DNA nicks in both leading and lagging strand templates can trigger break-induced replication

Abstract

Encounters between replication forks and unrepaired DNA single-strand breaks (SSBs) can generate both single-ended and double-ended double-strand breaks (seDSBs and deDSBs). seDSBs can be repaired by break-induced replication (BIR), which is a highly mutagenic pathway that is thought to be responsible for many of the mutations and genome rearrangements that drive cancer development. However, the frequency of BIR's deployment and its ability to be triggered by both leading and lagging template strand SSBs were unclear. Using site- and strand-specific SSBs generated by nicking enzymes, including CRISPR-Cas9 nickase (Cas9n), we demonstrate that leading and lagging template strand SSBs in fission yeast are typically converted into deDSBs that are repaired by homologous recombination. However, both types of SSBs can also trigger BIR, and the frequency of these events increases when fork convergence is delayed and the non-homologous end joining protein Ku70 is deleted.

Keywords: CRISPR-Cas9n; DNA replication; Flp-nick; break-induced replication; double-strand break; homologous recombination; replication fork; replication fork collapse; replication restart; single-strand break.

Graphical abstract

Figure 1

Cas9n-induced SSBs are converted into deDSBs that induce direct repeat recombination (A) Diagram showing the location of the recombination reporter and gRNA1 binding site (red arrow) on chromosome 3. It includes the centromere (blue oval), replication origins (brown circles), probes (gray rectangles), relevant restriction sites, and genes (orange and green rectangles with transcription direction indicated). The replication origins are sourced from the S. pombe DNA replication origin database: http://www.pombe.oridb.org. Asterisks mark the positions of loss-of-function mutations in the two ade6 alleles. The bottom panel displays the two types of Ade+ recombinants. (B) Detection of DSBs in genomic DNA by Southern blot analysis. DNA was extracted from cells expressing the indicated version of Cas9 with and without gRNA1. The DNA was cut with PstI and NheI, and the relevant restriction fragment was detected using probes A and S (see A). (C) Quantification of the DSB signals detected by Southern blot analysis. Data are presented as mean values ± SD. Individual data points are shown as colored dots, with the color denoting which experiment the data are from. (D) Frequency of Ade+ recombinants in strains containing the ade6⁻ direct repeat substrate shown in (A). The presence of gRNA1 and version of Cas9 is indicated. Data are presented as median values ± 95% confidence interval with individual data points shown as gray dots. Fold changes and p values refer to the comparison of total Ade+ frequencies between the same strains with and without gRNA1. The p values were calculated by the two-tailed Mann-Whitney test. ^∗∗∗∗p < 0.0001. See also Table S1.

Figure 2

DSBs arising from Cas9n-induced SSBs are primarily repaired through Rad51-dependent SCR Effect of Cas9/Cas9n expression, without (A) and with (B) gRNA1, on the frequency of Ade+ recombinants in strains containing the ade6⁻ direct repeat substrate shown in Figure 1A. The top panel shows the frequency of Ade+ recombinants for each strain with data presented as median values ± 95% confidence interval with individual data points shown as gray dots. The bottom panel shows the percentage of deletions and gene conversions among Ade+ recombinants. Fold changes and p values are relative to the recombination value for the corresponding wild-type strain. The p values were calculated by the Kruskal-Wallis test with Dunn’s multiple comparisons post-test. ^∗∗∗∗p < 0.0001; ^∗∗∗p < 0.001; ^∗∗p < 0.01; n.s., not significant. See also Table S1.

Figure 3

The cytotoxicity and mutagenicity of Cas9/Cas9n-induced DNA breaks (A) Diagram depicting the location of kan^R gene (purple rectangle) and the gRNA2 binding site (red arrow) on chromosome 3. An ade6⁻ direct repeat recombination reporter, similar to Figure 1A, is positioned 0.7 kb downstream of kan^R. It also shows the centromere (blue oval), replication origins (brown circles), ade6 genes (orange rectangles), and the his3 gene (green rectangle), with arrows indicating transcription direction. Asterisks mark the positions of loss-of-function mutations in the two ade6 alleles. (B) Spot assay showing the sensitivity of wild-type and indicated mutant strains to Cas9 cleavage of the kan^R gene shown in (A). In each case, sensitivity is compared with and without gRNA2, and with Cas9 expression repressed (+thiamine) or induced (−thiamine). (C) The same as (B) except sensitivity to Cas9n^H840A-induced nicking of kan^R is shown. (D) The same as (B) except sensitivity to Cas9n^D10A-induced nicking of kan^R is shown. (E) Comparison of kan^R gene mutation by Cas9 and Cas9n. Colonies, expressing the indicated version of Cas9, were grown with and without gRNA2. The colonies were then replica plated onto media with and without G418. The images show example replica plates after incubation at 30°C for 24 h. (F) Percentage of G418-sensitive colonies obtained from the plating assay shown in (E). Data from 5 independent experiments are presented as mean values ± SD. Individual data points are shown as gray dots.

