Two-ended recombination at a Flp-nickase-broken replication fork

Abstract

Replication fork collision with a DNA nick can generate a one-ended break, fostering genomic instability. The opposing fork's collision with the nick could form a second DNA end, enabling conservative repair by homologous recombination (HR). To study mechanisms of nickase-induced HR, we developed the Flp recombinase "step arrest" nickase in mammalian cells. A Flp-nick induces two-ended, BRCA2/RAD51-dependent short tract gene conversion (STGC), BRCA2/RAD51-independent long tract gene conversion, and discoordinated two-ended invasions. HR pathways induced by a replication-independent break and the Flp-nickase differ in their dependence on BRCA1, MRE11, and CtIP. To determine the origin of the second DNA end during Flp-nickase-induced STGC, we blocked the opposing fork using a Tus/Ter replication fork barrier (RFB). Flp-nickase-induced STGC remained robust and two ended. Thus, a single replication fork's collision with a Flp-nick triggers two-ended HR, possibly reflecting replicative bypass of lagging strand nicks. This response may limit genomic instability during replication of nicked DNA.

Keywords: BRCA1; Camptothecin; DNA nick; DNA-protein crosslink; Flp recombinase; Tus/Ter; break-induced replication; homologous recombination; replication fork breakage; replication stress.

Figure 1.. FlpH305L-induced HR products.

A. FlpH305L binding to the F13-FRT site generates DPC nicks on either strand. Pink: non-incising Flp monomers. Dark red: incising Flp monomer; active site tyrosine (Y) is shown. Flp monomers a and a′, but not b, mediate recombination at FRT. Lower panel: sequence of F13 FRT site in HR reporter, with incision sites of Flp monomers a and a′ shown. B. HR outcomes detected by reporter. STGC generates GFP⁺RFP^– outcome; LTGC generates GFP⁺RFP⁺ outcome. C. Orientation of the Rosa26 HR reporter with respect to a replication fork TEL to CEN. D. STGC frequencies induced by I-SceI and Flp mutants. Flp-YF/HL: Y343F/H305L double mutant. Data shows mean and s.e.m. of 6 independent experiments (n=6). Unpaired t-test: ** P < 0.01; *** P < 0.001. E. and F. LTGC products (E) and ratio of LTGC:Total GFP⁺ frequencies (F) from the same experiments as in D (n=6). * P < 0.05; ** P < 0.01; **** P < 0.0001. See also Supplemental Figure S1.

Figure 2.. Rad51 dependence of FlpH305L-induced repair products.

A and B. I-SceI induced (A) and FlpH305L-induced (B) STGC in parental Xrcc4^fl/fl and Xrcc4^−/− reporter cells. Data shows mean and s.e.m. of 4 independent experiments (n=4). Unpaired t-test: ns: not significant; *** P < 0.001. C-F. Brca1^fl/hyg FRT-F13 HR reporter cells were co-transfected with FlpH305L and siRNAs shown. B2: Brca2. R51: Rad51. Data shows mean and s.e.m. of 8 independent experiments (n=8). Unpaired t-test: * P < 0.05; **** P < 0.0001. C. STGC. D. LTGC. E. Ratio of LTGC:Total GFP⁺. F. GFP^–RFP⁺ outcome. See also Supplemental Figure S2.

Figure 3.. Analysis of FlpH305L-induced GFP^–RFP⁺ products.

A. Southern blot analysis of FlpH305L-induced GFP^–RFP⁺ clones. Genomic DNA was digested in vitro with BglII (B) or with BglII+I-SceI (B+I), GFP probe. Note: all clones except #3 and #4 (red *) contain two I-SceI sites. Clones #3 and #4 may contain three I-SceI sites, generating double intensity 3538 bp band. B. Deduced structure of major FlpH305L-induced GFP^–RFP⁺ products. Green lines: sequences hybridizing with GFP probe. C. Hypothetical dual invasion mechanism underlying FlpH305L-induced GFP^–RFP⁺ outcome. Original sites of left and right DNA ends are marked L and R, respectively. See also Supplemental Figure S3.

