PARP-mediated repair, homologous recombination, and back-up non-homologous end joining-like repair of single-strand nicks

Abstract

Double-strand breaks (DSBs) in chromosomal DNA can induce both homologous recombination (HR) and non-homologous end-joining (NHEJ). Recently we showed that single-strand nicks induce HR with a significant reduction in toxicity and mutagenic effects associated with NHEJ. To further investigate the differences and similarities of DSB- and nick-induced repair, we used an integrated reporter system in human cells to measure HR and NHEJ produced by the homing endonuclease I-AniI and a designed 'nickase' variant that nicks the same target site, focusing on the PARP and HR repair pathways. PARP inhibitors, which block single-strand break repair, increased the rate of nick-induced HR up to 1.7-fold but did not affect DSB-induced HR or mutNHEJ. Additionally, expression of the PALB2 WD40 domain in trans acted as a dominant-negative inhibitor of both DSB- and nick-induced HR, sensitized cells to PARP inhibition, and revealed an alternative mutagenic repair pathway for nicks. Thus, while both DSB- and nick-induced HR use a common pathway, their substrates are differentially processed by cellular factors. These results also suggest that the synthetic lethality of PARP and BRCA may be due to repair of nicks through an error prone, NHEJ-like mechanism that is active when both PARP and HR pathways are blocked.

Figure 1. PARP inhibitors increase the rate of nick-induced HR, but do not affect DSB repair

(A) Structure of the Traffic Light Reporter (TLR) is depicted in the center, with resulting GFP-expressing HR products (above) and mCherry-expressing mutNHEJ products (below). (B) 293/TLR-AIN cells were transfected with 0.5 μg donor plasmid and 0.5 μg of a plasmid expressing a variant of I-AniIY2. In PARP inhibition experiments, 1d after transfection the culture medium was replaced with DMEM 10% FBS containing a PARP inhibitor in DMSO, or solvent alone (DMSO). The frequency of HR (% GFP+ cells) and a corresponding estimate of mutNHEJ events (derived from 3 times the frequency of mCherry+ cells; see text) were detected and quantified by flow cytometry 3d after transfection with I-AniIY2 cleavase or nickase. The effect of PARP inhibitors on HR and mutNHEJ was determined as described above using TLR cells transfected with I-AniIY2 cleavase (C) or nickase (D) in the presence of 5 mM 3-aminobenzamide (3-ABA), 50 μM BSI-201 (Iniparib), 0.5 μM ABT-888 (Veliparib), or 0.5 μM AZD2281 (Olaparib). Each data point represents the mean (or geometric mean of normalized data) of three separate experiments ± s.e.m. Statistical significance was determined by two-tailed t test of paired values.

Figure 2. Expression of the PALB2 WD40 domain in trans blocks both nick- and DSB-induced HR

293/TLR-AIN cells were transfected with 0.4 μg donor plasmid, 0.2 μg of pUC19 or constructs expressing PALB2 C-terminal WD40 domain (PALB2C), or PALB2 C-terminal WD40 domain A1025R mutant (PALB2CR) and 0.2 μg of either I-AniIY2 cleavase (A) or nickase (B). HR and mutNHEJ were detected and quantified by flow cytometry 3d after transfection. All data points represent the mean of three separate experiments (± s.e.m.). (C) Western blot analysis demonstrates expression of HA-tagged PALB2 C-terminal domains (top) and β-actin loading control (bottom) using cell lysates (5 μg total protein/lane) prepared at the time of flow cytometry. (D) Sensitivity of 293T cells stably expressing PALB2C and PALB2CR to PARP inhibitor was determined by detecting colony forming ability in the presence of AZD2281 for 8–10 days. Data points were normalized to cells only treated with DMSO and represent the geometric mean from three separate experiments (± s.e.m). Asterisks (*) indicate p values of <0.05 compared independently to both cell lines with PALB2CR and empty vector (two-tailed unpaired t test of log transformed normalized data).

Figure 3. Sequences of nick-induced NHEJ-like mutations after block of HR

Sequences of 24 mutated clones from cells expressing I-AniIY2 nickase and the PALB2 C-terminal WD40 domain. The I-AniI target sequence in the pTLR-AIN plasmid is shown underlined in the top row, with the I-AniI binding site in capital letters. I-AniI nickase cleaves the site marked by the filled arrowhead, I-AniI cleavase cleaves the sites marked by both arrowheads. Microhomologies are underlined, dashes mark deleted bases in individual clones and new base insertions in angled brackets.

Figure 4. Model of DSB and single-strand nick repair pathways

DSBs can be repaired through either the NHEJ or HR pathways in normal cells (left panel), and single-strand nicks by SSBR (single strand break repair) and HR pathways (right panel) in normal cells with no detectable nick-induced mutagenic NHEJ. HR may be triggered by a nick that leads to fork collapse to form a single-ended break. If HR is blocked, nick-induced fork collapse can be repaired through a latent error-prone repair pathway (dotted line), which may be triggered by a converging second replication fork converting the single-ended DSB to a double-ended DSB.