4762–4774 Nucleic Acids Research, 2005, Vol. 33, No. 15 doi:10.1093/nar/gki780 Characterization of SpPol4, a unique X-family DNA polymerase in Schizosaccharomyces pombe Sergio Gonza´ lez-Barrera, Arancha Sa´ nchez, Jose´ F. Ruiz, Raquel Jua´ rez, Angel J. Picher, Gloria Terrados, Paula Andrade and Luis Blanco* Centro de Biolog´ıa Molecular Severo Ochoa (CSIC-UAM), Universidad Auto´ noma, Madrid, Spain Received May 25, 2005; Revised and Accepted August 3, 2005 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 30!50 proofreading activity and its preferred substrates are small gaps with a 50- phosphate group. Similarly to Polm, 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 Polm, SpPol4 lacks terminal deoxynucleotidyl- transferase activity and realigns the primer terminus to alternative template bases only under certain sequence contexts and, therefore, it is less error- prone than Polm. Nonetheless, the biochemical prop- erties of this gap-filling DNA polymerase are suitable for a possible role of SpPol4 in non-homologous end-joining. Unexpectedly based on sequence ana- lysis, SpPol4 has deoxyribose phosphate lyase activ- ity like Polb and Poll, and unlike Polm, suggesting also a role of this enzyme in base excision repair. Therefore, SpPol4 is a unique enzyme whose enzym- atic properties are hybrid of those described for mammalian Polb, Poll and Polm. INTRODUCTION Efﬁcient DNA repair is essential to maintain genome stability and cell viability (1,2). In spite of a variety of DNA repair mechanisms, one common step is DNA synthesis, carried out by specialized DNA polymerases. DNA polymerases are clas- siﬁed into four different groups according to their biochemical properties and to the biological processes in which they are involved. Among them, only family X DNA polymerases (PolX) are devoted to DNA repair, being evolutionarily conserved in prokaryotes, eukaryotes and archaea (3–5). However, their number ranges from ﬁve members in mammals [Polb, Poll, Polm, terminal deoxynucleotidyltransferase (TdT) and Pols] to one member in yeasts, plants, and some bacteria and viruses, i.e. Saccharomyces cerevisiae (ScPol4), Arabidopsis thaliana (AthPolX), Bacillus subtilis (BsPolX) and African swine fever virus (ASFVPolX). Interestingly, two model organisms, Caenorhabditis elegans and Drosophila melanogaster, whose genomes have been completely sequenced, have no putative PolX (3). PolX enzymes most probably share a common modular organization (Polb core) consisting of an 8 kDa domain and a 31 kDa polymerization domain comprising ‘ﬁngers’, ‘palm’ and ‘thumb’ subdomains. Such a structural organization has been demonstrated for Polb (6,7), TdT (8), Poll (9,10) and ASFVPolX (11,12). Unlike Polb, ASFVPolX, bacterial and archaea PolX members, other family enzymes (Poll, Polm, TdT and ScPol4) have an additional domain, the Brca1 C-terminal, named BRCT, which has been suggested to take part in protein–protein and protein–DNA interactions (13). Besides this BRCT domain, Poll AthPolX and ScPol4 have a proline/serine-rich region in their central part with a yet unknown function (3). Regarding their biochemical properties, all DNA poly- merases from this family are single-subunit enzymes, lacking the 30!50 exonuclease activity and displaying very low pro- cessivity during primer extension reactions [reviewed in (14)]. Polb, the paradigm of the PolX family, inserts nucleotides in a template-dependent manner and is moderately accurate (15,16). Its preference for small gaps with a 50-phosphate group (17) and its deoxyribose phosphate (dRP) lyase activity that relies on the 8 kDa domain (18) are properties consistent with a role in base excision repair (BER), a major pathway *To whom correspondence should be addressed. Tel: +34 91 497 8493; Fax: +34 91 497 4799; Email: lblanco@cbm.uam.es Present address: Jose´ F. Ruiz, Departamento de Gene´tica, Facultad de Biolog´ıa, Universidad de Sevilla, Spain The Author 2005. Published by Oxford University Press. All rights reserved. The online version of this article has been published under an open access model. 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