different activities of PolXBs. Introduction of an arginine in position 192 enhanced the DNA binding efﬁciency of the polymerase, mutant K192R showing a wild-type polymerization activity on gapped substrates. Interestingly, this mutant is endowed with the ability to displace partially the downstream strand. Contrarily to other DNA polymerases, such as Pold, whose 30-50 exo- nuclease activity prevents strand displacement synthesis during Okazaki fragment maturation (40), the reduced exonuclease activity exhibited by mutant K192R does not seem to be responsible for its strand displacement capacity. Thus, these results could be pointing to a stronger stabilization of the primer-terminus in an orien- tation prone to polymerization that would impede dissoci- ation of mutant PolXBs once the nucleotide has been inserted, compelling the polymerase to advance. In this sense, such an over-stabilization could hamper the access of the substrates to the PHP active site, explaining the reduced 30-50 exonuclease and AP endonuclease activities displayed by mutant K192R. Although this residue does not seem to be instrumental in general PolXBs–DNA complex stabilization, a fact that otherwise would under- mine dissociation after gap-ﬁlling completion, compromising the ﬁnal sealing step of the resulting adjacent 30-OH and 50-phosphate groups (17,26), the results support a role for Lys192 in the proper orientation of the DNA at both active sites to be functionally coordinated. SUPPLEMENTARY DATA Supplementary Data are available at Supplementary Figures 1 and 2. NAR Online: FUNDING Spanish Ministry of Economy and Competitiveness [BFU2011-23720 to M.V.]; the Spanish Research Council [200920I012 to M.V.]; the Spanish Ministry of Science and Innovation [BFU2008-00215 and Consolider-Ingenio CSD2007-00015 to M.S.]; Institu- tional grant from Fundacio´ n Ramo´ n Areces to the Centro de Biologı´ a Molecular ‘Severo Ochoa.’ Funding for open access charge: Spanish Ministry of Economy and Competitiveness [BFU2011-23720]. Conﬂict of interest statement. None declared. REFERENCES 1. Hoeijmakers,J.H. (2001) Genome maintenance mechanisms for preventing cancer. Nature, 411, 366–374. 2. Hu¨ bscher,U., Maga,G. and Spadari,S. (2002) Eukaryotic DNA polymerases. Annu. Rev. Biochem., 71, 133–163. 3. Braithwaite,E.K., Prasad,R., Shock,D.D., Hou,E.W., Beard,W.A. and Wilson,S.H. (2005) DNA polymerase lambda mediates a back-up base excision repair activity in extracts of mouse embryonic ﬁbroblasts. J. Biol. Chem., 280, 18469–18475. 4. Garc ı´a-Dı´ az,M., Bebenek,K., Kunkel,T.A. and Blanco,L. (2001) Identiﬁcation of an intrinsic 50-deoxyribose-5-phosphate lyase activity in human DNA polymerase lambda: a possible role in base excision repair. J. Biol. Chem., 276, 34659–34663. Nucleic Acids Research, 2012, Vol. 40, No. 19 9761 5. Matsumoto,Y. and Kim,K. (1995) Excision of deoxyribose phosphate residues by DNA polymerase beta during DNA repair. Science, 269, 699–702. 6. Moon,A.F., Garc ı´a-Dı´ az,M., Batra,V.K., Beard,W.A., Bebenek,K., Kunkel,T.A., Wilson,S.H. and Pedersen,L.C. (2007) The X family portrait: structural insights into biological functions of X family polymerases. DNA Repair (Amst), 6, 1709–1725. 7. Fan,W. and Wu,X. (2004) DNA polymerase lambda can elongate on DNA substrates mimicking non-homologous end joining and interact with XRCC4-ligase IV complex. Biochem. Biophys. Res. Commun., 323, 1328–1333. 