9750–9762 Nucleic Acids Research, 2012, Vol. 40, No. 19 doi:10.1093/nar/gks702 Published online 25 July 2012 DNA stabilization at the Bacillus subtilis PolX core—a binding model to coordinate polymerase, AP-endonuclease and 30-50 exonuclease activities Benito Ba n˜os, Laurentino Villar, Margarita Salas and Miguel de Vega* Instituto de Biologı´a Molecular ‘‘Eladio Vin˜ uela’’ (CSIC), Centro de Biologı´a Molecular ‘‘Severo Ochoa’’ (CSIC-UAM), C/Nicola´ s Cabrera 1, Universidad Aut o´noma, Cantoblanco, 28049 Madrid, Spain Received May 18, 2012; Revised June 25, 2012; Accepted June 26, 2012 ABSTRACT Family X DNA polymerases (PolXs) are involved in DNA repair. Their binding to gapped DNAs relies on two conserved helix-hairpin-helix motifs, one located at the 8-kDa domain and the other at the fingers subdomain. Bacterial/archaeal PolXs have a specifically conserved third helix-hairpin-helix motif (GFGxK) at the fingers subdomain whose putative role in DNA binding had not been estab- lished. Here, mutagenesis at the corresponding residues of Bacillus subtilis PolX (PolXBs), Gly130, Gly132 and Lys134 produced enzymes with altered DNA binding properties affecting the three enzym- atic activities of the protein: polymerization, located at the PolX core, 30-50 exonucleolysis and apurinic/ apyrimidinic (AP)-endonucleolysis, placed at the so-called polymerase and histidinol phosphatase domain. Furthermore, we have changed Lys192 of PolXBs, a residue moderately conserved in the palm subdomain of bacterial PolXs and immediately preceding two catalytic aspartates of the polymer- ization reaction. The results point to a function of residue Lys192 in guaranteeing the right orientation of the DNA substrates at the polymerization and histidinol phosphatase active sites. The results pre- sented here and the recently solved structures of other bacterial PolX ternary complexes lead us to propose a structural model to account for the ap- propriate coordination of the different catalytic activities of bacterial PolXs. INTRODUCTION Maintenance of genome stability is essential for the survival of the organisms (1). The presence of DNA damage triggers the activation of speciﬁc DNA repair pathways including multiple enzymatic activities specialized in dealing with these lesions that otherwise could have deleterious effects in the replication and tran- scription processes. Among the varied enzymes involved in DNA repair, family X DNA polymerases (PolX), highly conserved in all the kingdoms of life, from human to viruses, stand out (2). These enzymes play a role in DNA synthesis during the base excision repair (3–6) and non-homologous end joining (7–11) pathways, acting on the short gapped intermediates that arise during these events. Their ability to ﬁll a gapped DNA molecule relies on a common structural organization shared by most PolXs: the C-terminal polymerization domain formed by the ﬁngers, palm and thumb subdomains, present in nearly all DNA-dependent DNA polymerases and responsible for the binding and further elongation of the 30 terminus of the upstream primer strand and the N-terminal 8-kDa domain that contacts the downstream strand (6,12,13). In some cases, such as in mammalian Polb and Pol and yeast Pol4 and Trf4, the 8-kDa domain also contains an intrinsic 50-deoxyribose 50-phosphate lyase activity responsible for the release of the 50-deoxyribose 50-phosphate moiety during short patch base excision repair (4,6,14–16). Besides this common PolX core, several members of this family are endowed with accessory domains provided either with additional enzymatic activities, such as the 30- 50 exonuclease and AP-endonuclease present in the C-terminal polymerase and histidinol phosphatase (PHP) domain of bacterial/archaeal PolXs (17–19), or with residues responsible for protein–protein/protein–DNA interactions, such as the N-terminal BRCT domain of Pol , Polm and TdT through which these polymerases interact with other non-homologous end joining factors, such as Ku, XRCC4 and ligase IV, placed at the ends of double strand breaks (20–22). Binding of PolXs to DNA largely depends on the presence of two helix-hairpin-helix (HhH) motifs, one located at the 8-kDa domain and the other at the ﬁngers subdomain of the polymerization domain. These motifs *To whom correspondence should be addressed. Tel: +34 91 196 4717; Fax: +34 91 196 4420; Email: mdevega@cbm.uam.es The Author(s) 2012. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.