Evidence that base stacking potential in annealed 3′ overhangs determines polymerase utilization in yeast nonhomologous end joining

Abstract

Nonhomologous end joining (NHEJ) directly rejoins DNA double-strand breaks (DSBs) when recombination is not possible. In Saccharomyces cerevisiae, the DNA polymerase Pol4 is required for gap filling when a short 3' overhang must prime DNA synthesis. Here, we examined further end variations to test specific hypotheses regarding Pol4 usage in NHEJ in vivo. Surprisingly, Pol4 dependence at 3' overhangs was reduced when a nonhomologous 5' flap nucleotide was present across from the gap, even though the mismatched nucleotide was resynthesized, not incorporated. In contrast, a gap with a 5' deoxyribosephosphate (dRP) was as Pol4-dependent as a gap with a 5' phosphate, demonstrating the importance of the downstream base in relaxing the Pol4 requirement. Combined with prior observations of Pol4-independent NHEJ of nicks with 5' hydroxyls, we suggest that base stacking interactions across the broken strands can stabilize a joint, allowing another polymerase to substitute for Pol4. This model predicts that a unique function of Pol4 is to actively stabilize template strands that lack stacking continuity. We also explored whether NHEJ end processing can occur via short and long patch pathways analogous to base excision repair. Results demonstrated that 5' dRPs could be removed in the absence of Pol4 lyase activity. The 5' flap endonuclease Rad27 was not required for repair in this or any situation tested, indicating that still other NHEJ 5' nucleases must exist.

Consider the workhorse: Nonhomologous end-joining in budding yeast

“…This model of Pol4 function is supported by the finding that stabilizing 3′ overhangs, either by extending the overhang, changing overhang polarity, or adding a 5′ nucleotide flap, results in reduced dependence on Pol4 for repair (Daley et al 2005; Daley and Wilson 2008). Pol4 synthesis activity is stimulated by interaction of the Pol4 BRCT domain with the Dnl4-Lif1 complex, and this interaction also weakly stimulates ligation efficiency (Tseng and Tomkinson 2002).…”

Role of the yeast DNA repair protein Nej1 in end processing during the repair of DNA double strand breaks by non-homologous end joining

“…Genetic studies in yeast have implicated a DNA replication protein, Rad27, a homolog of mammalian flap endonuclease 1 (29), in the processing of a subset of DSBs with incompatible DNA ends (30). A later study using a different assay failed to reproduce these findings, suggesting that Rad27 either is not involved in end processing or is functionally redundant with other nucleases in the end processing step of this repair pathway (31). In support of the idea that Rad27 participates in the end processing reactions, we previously characterized physical and functional interactions among Pol4, Rad27 and Dnl4/Lif1 that coordinate end processing and ligation (32,33).…”

Nonhomologous DNA end-joining for repair of DNA double-strand breaks

“…TdT is only expressed in early B-and T-lymphocytes, making it most relevant to the NHEJ repair that occurs during V(D)J recombination, where it has a major role in promoting immunoglobulin diversity. DNA polymerases outside the Pol X family are able to incorporate nucleotides during NHEJ, but only in a template-dependent manner (16,17,(57)(58)(59).…”