Figure 4.. Brca1 exon 11 is dispensable for FlpH305L-induced STGC.

Data pooled from 6 independent Cre-exposed Brca1^fl/hyg clones and 6 independent Cre-exposed Brca1^Δ11/hyg clones. Data shows mean and s.e.m. of 6 independent experiments (n=6). Unpaired t-test: ns: not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. A. Left panel: STGC. Right panel: Western blot of full-length p220 BRCA1 in Brca1^fl/hyg cells. Note absence of p220 BRCA1 in Brca1^Δ11/hyg cells. *: background bands. B. LTGC. C. Ratio of LTGC:Total GFP⁺. D. GFP^–RFP⁺ outcome. E. Brca1^fl/hyg clone #12 and Brca1^Δ11/hyg clone #8 (see Supplemental Figure S4) were co-transfected with siRNAs shown, together with either FlpH305L or I-SceI. Data shows mean and s.e.m. from 4 independent experiments (n=4), with analysis by unpaired t-test. Right panel: western blot showing depletion of full-length p220 BRCA1 in Brca1^fl/hyg cells transfected with siBrca1 vs. siLuc control. B1: Brca1. See also Supplemental Figure S5.

Figure 5.. Impact of MRE11 and CtIP on FlpH305L-induced HR.

A. and B. Brca1^fl/hyg FRT-F13 HR reporter cells were co-transfected with either FlpH305L (A) or I-SceI (B), together with siRNAs shown. Data shows mean and s.e.m. of 5 independent experiments (n=5). Unpaired t-test: ns: not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. C. Western blot of MRE11 in cells treated with the siRNAs shown. β-tubulin: loading control. D. Western blot of CtIP in cells treated with the siRNAs shown. *: background band. β-tubulin: loading control.

Figure 6.. FlpH305L-induced STGC is the product of a single fork collision with the Flp-nick.

A. Upper panel: Classical model predicts that only one-ended breaks form at the Flp-nick site. Collision of each converging fork with the nick generates two DNA ends, enabling repair by two-ended HR. Lower panel: Classical model predicts that Tus/Ter RFB will prevent formation of a second DNA end at the Flp-nick site. B.–E. Data pooled from 5 independent Brca1^fl/hyg clones targeted with the F13FRT-HR reporter carrying an additional 6xTer array positioned to establish a Tus-mediated RFB to block the leftward fork. Data shows mean and s.e.m. of 5 independent experiments (n=5). Unpaired t-test: ns: not significant; **** P < 0.0001. B. FlpH305L-induced STGC is unaffected by activation of the Tus/Ter RFB. C. and D. FlpH305L-induced LTGC frequencies (C), and ratio of LTGC:Total GFP⁺ (D). E. FlpH305L-induced GFP^– RFP⁺ products. F. Southern blot analysis of FlpH305L-induced STGC products generated in presence of the Tus/Ter RFB. gDNA digested with BglII; see Supplemental Figure S1F for expected fragment sizes. M: MW markers. P: parental reporter cells. Note that all FlpH305L-induced STGCs are the product of two-ended HR in the presence of the Tus/Ter RFB. See also Supplemental Figure S6.

Figure 7.. Model: distinct outcomes of fork collision with a lagging or leading strand Flp-nick.

A. Lagging strand Flp-nick. The CMG replicative helicase bypasses a lagging strand Flp-nick, allowing replication to continue beyond the nick site. The two-ended break is generated with no contribution from the opposing (leftward) replication fork and is repaired by STGC. Non-incising Flp monomers: pink. Incising Flp monomer: dark red, active site tyrosine (Y) shown. B. Leading strand Flp-nick. A Flp-nick on the leading strand exposes a free 5’ end of the leading parental strand, leading to loss of CMG from chromatin and formation of a one-ended break, favoring LTGC or chromosome rearrangement.