequences of DNA. It is employed during meiosis in sexually reproducing organisms or in somatic cells to accurately repair toxic DNA lesions like double-strand breaks and stalled replication forks. In these separate roles, recombination drives genetic diversity by enabling reshuffling of parental genetic information while also serving as a molecular safeguard against the deleterious effects of gross chromosomal rearrangements or mutagenic insults arising for either endogenous or exogenous reasons. In both cases, efficient recombination ensures faithful transmission of genetic information to subsequent generations. In this review, we provide an exploration of the biochemical mechanisms driving genetic recombination, elucidating the molecular intricacies of fundamental processes involved therein with a focus on mechanistic insights gained into these processes using biochemical and single-molecule techniques.

Keywords: DNA end resection; crossover; homologous recombination; homology search; meiotic recombination; noncrossover.

Figure 1

DNA end resection. (a) DSBs occur on the DNA. (b) Ku and MRN/X are recruited. (c) Phosphorylated CtIP–Sae2 is recruited by MRN/X and stimulates the nuclease activity of MRE11, leading to the introduction of DNA nicks and the initiation of short-range resection. (d) EXO1 or DNA2 plus BLM or WRN initiate bidirectional resection. (e) Long-range resection yields large stretches of single-stranded DNA that are rapidly bound by RPA. (f) RPA phosphorylation provides negative feedback to the end resection machinery. Abbreviations: BLM, Bloom helicase; CtIP, CtBP-interacting protein; DSB, double-strand break; EXO1, exonuclease I; MRN/X, MRE11–RAD50–NBS1/Xrs2 complex; RPA, replication protein A; WRN, Werner helicase.

Figure 2

Presynaptic filament formation and homology search. (a) RAD51 replaces RPA on the single-stranded DNA leading to the formation of a RAD51–nucleoprotein filament, and this process is subject to both positive and negative regulatory factors. (b) The presynaptic filament can search for homology through a combination of diffusion-based processes (intersegmental transfer, 1D sliding). (c) Homology search is augmented by the motor protein RAD54, which can drive ATP-dependent translocation of the presynaptic filament along the double-stranded DNA. Abbreviations: 1D, one-dimensional; BLM, Bloom helicase; EXO1, exonuclease I; RPA, replication protein A; WRN,Werner helicase.

Figure 3

Synthesis-dependent strand annealing. (a) The single-stranded DNA formed after end resection is paired with the homologous template by Rad51 to yield a D-loop; note that Rad51 protein is not shown in the figure. (b) The invading 3′ end is extended by DNA synthesis. (c) The D-loop is then disrupted, allowing the newly synthesized end to be paired with the second DNA end. (d) The resulting intermediate is further processed by gap filling and ligation leading to the repair of the double-strand break. Abbreviation: D-loop, displacement loop.

Figure 4

The DSBR pathway. (a) During DSBR, D-loop formation is followed by second-end capture. (b) DNA synthesis leads to the formation of a dHJ. (c) dHJs can be processed via dissolution and resolution. (d) In dissolution, helicases and topoisomerases coordinate to enable convergent migration of the two HJs, which are processed to give rise to noncrossover products. (e) In resolution, structure-specific endonucleases cleave the HJs to yield either a crossover or noncrossover product. Abbreviations: BLM, Bloom helicase; dHJ, double Holliday junction; D-loop, displacement loop; DSBR, double-strand break repair; HJ, Holliday junction.

Figure 5

Break-induced replication (BIR). (a) BIR is initiated when there is homology present only to one end of the double-strand break (DSB). (b) Displacement loop (D-loop) formation takes place followed by extension. (c) PCNA, RFC, Polδ, and PIF1 coordinate bubble migration along with synthesis of long tracts of DNA. (d) The newly synthesized DNA is used as a template for replicating the lagging strand. (e) The newly formed double-stranded DNA is ligated, leading to the repair of the DSB.

Figure 6

Meiotic DSB formation. (a) Meiotic chromosomes are organized into a series of chromatin loops that emanate from the proteinaceous axis, which consists of multiple proteins including those directly involved in catalyzing the DSBs. (b) Interactions between different proteins and DNA signatures help bring the DNA sequence on the loop into close proximity to the axis where DSBs are catalyzed. Abbreviation: DSB, double-strand break.