Characterization of SpPol4, a unique X-family DNA polymerase in Schizosaccharomyces pombe

Abstract

As predicted by the amino acid sequence, the purified protein coded by Schizosaccharomyces pombe SPAC2F7.06c is a DNA polymerase (SpPol4) whose biochemical properties resemble those of other X family (PolX) members. Thus, this new PolX is template-dependent, polymerizes in a distributive manner, lacks a detectable 3′→5′ proofreading activity and its preferred substrates are small gaps with a 5′-phosphate group. Similarly to Polμ, SpPol4 can incorporate a ribonucleotide (rNTP) into a primer DNA. However, it is not responsible for the 1–2 rNTPs proposed to be present at the mating-type locus and those necessary for mating-type switching. Unlike Polμ, SpPol4 lacks terminal deoxynucleotidyltransferase activity and realigns the primer terminus to alternative template bases only under certain sequence contexts and, therefore, it is less error-prone than Polμ. Nonetheless, the biochemical properties of this gap-filling DNA polymerase are suitable for a possible role of SpPol4 in non-homologous end-joining. Unexpectedly based on sequence analysis, SpPol4 has deoxyribose phosphate lyase activity like Polβ and Polλ, and unlike Polμ, suggesting also a role of this enzyme in base excision repair. Therefore, SpPol4 is a unique enzyme whose enzymatic properties are hybrid of those described for mammalian Polβ, Polλ and Polμ.

Microhomology-Mediated End Joining in Fission Yeast Is Repressed by Pku70 and Relies on Genes Involved in Homologous Recombination

“…Deletion of both exo1 1 and rhp51 1 genes completely abolished m8 circularization, suggesting that distinct HR mechanisms, both dependent on rad22 1 , may be involved in MMEJ. The S. pombe pol4 1 gene encodes a DNA polymerase belonging to the PolX family of polymerases devoted to DNA repair (Gonzalez-Barrera et al 2005). Pol4 is template dependent, lacks a detectable 39 / 59 proofreading activity, and has a preference for small gaps.…”

“…Because of the gap-filling activity of fission yeast Pol4 (Gonzalez-Barrera et al 2005), the MMEJ efficiency was also tested in lig4Dpol4D cells. As shown in Figure 1B, deletion of the pol4 1 gene abolished MMEJ-dependent repair of EC DSBs with discontinuous microhomologous ends (circularization efficiency ,0.01% with 95% confidence).…”

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

“…Pol4 Gonzalez-Barrera et al, 2005], terminal deoxynucleotidyl transferase (TdT) [Kato et al, 1967;Boule et al, 2001], and polymerase l (Pol l) [Nick McElhinny and Ramsden, 2003;Ruiz et al, 2003] do not efficiently exclude ribonucleotide triphosphates, and thus extend DNA primers with deoxynucleotides or ribonucleotides with similar efficiency (low-sugar selectivity). Low-sugar selectivity is also observed in members of the archaeo-eukaryotic primase family implicated in synthesis during bacterial NHEJ [Della et al, 2004;Pitcher et al, 2007;Zhu and Shuman, 2008], suggesting that it might be generally important for NHEJ function.…”

“…A third domain, the breast cancer carboxy-terminal (BRCT)-associated domain is also found in many family X members, including the single Pol X members in S. cerevisiae [Tseng and Tomkinson, 2002] and Schizosaccharomyces pombe [Gonzalez-Barrera et al, 2005] and three out of the four Pol X members (not Pol b) in vertebrates [Aoufouchi et al, 2000]. Pol X members have a single BRCT domain, unlike many BRCT domain-containing proteins (including ligase IV) where the domains are present as one or more tandemly repeated pairs [Leung and Glover, 2011].…”

“…pombe have a single Pol X member, Pol4, with similar (but not identical) biochemical characteristics Gonzalez-Barrera et al, 2005]. Both polymerases possess 5 0 -dRP lyase activity, lack exonuclease activity, and have low-sugar selectivity.…”