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

Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma, Madrid, Spain.
Nucleic Acids Research (Impact Factor: 9.11). 02/2005; 33(15):4762-74. DOI: 10.1093/nar/gki780
Source: PubMed


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.

Download full-text


Available from: Arancha Sánchez
  • Source
    • "Both Pol4 from S. cerevisiae and S. pombe possess two additional domains at their N-terminus: a BRCT domain followed by a regulatory Ser/Pro domain (Fig. 2). In addition, both Pol4 have a dRP-lyase activity associated with the 8 kDa domain suggesting a role in repair processes such as BER [25] [26]. Although both Pol4 enzymes share a common structural organization, they differ in terms of sequence similarity with their human counterparts. "

    Full-text · Chapter · May 2013
  • Source
    • "In the light of all this evidence, we decided to use S. pombe as a model organism, as it provides us with a simplified system to test the usage of ribonucleotide substrates by SpPol4, Pol µ and Polλ (over-expressed in this system) with a more physiological approach: using whole cell extracts instead of purified recombinant proteins. To eliminate the background of an endogenous SpPol4 activity, we used an S. pombe mutant strain lacking the pol4 gene, ΔPol4 (22). The level of over-expression of the three polymerases in the whole cell extracts was very similar, as assessed by WB (Figure 5A). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Human DNA polymerase mu (Polμ), a family X member involved in DNA repair, has both template-directed and terminal transferase (template-independent) activities. In addition to their ability to incorporate untemplated nucleotides, another similarity between Polµ and terminal deoxynucleotidyl transferase (TdT) is their promiscuity in using ribonucleotides (NTPs), whose physiological significance is presently unknown. As shown here, Polµ can use NTPs instead of deoxynucleotides (dNTPs) during non-homologous end joining (NHEJ) of non-complementary ends, a Polµ-specific task. Moreover, a physiological concentration of Mn2+ ions did benefit Polµ-mediated NHEJ by improving the efficiency and accuracy of nucleotide insertion. Analysis of different mutations in the ‘steric gate’ of the active site indicated that Polµ is taking advantage of an open active site, valid for selecting alternative activating metal ions and nucleotides as substrates. This versatility would allow ad hoc selection of the most appropriate nucleotide/metal ion combination for individual NHEJ events to gain efficiency without a cost in terms of fidelity, thus widening the spectrum of available solutions to position a discontinuous template strand in proper register for connection.
    Full-text · Article · Dec 2012 · Nucleic Acids Research
  • Source
    • "Pol4 [Bebenek et al., 2005; Gonzalez-Barrera et al., 2005] "
    [Show abstract] [Hide abstract]
    ABSTRACT: Chromosome breaks, often with damaged or missing DNA flanking the break site, are an important threat to genome stability. They are repaired in vertebrates primarily by nonhomologous end joining (NHEJ). NHEJ is unique among the major DNA repair pathways in that a continuous template cannot be used by DNA polymerases to instruct replacement of damaged or lost DNA. Nevertheless, at least 3 out of the 17 mammalian DNA polymerases are specifically employed by NHEJ. Biochemical and structural studies are further revealing how each of the polymerases employed by NHEJ possesses distinct and sophisticated means to overcome the barriers this pathway presents to polymerase activity. Still unclear, though, is how the resulting network of overlapping and nonoverlapping polymerase activities contributes to repair in cells. Environ. Mol. Mutagen., 2012. © 2012 Wiley Periodicals, Inc.
    Preview · Article · Dec 2012 · Environmental and Molecular Mutagenesis
Show more