Homology-directed repair of DNA nicks via pathways distinct from canonical double-strand break repair

Abstract

DNA nicks are the most common form of DNA damage, and if unrepaired can give rise to genomic instability. In human cells, nicks are efficiently repaired via the single-strand break repair pathway, but relatively little is known about the fate of nicks not processed by that pathway. Here we show that homology-directed repair (HDR) at nicks occurs via a mechanism distinct from HDR at double-strand breaks (DSBs). HDR at nicks, but not DSBs, is associated with transcription and is eightfold more efficient at a nick on the transcribed strand than at a nick on the nontranscribed strand. HDR at nicks can proceed by a pathway dependent upon canonical HDR factors RAD51 and BRCA2; or by an efficient alternative pathway that uses either ssDNA or nicked dsDNA donors and that is strongly inhibited by RAD51 and BRCA2. Nicks generated by either I-AniI or the CRISPR/Cas9(D10A) nickase are repaired by the alternative HDR pathway with little accompanying mutagenic end-joining, so this pathway may be usefully applied to genome engineering. These results suggest that alternative HDR at nicks may be stimulated in physiological contexts in which canonical RAD51/BRCA2-dependent HDR is compromised or down-regulated, which occurs frequently in tumors.

Keywords: cancer; gene conversion; loss of heterozygosity; recombination; targeted gene correction.

Fig. 1.

HDR occurs preferentially at a nick on the transcribed strand and is stimulated by transcription. (A) The chromosomal I-AniI TL reporter (16) consists of a defective GFP gene containing an I-AniI site and two stop codons (asterisks) near the 5′ end, joined by a T2A translational linker to the mCherry coding sequence in the +2 ORF. The I-AniI site is oriented to nick the transcribed strand in the TL^TS reporter and the nontranscribed strand in the TL^NT reporter, as indicated by the two arrowheads. GFP⁺ (green) cells will result from HDR of the chromosomal reporter using an exogenous donor, whereas mCherry⁺ (red) cells will result from cleavage at the I-AniI site followed by mutEJ that puts mCherry in the +2 reading frame. Details in Fig. S1. (B) HDR (GFP⁺) and mutEJ (mCherry⁺) frequencies calculated from independent transient transfections of 293T-TL^TS or 293T-TL^NT cells with indicated I-AniI derivatives (Dead, n = 2; Nick, n = 4; DSB, n = 4), one example of which is shown in Fig. S2. Mean and SEM are shown. Differences in HDR between the TL^TS and TL^NT cell populations are significant at nicks (**P < 0.005) but not at DSBs (P = 0.23). (C) HDR (GFP⁺) frequencies, based on pooled results from a total of 11 independent transfections of three different clonal 293-P-Tet-TL^TS or 293-P-Tet-TL^NT cell lines carried out in the absence (OFF) or presence (ON) of 1 μg/mL doxycycline and normalized to frequencies for cells cultured without inducer. Cell lines were analyzed individually, and frequencies for HDR at nicks or DSBs in each were normalized to frequencies for cells cultured without doxycycline (Dataset S1). (D) The model diagrams how transcription may stimulate repair at a nick on the transcribed strand (TL^TS reporter) by unwinding DNA to expose the recombinogenic 3′ end, but inhibit repair of a nick on the nontranscribed strand (TL^NT reporter) by occluding the 3′ end and exposing the less recombinogenic 5′ end.

Fig. 2.

Donor use and donor strand bias in HDR at nicks. (A) TL^TS and TL^NT reporters. Blue, transcribed strand; green, nontranscribed strand. ssDNA oligonucleotide donors complementary to the nicked (cN) or intact (cI) strand are shown below. Donors were 99 nt in length, centered at the nick, and complementary to the indicated strand except for a 17-nt region of heterology, shown in orange. This region of heterology contains the GFP coding sequence that replaces the stop codons and I-AniI site in the reporter to enable GFP expression (Fig. S1). (B) HDR (GFP⁺) frequencies at nicks or DSBs, using ssDNA donors shown in A, in 293T-TL7^TS and HT1080 TL4^TS clonal lines. Mean and SEM calculated from at least six transfections; **P < 0.005. (C) HDR (GFP⁺) frequencies at nicks (Left) or DSBs (Right), using ssDNA donors shown in A, in 293T-TL^NT and 293T-TL^TS cell populations. Mean and SEM calculated from at least five transfections; *P < 0.05, **P < 0.005.

Fig. 3.

