DNA Joint Dependence of Pol X Family Polymerase Action in Nonhomologous End Joining

Abstract

DNA double strand breaks (DSBs) can be rejoined directly by the nonhomologous end-joining (NHEJ) pathway of repair. Nucleases and polymerases are required to promote accurate NHEJ when the terminal bases of the DSB are damaged. The same enzymes also participate in imprecise rejoining and joining of incompatible ends, important mutagenic events. Previous work has shown that the Pol X family polymerase Pol4 is required for some but not all NHEJ events that require gap filling in Saccharomyces cerevisiae. Here, we systematically analyzed DSB end configurations and found that gaps on both strands and overhang polarity are the principal factors that determine whether a joint requires Pol4. DSBs with 3-overhangs and a gap on each strand strongly depended on Pol4 for repair, DSBs with 5-overhangs of the same sequence did not. Pol4 was not required when 3-overhangs contained a gap on only one strand, however. Pol4 was equally required at 3-overhangs of all lengths within the NHEJ-dependent range but was dispensable outside of this range, indicating that Pol4 is specific to NHEJ. Loss of Pol4 did not affect the rejoining of DSBs that utilized a recessed microhomology or DSBs bearing 5-hydroxyls but no gap. Finally, mammalian Pol X polymerases were able to differentially complement a pol4 mutation depending on the joint structure, demonstrating that these polymerases can participate in yeast NHEJ but with distinct properties.

DNA polymerases in adaptive immunity

“…The possible use of these enzymes during NHEJ seems to be due to their capacity to synthesize DNA from less stable primer structures compared to replicative polymerases, such as primers that encompass a DNA template with a gap 19 (Fig.3c). Lieber and colleagues, using purified polymerase proteins in vitro, showed that a competitive hierarchy exists in the recruitment of polymerases to the DNA during NHEJ, with Polµ being preferentially recruited over Polλ in the absence of TdT, whereas the reverse was true when TdT was present 12 .…”

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

“…Pol4, the only Pol X polymerase in S. cerevisiae, is dispensable for simple religation NHEJ [9], but is strictly required when a gap in the joint must be filled using an unstable primertemplate pair, as is the case with 3' overhangs [10]. Other polymerases can compensate for the loss of Pol4 when a stably paired primer is available, for example at 5' overhangs [10].…”

“…Other polymerases can compensate for the loss of Pol4 when a stably paired primer is available, for example at 5' overhangs [10]. However, Pol4 is surprisingly not required for joining of nicked 3' overhang DSBs with 5' hydroxyls [10], even though yeast lack a 5' kinase and thus demand resynthesis of the damaged 5' nucleotide [11].…”

DNA polymerases in nonhomologous end joining: Are there any benefits to standing out from the crowd?

“…Pol b, consequently, does not stably associate with NHEJ core factors at ends, nor does it effectively promote NHEJ in many contexts in vitro [Mahajan et al, 2002;Ma et al, 2004;Nick McElhinny et al, 2005]. Nevertheless, Pol b is able to partly compensate for Pol4 deficiency during NHEJ when overexpressed in yeast [Daley et al, 2005] and Pol b-deficient mouse embryonic fibroblasts (MEFs) are moderately sensitive to ionizing radiation [Horton et al, 2008]. Unlike other vertebrate Pol X members, Pol b deficiency results in embryonic lethality in mice [Gu et al, 1994;Bertocci et al, 2006].…”

“…This is apparent from the analysis of repair events from cells that survive (and thus inaccurately repair) chromosome breaks generated after constitutive induction of the HO endonuclease [Wilson and Lieber, 1999;Li et al, 2012]. More systematic analysis of substrate specificity [Daley et al, 2005;Daley and Wilson, 2008] indicates S. cerevisiae Pol4 is essential for synthesis required after alignment of 3 0 overhangs leaves behind gaps in both strands. By comparison, synthesis in many other contexts (e.g., after alignment leaves a gap in only one strand, or after alignment of 5 0 overhangs) is independent of Pol4, indicating that other polymerases are sufficient for these substrates.…”