All who wander are not lost: the search for homology during homologous recombination

Abstract

Homologous recombination (HR) is a template-based DNA double-strand break repair pathway that functions to maintain genomic integrity. A vital component of the HR reaction is the identification of template DNA to be used during repair. This occurs through a mechanism known as the homology search. The homology search occurs in two steps: a collision step in which two pieces of DNA are forced to collide and a selection step that results in homologous pairing between matching DNA sequences. Selection of a homologous template is facilitated by recombinases of the RecA/Rad51 family of proteins in cooperation with helicases, translocases, and topoisomerases that determine the overall fidelity of the match. This menagerie of molecular machines acts to regulate critical intermediates during the homology search. These intermediates include recombinase filaments that probe for short stretches of homology and early strand invasion intermediates in the form of displacement loops (D-loops) that stabilize paired DNA. Here, we will discuss recent advances in understanding how these specific intermediates are regulated on the molecular level during the HR reaction. We will also discuss how the stability of these intermediates influences the ultimate outcomes of the HR reaction. Finally, we will discuss recent physiological models developed to explain how the homology search protects the genome.

Keywords: DNA repair; Rad51; helicases; homologous recombination; homology search.

Figure 1.. Illustrating the HR process in Saccharomyces cerevisiae.

Homologous recombination (HR) initiates with DNA end resection of a DSB, creating 3′ overhangs of ssDNA. The ssDNA is initially bound by replication protein A (RPA) and (A) followed by recombinase binding which is regulated by a series of mediators and anti-recombinases, culminating in the formation of an active presynaptic complex (PSC). The PSC gains the capability to perform (B) the homology search among the genome by microhomology kinetic search, intersegmental search, and local 1D sliding search. Once the homologous sequence is identified, the ssDNA invades the dsDNA to form a D-loop. (C) Zoom in the black box in (B). The D-loop is reversible and regulated by several helicases and a topoisomerase, which eventually leads to different (D) HR outcomes.

Figure 2.. Cytological models of the homology search.

(A) The RecA bundle model proposes that RecA nucleates at sites of double-strand breaks and then forms a bundle of filaments to promote the homology search. This allows the identification of distant sister sites. (B) The dimensionality reduction model proposes that the growth of the RecA filament allows a matching sequence of the donor strand to dimensionally match at least a small portion of the RecA filament, which can then recognize a matching sequence in 2D space. (C) The direct pole-to-pole movement of RecA filaments observed in C. crescentus. Movement is dependent on the SMC motor protein RecN. (D) An expansion and contraction model were developed from in vivo imaging of Rad51. This model proposes that rounds of expansion and contraction help to promote pairing during the homology search.