DNA Repair 4 (2005) 1347–1357 Structural insight into the DNA polymerase  deoxyribose phosphate lyase mechanism Rajendra Prasad, Vinod K. Batra, Xiao-Ping Yang, Joseph M. Krahn, Lars C. Pedersen, William A. Beard, Samuel H. Wilson ∗ Laboratory of Structural Biology, NIEHS, National Institutes of Health, Research Triangle Park, NC 27709, USA Available online 19 September 2005 Abstract A large number of biochemical and genetic studies have demonstrated the involvement of DNA polymerase  (Pol ) in mammalian base excision repair (BER). Pol  participates in BER sub-pathways by contributing gap filling DNA synthesis and lyase removal of the 5 ′ -deoxyribose phosphate (dRP) group from the cleaved abasic site. To better understand the mechanism of the dRP lyase reaction at an atomic level, we determined a crystal structure of Pol  complexed with 5 ′ -phosphorylated abasic sugar analogs in nicked DNA. This DNA ligand represents a potential BER intermediate. The crystal structure reveals that the dRP group is bound in a non-catalytic binding site. The catalytic nucleophile in the dRP lyase reaction, Lys72, and all other potential secondary nucleophiles, are too far away to participate in nucleophilic attack on the C1 ′ of the sugar. An approximate model of the dRP group in the expected catalytic binding site suggests that a rotation of 120 ◦ about the dRP 3 ′ -phosphate is required to position the -amino Lys72 close to the dRP C1 ′ . This model also suggests that several other side chains are in position to facilitate the -elimination reaction. From results of mutational analysis of key residues in the dRP lyase active site, it appears that the substrate dRP can be stabilized in the observed non-catalytic binding conformation, hindering dRP lyase activity. Published by Elsevier B.V. Keywords: DNA polymerase ; DNA repair; Base excision repair; dRP lyase; -Elimination; Schiff base nucleophile 1. Introduction Cellular genomic DNA suffers damage from a variety of physical and chemical agents leading to base loss or alteration. Failure to repair such lesions can lead to deleterious mutations, genomic instability, or cell death. In higher eukaryotes, damage in genes responsible for DNA repair and/or cell cycle regulation can lead to life threatening diseases such as cancer. To remove such DNA damage and maintain genomic stability, a number of DNA repair pathways operate within cells. The major repair pathway protecting cells against single-base damage and loss is termed base excision repair (BER) [1,2]. The current and gen- erally accepted working model for mammalian BER involves two sub-pathways, “single-nucleotide” and “long-patch” BER, differentiated by the repair patch size and the enzymes involved [3–8]. Abbreviations: APE, apurinic/apyrimidinic endonuclease; BER, base exci- sion repair; dRP, 5 ′ -deoxyribose phosphate; EndoIII, exonuclease III; HhH, helix–hairpin–helix; Pol , DNA polymerase ; Pol , DNA polymerase  ∗ Corresponding author. Tel.: +1 919 541 3267; fax: +1 919 541 3592. E-mail address: wilson5@niehs.nih.gov (S.H. Wilson). BER is a sequential process initiated by recognition and removal of a damaged or inappropriate base by a damage specific DNA-glycosylase [9]. In cases of the monofunctional glycosy- lases, the resulting apurinic/apyrimidinic (AP) site is cleaved by AP endonuclease (APE), which incises the phosphodiester backbone 5 ′ to the AP site, generating a single-nucleotide gap with 3 ′ hydroxyl and 5 ′ -2-deoxyribose-5 ′ -phosphate (dRP) ter- mini [10]. Following this AP endonuclease cleavage, repair can proceed through either the single-nucleotide or long-patch BER (2–10 nucleotide patch) sub-pathways. Sub-pathway choice in BER appears to be influenced by a rate-limiting step in single- nucleotide BER, i.e., removal of the 5 ′ -dRP group by the dRP lyase activity of DNA polymerase  (Pol ) [11–13]. For exam- ple, if the 5 ′ -dRP group is not efficiently removed, continued DNA synthesis can lead to the long-patch BER sub-pathway [8]. Both sub-pathways appear to operate simultaneously to repair the same types of lesions in a cell [14,15]. It has been pro- posed that the sequential steps in BER are coordinated through protein–protein and/or DNA–protein interactions [16–20]. Pol  is a constitutively expressed “housekeeping” enzyme in vertebrates [7,21–23]. Mammalian Pol s characterized to date are highly conserved at the primary and secondary structure 1568-7864/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.dnarep.2005.08.009