Rad51-mediated double-strand break repair and mismatch correction of divergent substrates

Abstract

The Rad51 (also known as RecA) family of recombinases executes the critical step in homologous recombination: the search for homologous DNA to serve as a template during the repair of DNA double-strand breaks (DSBs)1,2,3,4,5,6,7. Although budding yeast Rad51 has been extensively characterized in vitro3,4,6,7,8,9, the stringency of its search and sensitivity to mismatched sequences in vivo remain poorly defined. Here, in Saccharomyces cerevisiae, we analysed Rad51-dependent break-induced replication in which the invading DSB end and its donor template share a 108-base-pair homology region and the donor carries different densities of single-base-pair mismatches. With every eighth base pair mismatched, repair was about 14% of that of completely homologous sequences. With every sixth base pair mismatched, repair was still more than 5%. Thus, completing break-induced replication in vivo overcomes the apparent requirement for at least 6–8 consecutive paired bases that has been inferred from in vitro studies6,8. When recombination occurs without a protruding nonhomologous 3′ tail, the mismatch repair protein Msh2 does not discourage homeologous recombination. However, when the DSB end contains a 3′ protruding nonhomologous tail, Msh2 promotes the rejection of mismatched substrates. Mismatch correction of strand invasion heteroduplex DNA is strongly polar, favouring correction close to the DSB end. Nearly all mismatch correction depends on the proofreading activity of DNA polymerase-δ, although the repair proteins Msh2, Mlh1 and Exo1 influence the extent of correction.

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Figure 1: Stringency and sensitivity of Rad51-mediated recombination.
Figure 2: Influence of nonhomologous tail on recombination.
Figure 3: Mismatch correction during BIR depends on the placement of the mismatch along the heteroduplex.
Figure 4: Mismatch correction in the heteroduplex.

Change history

  • 25 May 2017

    The DOI in ref. 29 was corrected.

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Acknowledgements

We thank W.-D. Heyer, T. Petes, M. Prentiss, S. Keeney and members of the Haber laboratory for their comments; V. Vyas for providing the S. cerevisiae codon optimized Cas9 plasmid; J. E. DiCarlo for providing the hCas9 plasmid; and the makers of the freely available DNA analyses software Serial Cloner 2.6.1 (F. Perez). R.A. is a former recipient of an NRSA award (1F32GM096690). This work was supported by NIH grants GM20056 and GM76020 to J.H.

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Contributions

R.A. and J.H. conceived, designed and developed the genetic assays to measure BIR efficiency. R.A. designed synthetic constructs (‘gBlocks’) with defined mismatches. R.A. designed and developed Cas9-based protocols. R.A and A.B. carried out experiments that measured BIR efficiency. R.A. did the data analyses, including data compilation, statistical testing, and DNA sequence analyses of the break repair junctions. K.L. and J.H. did the theoretical modelling. R.A. and J.H. wrote the paper.

Corresponding author

Correspondence to James Haber.

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The authors declare no competing financial interests.

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Reviewer Information Nature thanks M. Cox, H. Klein and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Sensitivity of BIR to mismatches.

a, Donors differing by a single base pair. b, Donors containing 0, 1, 2, 3 or 4 mismatches. Experiments were independently repeated three times and averaged to arrive at the experimental mean. Error bars represent s.e.m. **P < 0.01, Student’s t-test. Blue and red letters represent the mismatches in the recipient and donor, respectively.

Extended Data Figure 2 Sensitivity of BIR to mismatches.

In the strains carrying mismatches at every sixth, fifth or fourth position, all of the residual recombinants were Rad51-dependent. BIR data for wild type and rad51Δ are shown. The mean of each of the experiments is shown at the top of the respective histogram. Error bars represent s.e.m based on a minimum of three independent experiments.

Extended Data Figure 3 Theoretical modelling of recombination in vivo.

Fraction of possible alignments of Rad51 multimers that meet the criteria for an initial, stable strand invasion between ssDNA and homeologous donor sequences. Blue symbols show a model in which three Rad51 monomers can bind if the first five sites are perfectly base-paired and the remaining four sites can tolerate a single mismatch, plotted for each possible donor with donors having uniformly spaced mismatches with 1–54-bp spacings, compared to the measured data (red symbols) derived from Fig. 1. Purple crosses show the expected fraction of possible alignments based on a dimer of Rad51 that must complete all six consecutive base pairs.

Extended Data Figure 4 Presence of a nonhomologous tail affects recombination.

a, Schematic of the chromosomal construct. A DSB is induced by the galactose-inducible HO endonuclease adjacent to the UR segment, located at the CAN1 locus in a non-essential terminal region of chromosome 5. This break can be repaired by a BIR mechanism using donor sequences that share 300 bp of homology (R) located on the opposite arm of chromosome 5 and situated about 30 kb proximal to the telomere. DSB induction by HO generates a 68-bp nonhomologous tail that is removed before primer extension by DNA polymerase. DSB induction by various Cas9 constructs generates 42-, 30-, 24-, 10-, 3- or 0-nucleotide tails. b, Invasion intermediates (D-loops) with or without a nonhomologous tail (red arrow). c, Influence of nonhomologous tail on BIR.

Extended Data Figure 5 Nonhomologous tails generated by HO and Cas9 endonuclease.

a, Schematic of the invasion intermediates (D-loop) with or without a nonhomologous tail (red arrow). b, To use pGAL1–Cas9, the HO cleavage site was first removed by selecting NHEJ survivors that had a CA insertion at the HOcs after induction of pGAL1-HO endonuclease30. Such NHEJ survivors are immune to cutting by HO endonuclease. pGAL1–Cas9 constructs were then used to generate nonhomologous tails of various lengths. Protospacer adjacent motif (PAM) sequences are in bold. c, To generate a 0-nt tail, a strain with CAACGG adjacent to the UR region was constructed that could be cut with Cas9. See Supplementary Table 1.

Extended Data Figure 6 Mismatch correction of multiple, evenly-spaced mismatches.

Donors differ at every sixth, seventh, eighth or ninth position. A minimum of 24 samples were sequence analysed for each of the constructs.

Extended Data Figure 7 Series of events that take place in BIR compared to gene conversion.

Left, in BIR, we exclusively measure mismatch correction of heteroduplex DNA (dashed box) formed during the initial strand invasion. Right, in gene conversion, sequences copied from the donor by extending the invading strand may extend well beyond half the length of the homology on the second end. Annealing between this extended end and the resected second end (second-end capture) would result in heteroduplex DNA (dashed box). For simplicity, only two mismatches are shown. Mismatch correction in gene conversion studies therefore could be a combination of correction in the context of invasion and second-end capture.

Extended Data Table 1 Mismatch correction of a single-base-pair mismatch in various repair-defective mutants

Supplementary information

Supplementary Information

This file contains Supplementary Tables 1-2 and the computer code. (PDF 378 kb)

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Anand, R., Beach, A., Li, K. et al. Rad51-mediated double-strand break repair and mismatch correction of divergent substrates. Nature 544, 377–380 (2017). https://doi.org/10.1038/nature22046

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