Figure 4

Cas9n-induced lead- and lag-SSBs trigger BIR (A) Effect of Cas9/Cas9n/Cas9d expression, with and without gRNA2, on the frequency of Ade+ recombinants in strains containing the ade6⁻ direct repeat recombination reporter and kan^R gene shown in Figure 3A. (B) Frequency of Cas9n-induced Ade+ recombinants in the indicated mutant strains containing the recombination reporter and kan^R gene shown in Figure 3A. In both (A) and (B), data are presented as median values ± 95% confidence interval with individual data points shown as gray dots. In (A), the indicated fold changes and p values relate to the comparison of total Ade+ frequencies between the same strains with and without gRNA2, while in (B), they are relative to the equivalent wild-type strain value. The p values in (A) were calculated by the two-tailed Mann-Whitney test, while those in (B) were derived from the Kruskal-Wallis test with Dunn’s multiple comparisons post-test. ^∗∗∗∗p < 0.0001; ^∗p < 0.05; n.s., not significant. See also Table S1.

Figure 5

BIR is limited by fork convergence and suppressed by Ku70 (A) Diagram showing the location of kan^R gene (purple rectangle) and gRNA2 binding site (red arrow) ∼14 kb upstream of the same ade6⁻ direct repeat recombination reporter shown in Figure 3A. (B) Frequency of Cas9n-induced Ade+ recombinants in wild-type, ori1253Δ, and ori1253Δ ku70Δ strains containing the recombination reporter and kan^R gene shown in (A). The presence of gRNA2 and version of Cas9 is indicated. Data are presented as median values ± 95% confidence interval with individual data points shown as gray dots. The indicated fold changes and p values pertain to the comparison of recombination values between the same strains with and without gRNA2. The p values were calculated by the two-tailed Mann-Whitney test. ^∗∗∗∗p < 0.0001; ^∗∗p < 0.01. See also Table S1.

Figure 6

Lead- and lag-SSBs induced by Flp^H305L and gpII trigger BIR (A) Diagram showing the different site-specific-nicking enzymes used to generate lead- and lag-SSBs (top) and their cleavage sites located upstream of the ade6⁻ direct repeat recombination reporter on chromosome 3 (bottom). The recombination reporter and its location are the same as shown in Figures 3A and 5A. (B) Frequency of Ade+ recombinants induced by Cas9n, Flp-nick, and gpII in ori1253Δ strains containing the recombination reporter shown in (A). The position of the cleavage site in either the leading template (top) strand (TS) or lagging template (bottom) strand (BS) is indicated. For Cas9n, its expression without gRNA2 acts as a no SSB control. Absence of the nicking enzyme acts as the no SSB control for Flp-nick and gpII. (C) Frequency of Flp-nick-induced Ade+ recombinants in pfh1⁺ori1253Δ, pfh1-m21 ori1253Δ, and pfh1-mt^∗ ori1253Δ strains containing the ade6⁻ direct repeat recombination reporter shown in (A). The data in (B) and (C) are presented as median values ± 95% confidence interval with individual data points shown as gray dots. The fold changes and p values in (B) refer to the comparison of recombination values between equivalent strains with and without SSB, with p values calculated using the two-tailed Mann-Whitney test. The p values in (C) relate to the comparison of recombination values with the equivalent pfh1⁺ strain and were calculated by the Kruskal-Wallis test with Dunn’s multiple comparisons post-test. ^∗∗∗∗p < 0.0001; ^∗∗p < 0.01. See also Table S1.

Figure 7

Model for the repair of DSBs that arise from replication fork encounters with SSBs (A) Diagram of replication run-off at a lead-SSB leading to the formation of a seDSB/deDSB and subsequent repair by SCR or BIR. (B) Diagram of replication bypass or CMG unloading at a lag-SSB leading to the formation of a seDSB/deDSB and subsequent repair by SCR or BIR. Parental DNA strands are shown in dark blue and nascent strands in light blue. Light and dark blue arrowheads indicate the direction of DNA synthesis.