8. Lecointe,F., Shevelev,I.V., Bailone,A., Sommer,S. and Hu¨ bscher,U. (2004) Involvement of an X family DNA polymerase in double-stranded break repair in the radioresistant organism Deinococcus radiodurans. Mol. Microbiol., 53, 1721–1730. 9. Lee,J.W., Blanco,L., Zhou,T., Garc ı´a-Dı´ az,M., Bebenek,K., Kunkel,T.A., Wang,Z. and Povirk,L.F. (2004) Implication of DNA polymerase lambda in alignment-based gap ﬁlling for nonhomologous DNA end joining in human nuclear extracts. J. Biol. Chem., 279, 805–811. 10. Mahajan,K.N., Nick McElhinny,S.A., Mitchell,B.S. and Ramsden,D.A. (2002) Association of DNA polymerase mu (pol mu) with Ku and ligase IV: role for pol mu in end-joining double-strand break repair. Mol. Cell Biol., 22, 5194–5202. 11. Nick McElhinny,S.A., Havener,J.M., Garc ı´a-Dı´ az,M., Ju a´rez,R., Bebenek,K., Kee,B.L., Blanco,L., Kunkel,T.A. and Ramsden,D.A. (2005) A gradient of template dependence deﬁnes distinct biological roles for family X polymerases in nonhomologous end joining. Mol. Cell, 19, 357–366. 12. Beard,W.A. and Wilson,S.H. (2006) Structure and mechanism of DNA polymerase beta. Chem. Rev., 106, 361–382. 13. Yamtich,J. and Sweasy,J.B. (2010) DNA polymerase family X: function, structure, and cellular roles. Biochim. Biophys. Acta, 1804, 1136–1150. 14. Bebenek,K., Garcı´ a-Dı´ az,M., Patishall,S.R. and Kunkel,T.A. (2005) Biochemical properties of Saccharomyces cerevisiae DNA polymerase IV. J. Biol. Chem., 280, 20051–20058. 15. Garcı´ a-Dı´ az,M., Bebenek,K., Krahn,J.M., Kunkel,T.A. and Pedersen,L.C. (2005) A closed conformation for the Pol lambda catalytic cycle. Nat. Struct. Mol. Biol., 12, 97–98. 16. Saxowsky,T.T., Matsumoto,Y. and Englund,P.T. (2002) The mitochondrial DNA polymerase beta from Crithidia fasciculata has 50-deoxyribose phosphate (dRP) lyase activity but is deﬁcient in the release of dRP. J. Biol. Chem., 277, 37201–37206. 17. Ban˜ os,B., La´ zaro,J.M., Villar,L., Salas,M. and de Vega,M. (2008) Editing of misaligned 30-termini by an intrinsic 30-50 exonuclease activity residing in the PHP domain of a family X DNA polymerase. Nucleic Acids Res., 36, 5736–5749. 18. Ban˜ os,B., Villar,L., Salas,M. and de Vega,M. (2010) Intrinsic apurinic/apyrimidinic (AP) endonuclease activity enables Bacillus subtilis DNA polymerase X to recognize, incise, and further repair abasic sites. Proc. Natl. Acad. Sci. USA, 107, 19219–19224. 19. Nakane,S., Nakagawa,N., Kuramitsu,S. and Masui,R. (2009) Characterization of DNA polymerase X from Thermus thermophilus HB8 reveals the POLXc and PHP domains are both required for 30-50 exonuclease activity. Nucleic Acids Res., 37, 2037–2052. 20. Ma,Y., Lu,H., Schwarz,K. and Lieber,M.R. (2005) Repair of double-strand DNA breaks by the human nonhomologous DNA end joining pathway: the iterative processing model. Cell Cycle, 4, 1193–1200. 21. Nick McElhinny,S.A. and Ramsden,D.A. (2004) Sibling rivalry: competition between Pol X family members in V(D)J recombination and general double strand break repair. Immunol. Rev., 200, 156–164. 22. Rooney,S., Chaudhuri,J. and Alt,F.W. (2004) The role of the non-homologous end-joining pathway in lymphocyte development. Immunol. Rev., 200, 115–131. 23. Doherty,A.J., Serpell,L.C. and Ponting,C.P. (1996) The helix-hairpin-helix DNA-binding motif: a structural basis for non-sequence-speciﬁc recognition of DNA. Nucleic Acids Res., 24, 2488–2497. 24. Pelletier,H., Sawaya,M.R., Wolﬂe,W., Wilson,S.H. and Kraut,J. (1996) Crystal structures of human DNA polymerase beta