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). Although budding yeast Rad51 has been extensively characterized in vitro, 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 studies. 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.

Extended Data Figure 1

Sensitivity of BIR to mismatches. a, Donors differing by a single bp. b, Donors containing 0, 1, 2, 3 or 4 mismatches. Experiments were independently repeated 3 times and averaged to arrive at experimental mean. Error bars represent s.e.m. Asterisks indicate p < 0.01, student’s t test. Blue and red letters represent the mismatches in the recipient and donor.

Extended Data Figure 2

Sensitivity of BIR to mismatches. In the strains carrying mismatches at every 6^th, 5^th and 4^th position, all of the residual recombinants were Rad51-dependent. BIR data for WT and rad51Δ are shown. Mean of each of the experiments are shown at the top of the respective histogram. Error bars represent s.e.m based on a minimum of 3 independent experiments.

Extended Data Figure 3

Theoretical modeling 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 3 Rad51 monomers can bind if the first 5 sites are perfectly base-paired and the remaining 4 sites can tolerate a single mismatch, plotted for each possible donor with donors having uniformly spaced mismatches with 1 to 54 bp spacings, compared to the measured data (red symbols) derived from Fig. 1. Purple Xs show the expected fraction of possible alignments based on a dimer of Rad51 that must complete all 6 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 (Chr 5). This break can be repaired by a BIR mechanism using the donor sequences that share 300 bp of homology (R) located on the opposite arm of Chr 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 and 0 nt tails respectively. b, Invasion intermediates (D-loop) 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 nonhomologous end-joining (NHEJ) survivors that had a ‘CA’ insertion at the HOcs after induction of pGAL1-HO endonuclease. Such NHEJ survivors are immune to cutting by HO endonuclease. pGAL1-Cas9 constructs were then used to generate nonhomologous tails of various lengths. PAM sequences are in bold. c, For generating 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 6^th, 7^th, 8^th or 9^th position. A minimum of 24 samples were sequence analyzed for each of the construct.

Extended Data Figure 7

Series of events that take place in BIR vs. GC. a, In BIR, we exclusively measure mismatch correction of heteroduplex DNA (dashed box) formed during the initial strand invasion. b, In GC, 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 2 mismatches are shown. Mismatch correction in GC studies therefore could be a combination of correction in the context of invasion and second-end capture.

Fig. 1

Stringency and sensitivity of Rad51-mediated recombination. a, A DSB is repaired by BIR using homologous or homeologous donor (108 bp). UR: first 400 bp of the URA3 ORF. SD: splice donor. SA: splice acceptor. A3: the remaining 404 bp of the URA3 ORF. b, 108, 54 and 27 bp donors. c, Donors differing by a single bp. d, Donors containing 0, 1, 2, 3 or 4 mismatches. e, Donors containing mismatches every 12^th, 11^th, etc. position. Error bars represent s.e.m. Asterisks indicate p < 0.01, student’s t test.

Fig. 2

Influence of nonhomologous tail on recombination. a, Efficiencies of BIR between ‘UR’ and ‘RA3’ in the presence of varying lengths of nonhomologous tail. UR and RA3 share 300 bp. HO endonuclease generates a 68 bp nonhomologous tail. Cas9 endonuclease generates 42, 30, 24, 10, 3 or 0 nt tails. b, Effect of deleting mismatch repair genes when there is no nonhomologous tail (108 bp donor). c, Effect of introducing a 34 bp nonhomologous tail with 108-bp donor (Fig. 2b) in the presence or absence of mismatches. Cas9 endonuclease was used to generate a 34 bp tail. Error bars represent s.e.m.

Fig. 3

Mismatch correction during BIR is dependent on the placement of the mismatch along the heteroduplex. a, Predicted sequence-changes in the recipient during BIR with or without correction. b, Correction of a single bp mismatch at different positions. c, Correction of 1, 2, 3 and 4 evenly-spaced mismatches. d, Correction of multiple, evenly-spaced mismatches. e, Correction of transversion (top) and transition (bottom) mismatches. f, Correction of clustered mismatches. Percent correction (%) and positional information of mismatches are indicated.

Fig. 4

Mismatch-correction in the heteroduplex. a, Effect of nonhomologous tail on correcting a single mismatch. b, Effect of mutations on correction of multiple mismatches. c, Effect of nonhomologous tail compared to panel b. d, Model depicting correction in the heteroduplex. Left panel: Mlh1/Pms1-mediated correction may result in a repair patch (dashed box) whereas mismatches closer to the 3′ end are left unrepaired. No such instances were found. Right panel: DNA polymerase δ-mediated correction resulting in a co-corrected patch (dashed box). Predictions by each model are represented in bold letters.