Article

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

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.

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    • "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. "

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    • "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). "
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    • "Pol4 [Bebenek et al., 2005; Gonzalez-Barrera et al., 2005] "
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