DNA Sequence Alignment during Homologous Recombination

Abstract

Homologous recombination allows for the regulated exchange of genetic information between two different DNA molecules of identical or nearly identical sequence composition, and is a major pathway for the repair of double-stranded DNA breaks. A key facet of homologous recombination is the ability of recombination proteins to perfectly align the damaged DNA with homologous sequence located elsewhere in the genome. This reaction is referred to as the homology search and is akin to the target searches conducted by many different DNA-binding proteins. Here I briefly highlight early investigations into the homology search mechanism, and then describe more recent research. Based on these studies, I summarize a model that includes a combination of intersegmental transfer, short-distance one-dimensional sliding, and length-specific microhomology recognition to efficiently align DNA sequences during the homology search. I also suggest some future directions to help further our understanding of the homology search. Where appropriate, I direct the reader to other recent reviews describing various issues related to homologous recombination.

Keywords: DNA recombination; DNA repair; Dmc1; Rad51; RecA; biochemistry; biophysics; homologous recombination; homology search; single-molecule biophysics.

FIGURE 1.

Overview of homologous recombination. A, simplified schematic highlighting the early stages of recombination. DSBs are resected to yield long 3′ ssDNA overhangs that serve as the binding sites for Rad51/RecA DNA recombinases. The recombinases form long filaments on the ssDNA that are referred to as the presynaptic complex. The presynaptic complex then searches for a homologous DNA and pairs the processed ssDNA overhang with its homologous partner to generate a D-loop intermediate. B, RecA-ssDNA crystal structure highlighting the base triplet organization of the presynaptic ssDNA (34). Figure adapted with permission from Ref. .

FIGURE 2.

Single-molecule optical microscopy studies of the homology search. A, example illustrating how modulating dsDNA extension in a dual-beam optical trap can affect the efficiency of DNA pairing interactions during homologous recombination. Adapted with permission from Ref. . Error bars represent the standard error of the mean from multiple experiments. B, schematic illustration of a singe-molecule FRET measurement of the homology search. Presynaptic complexes labeled with an acceptor fluorophore (red) are anchored to a surface. Duplex DNA labeled with a donor fluorophore (green) is then injected into the sample chamber. Fluorescence from the acceptor is detected when the donor-labeled dsDNA binds to the presynaptic complex. Dynamic behaviors of the resulting intermediates are revealed as distance-dependent changes in the FRET signal. ATPγS, adenosine 5′-O-(thiotriphosphate). Adapted with permission from Ref. . C, examples of kymographs from a DNA curtain assay using fluorescently tagged dsDNA fragments. Transient binding events are detected with DNA fragments containing ≤7 nt of microhomology, and more stable events are observed for fragments with ≥8 nt of microhomology. Adapted with permission from Ref. .

FIGURE 3.

Homology search model. A, graphical representation of microhomology length versus abundance as calculated for the S. cerevisiae genome. Sequences ≤7 nt in length can be ignored during the homology search, whereas longer sequences, which are far less abundant, are more carefully scrutinized. Adapted with permission from Ref. . B, the search process involves multiple contact points between the presynaptic complex and the dsDNA molecule that is being interrogated for homology. The most stable contacts bear ≥8 nt of microhomology, allowing the presynaptic complex to probe flanking sequences for additional homology. Contacts with ≤7 nt of microhomology are rapidly released, ensuring that the search is focused only on sequences with a high probability of being the correct target. Each individual contact can slide short distances, and release of a single contact allows the presynaptic complex to sample a different region of the dsDNA for sequence homology. For clarity, the proteins and flanking dsDNA are omitted from the presynaptic complex.

FIGURE 4.

Theoretical studies and in vivo observations of the homology search. A, schematic of the duplet pairing interactions observed in molecular dynamics simulations and proposed transition leading to a more stable binding configuration. Adapted with permission from Ref. . B, live cell microscopy assay to visualize DSB-dependent motion of RFP- and YFP-tagged chromosomal loci. The yellow and red trajectories superimposed upon a differential interference contrast image of the cell reveal diffusive motions of the DNA after γ irradiation. Adapted with permission from Ref. . C, live cell microscopy revealing the existence of DSB-dependent RecA bundles (green). Adapted with permission from Ref. .