Repair of gaps opposite lesions by homologous recombination in mammalian cells

Abstract

Damages in the DNA template inhibit the progression of replication, which may cause single-stranded gaps. Such situations can be tolerated by translesion DNA synthesis (TLS), or by homology-dependent repair (HDR), which is based on transfer or copying of the missing information from the replicated sister chromatid. Whereas it is well established that TLS plays an important role in DNA damage tolerance in mammalian cells, it is unknown whether HDR operates in this process. Using a newly developed plasmid-based assay that distinguishes between the three mechanisms of DNA damage tolerance, we found that mammalian cells can efficiently utilize HDR to repair DNA gaps opposite an abasic site or benzo[a]pyrene adduct. The majority of these events occurred by a physical strand transfer (homologous recombination repair; HRR), rather than a template switch mechanism. Furthermore, cells deficient in either the human RAD51 recombination protein or NBS1, but not Rad18, exhibited decreased gap repair through HDR, indicating a role for these proteins in DNA damage tolerance. To our knowledge, this is the first direct evidence of gap-lesion repair via HDR in mammalian cells, providing further molecular insight into the potential activity of HDR in overcoming replication obstacles and maintaining genome stability.

Figure 1.

A plasmid-based assay for the repair of gaps opposite lesions. (A) Outline of the experimental system. See text for details. (B) Relevant sequences of the GPs and the homologous donors used in this work. X represents the synthetic abasic site, and the star underneath the G—the benzo[a]pyrene-guanine adduct. hDNA, homologous DNA. The site of the lesion and the appropriate recombination markers are indicated by long and short vertical rectangles, respectively.

Figure 2.

HDR repairs gaps opposite lesions in human cells. (A) Homology-dependent repair enhances repair of gaps opposite an abasic site or a BP-G adduct in the human H1299 lung cancer cell line. See Table 1 for details. (B) The percentage of repair events by HDR from the experiments depicted in (A), and presented in detail in Table 2.

Figure 3.

Gap-filling by HDR occurs predominantly by a strand transfer mechanism. (A) A scheme illustrating strand transfer and template switch homology-dependent repair (HDR; HRR) mechanisms for filling in gaps opposite a lesion (indicated by a star). See text for details. (B) Fraction of HDR events occurring via strand transfer (black columns) and template switch (gray columns). Shown are results in HCT116 (mismatch repair defective) and H1299 human cell lines, with a donor carrying either a G : T, A : C, C : C or G : G mismatch. Detailed data are presented in Table 4.

Figure 4.

Involvement of RAD51 and NBS1, but not Rad18, in gap-filling HDR. (A) Immunoblot analysis showing siRNA knock-down of RAD51 expression in H1299 cells either without (lanes 1 and 3) or with (lanes 2 and 4) transfection with the gapped DNAs. See methods for details. (B) Results of experiments in H1299 cells in which RAD51 was knocked-down show more then a 2-fold reduction in both HDR and TLS. (C) Experiments conducted in NBS1 cells show significant reduction in HDR without affecting TLS. (D) Experiments conducted in Rad18^−/− MEFs show strong decrease in TLS in the RAD18 deficient cells with no effect on HDR. See Tables 5–7 for details.