Down-regulation of canonical HDR stimulates HDR at nicks by ssDNA or nicked dsDNA donors. (A) HDR (GFP⁺) frequencies at nicks (Left) or DSBs (Right) in the 293T-TL7^TS clonal line, either untreated or treated with siRAD51, using the pCS14GFP dsDNA donor (n = 4) or cN (n = 2) or cI (n = 4) ssDNA donors, as indicated. Mean and SEM are presented; *P < 0.05, **P < 0.005. (B) HDR (GFP⁺) frequencies at nicks in 293T-TL^NT and 293T-TL^TS control cell populations or populations transiently expressing RAD51^K133R, using indicated donors (n = 4–6). (C) HDR (GFP⁺) frequencies at nicks using the cI ssDNA donor or at DSBs using the dsDNA donor, in the 293T-TL7^TS clonal line treated with the indicated siRNA (n = 6–12; siNT2 is a nontargeting control siRNA from Life Technologies). (D) (Left) Diagram of dsDNA donors, with no I-AniI site (pG-no) or carrying an I-AniI site oriented for intracellular nicking of the transcribed (pGAn-TS) or nontranscribed (pGAn-NT) strand. The I-AniI site in pGAn-TS and pGAn-NT is ∼500 bp downstream of the I-AniI site in the reporter GFP gene targeted for repair. The donors contain 100 bp of upstream and 500 bp of downstream homology with the chromosomal reporter, and the promoters are not homologous to the TL promoter (Fig. S1). (Right) HDR (GFP⁺) frequencies at nicks in the 293T-TL7^TS clonal line, either control cells or cells transiently expressing the RAD51^K133R dominant negative mutant, as indicated, with intact or nicked dsDNA donors (n = 5); **P < 0.005.

Fig. 4.

Nicks generated by CRISPR/Cas9^D10A initiate alternative HDR and are associated with less mutEJ than are DSBs. (A) Sequence of the portion of the TL^TS reporter containing CRISPR/Cas9 and I-AniI target sites (open and filled arrowheads). The insertion (lowercase) bearing the I-AniI site and stop codons (underlined) and a portion of the GFP coding sequence (uppercase) are shown. The CRISPR guide RNA and protospacer adjacent motif (PAM) sequence are indicated. (B) HDR (GFP⁺) frequencies at nicks (Left) or DSBs (Right) in the 293T-TL7^TS clonal line after transient transfection of a Cas9^D10A (Nick) or Cas9 (DSB) expression plasmid, a guide RNA expression plasmid, and either a dsDNA plasmid donor pCS14GFP or cI or cN ssDNA donors, as indicated. Mean and SEM calculated from three transfections; **P < 0.005. (C) MutEJ (mCherry⁺) frequencies at nicks or DSBs in the 293T-TL7^TS clonal line; same cells as in B. (D) The ratio of HDR to mutEJ (GFP⁺:mCherry⁺ cells) compiled from transfections of 293T-TL7^TS cells in B, analyzing HDR at a nick in the transcribed strand using the cI ssDNA donor in siBRCA2-treated cells; and HDR at DSBs using a dsDNA donor in untreated cells.

Fig. 5.

ssDNA donor homology and HDR at nicks. (A) 293T-TL^TS and (B) 293T-TL^NT cell populations, transiently expressing the I-AniI nickase and RAD51^K133R, were provided with a 75-nt ssDNA donor centered at the nick or extending either 3′ or 5′ of the nick, as diagrammed, and HDR assayed. Target: blue, transcribed strand; green, nontranscribed strand. ssDNA donors were complementary to the nicked (cN) or intact (cI) strands and carried a 17-nt region of heterology (orange) and homologous flanking sequences of indicated lengths. For each target/donor combination, the frequency of HDR (% HDR) is shown, and HDR relative to the donor centered on the nick is graphed. Data represent mean and SEM of at least seven transfections.

Fig. 6.

Working model for pathways of HDR at nicks. (Left) RAD51-dependent HDR using a dsDNA donor. A gap is exposed at the nicked target, and BRCA2 loads RAD51 on the free 3′ end, enabling invasion of a homologous dsDNA donor, as in canonical DSB repair. (Right) RAD51/BRCA2-independent HDR. A gap is exposed at the nicked target, and the donor anneals to either the nicked (Left) or intact (Right) strand of the duplex, independent of RAD51/BRCA2. Heterology (orange) and repair synthesis (dashed line) are shown. Arrowheads represent nucleolytic removal of DNA, either by excision or flap cleavage. Refer to Discussion for more detailed description; Fig. S5 for more complete diagrams of mismatch repair and ligation steps; and Figs. S6 and S7 for diagrams of how nicked dsDNA donors may participate in this pathway. MMR, mismatch repair.