Alternative-NHEJ Is a Mechanistically Distinct Pathway of Mammalian Chromosome Break Repair

Abstract

Characterizing the functional overlap and mutagenic potential of different pathways of chromosomal double-strand break (DSB) repair is important to understand how mutations arise during cancer development and treatment. To this end, we have compared the role of individual factors in three different pathways of mammalian DSB repair: alternative-nonhomologous end joining (alt-NHEJ), single-strand annealing (SSA), and homology directed repair (HDR/GC). Considering early steps of repair, we found that the DSB end-processing factors KU and CtIP affect all three pathways similarly, in that repair is suppressed by KU and promoted by CtIP. In contrast, both KU and CtIP appear dispensable for the absolute level of total-NHEJ between two tandem I-SceI–induced DSBs. During later steps of repair, we find that while the annealing and processing factors RAD52 and ERCC1 are important to promote SSA, both HDR/GC and alt-NHEJ are significantly less dependent upon these factors. As well, while disruption of RAD51 causes a decrease in HDR/GC and an increase in SSA, inhibition of this factor did not affect alt-NHEJ. These results suggest that the regulation of DSB end-processing via KU/CtIP is a common step during alt-NHEJ, SSA, and HDR/GC. However, at later steps of repair, alt-NHEJ is a mechanistically distinct pathway of DSB repair, and thus may play a unique role in mutagenesis during cancer development and therapy.

Differential contribution of HP1 proteins to DNA end resection and homology-directed repair

“…NHEJ protein KU80. 22 This was also observed under our experimental settings (Fig. S1D), thus validating the sensitivity of our method.…”

“…To this end, we initially exploited a system enabling us to assess the efficiency of homology directed repair by single-strand annealing (SSA). 22 In a similar fashion to the DR-GFP system, this assay relies on the restoration of a GFP gene that also presents an I-SceI cutting site, which is truncated by the insertion of 2.7 Kb DNA fragment. When the DSB is induced by I-SceI transfection, the 2.7 kb fragment is removed Herein, using specific DNA repair assays and exploring the recruitment of key repair proteins to sites of DNA damage in mammalian cells, we show that HP1 paralogs impact differentially on the DDR, leading both to inhibition and stimulation of the repair of double-stand breaks (DSBs) by homologous recombination.…”

Microhomology-mediated End Joining and Homologous Recombination share the initial end resection step to repair DNA double-strand breaks in mammalian cells

“…Further studies suggest that in yeast, Srs2 helicase, Sae2 nuclease [CtBP-interacting protein (CtIP) homologue], Tel1 [ataxia telangiectasia mutated (ATM) homologue], and DNA polymerases (Pol4, Rev3, and Pol32) are important for MMEJ (11). In mammalian cells, Mre11, CtIP, and DNA ligase III have critical roles in MMEJ (12)(13)(14)(15)(16)(17).…”

Development of Small Molecules that Specifically Inhibit the D-loop Activity of RAD51

“…SSA efficiency is known to be elevated in situations where RAD51 function has been generally disrupted. 27,28 Similarly, we previously demonstrated that both RI-1 and RI-2 induce elevated levels of SSA activity. 19 By contrast, we found that 9h does not stimulate SSA, using a relevant concentration range that inhibits HR (Figure 5b).…”

“…27 Briefly, cells containing the chromosomal DR-GFP reporter were transfected with a plasmid that expresses I-SceI, a rare-cutting endonuclease that makes a DSB within the DR-GFP cassette. Compounds were added at the time of transfection, and the cells were outgrown for 24 h. We noted that for longer outgrowth periods such as 48 h, cells regained some HR activity (data not shown).…”