Limiting the Persistence of a Chromosome Break Diminishes Its Mutagenic Potential

Abstract

To characterize the repair pathways of chromosome double-strand breaks (DSBs), one approach involves monitoring the repair of site-specific DSBs generated by rare-cutting endonucleases, such as I-SceI. Using this method, we first describe the roles of Ercc1, Msh2, Nbs1, Xrcc4, and Brca1 in a set of distinct repair events. Subsequently, we considered that the outcome of such assays could be influenced by the persistent nature of I-SceI-induced DSBs, in that end-joining (EJ) products that restore the I-SceI site are prone to repeated cutting. To address this aspect of repair, we modified I-SceI-induced DSBs by co-expressing I-SceI with a non-processive 3′ exonuclease, Trex2, which we predicted would cause partial degradation of I-SceI 3′ overhangs. We find that Trex2 expression facilitates the formation of I-SceI-resistant EJ products, which reduces the potential for repeated cutting by I-SceI and, hence, limits the persistence of I-SceI-induced DSBs. Using this approach, we find that Trex2 expression causes a significant reduction in the frequency of repair pathways that result in substantial deletion mutations: EJ between distal ends of two tandem DSBs, single-strand annealing, and alternative-NHEJ. In contrast, Trex2 expression does not inhibit homology-directed repair. These results indicate that limiting the persistence of a DSB causes a reduction in the frequency of repair pathways that lead to significant genetic loss. Furthermore, we find that individual genetic factors play distinct roles during repair of non-cohesive DSB ends that are generated via co-expression of I-SceI with Trex2.

Correct End Use during End Joining of Multiple Chromosomal Double Strand Breaks Is Influenced by Repair Protein RAD50, DNA-dependent Protein Kinase DNA-PKcs, and Transcription Context

“…Notably, co-expression of I-SceI and Trex2 leads to distal EJ products that are completely I-SceI resistant (7,37). The repair junctions following I-SceI/ Trex2 co-expression mostly show very short deletions of the I-SceI 3Ј overhang region, which is consistent with the low processivity of Trex2 (7,37). We also observed a minor class of larger deletions, but importantly none of these deletions disrupt GFP expression (7,37).…”

“…To examine this key aspect of genome maintenance, we used the EJ5-GFP reporter that contains two tandem I-SceI sites ( Fig. 1A) (7,37). Following I-SceI expression, distal EJ places a promoter adjacent to the rest of the GFP cassette, such that the distal EJ can be quantified as the percentage of GFP ϩ cells.…”

“…Proximal EJ is difficult to measure with I-SceI expression alone, because proximal EJ that restores the I-SceI site cannot be differentiated from the uncut reporter. For this, we used co-expression of I-SceI with the 3Ј-exonuclease Trex2, which has been shown to cause a high level of I-SceI-resistant proximal EJ products, as quantified by PCR amplification across the 3Ј I-SceI site, and subsequent I-SceI digestion analysis (7,37). Notably, co-expression of I-SceI and Trex2 leads to distal EJ products that are completely I-SceI resistant (7,37).…”

“…For this, we used co-expression of I-SceI with the 3Ј-exonuclease Trex2, which has been shown to cause a high level of I-SceI-resistant proximal EJ products, as quantified by PCR amplification across the 3Ј I-SceI site, and subsequent I-SceI digestion analysis (7,37). Notably, co-expression of I-SceI and Trex2 leads to distal EJ products that are completely I-SceI resistant (7,37). The repair junctions following I-SceI/ Trex2 co-expression mostly show very short deletions of the I-SceI 3Ј overhang region, which is consistent with the low processivity of Trex2 (7,37).…”

Redesign of extensive protein–DNA interfaces of meganucleases using iterative cycles of in vitro compartmentalization

“…Deletion, duplication, and insertion promoted by the MegaTALs were substantially suppressed by coexpression of Trex2 (Fig. 3A), in a good agreement with a previous study (39).…”