4770 Nucleic Acids Research, 2005, Vol. 33, No. 15 Figure 5. Characterization of SpPol4 dRP lyase activity and reconstitution of BER in vitro. (A) Multiple amino acid alignment of the 8 kDa domain of SpPol4 with other family X DNA polymerases members. Numbers between slashes indicate the amino acid position relative to the N-terminus of each polymerase. Residues described to be relevant to the dRP lyase function of HsPolb (Lys35, Tyr39, Lys60, Lys68) are indicated with dots. Residues that are essential for (HsPolb Lys72; HsPoll Lys312) or may be involved in (ScPol4 Lys248) dRP lyase activity are in bold type. The position of SpPol4 Lys240 that might substitute for HsPolb Lys72 is indicated with an asterisk. Invariant (in white letters over a black background) and conservative substitutions referred to SpPol4 residues are boxed in dark gray. Abbreviations used are: Hom.sa. (H.sapiens), Sch.po. (S.pombe), and Sac.ce. (S.cerevisiae). (B) In vitro analysis of the dRP lyase reaction. The scheme shows a 34mer double- stranded oligonucleotide containing an uracil residue (at position 16) in the strand which is 30 end labeled (asterisk). After treatment with UDG and hAPE, a dRP- containing nicked substrate (18mer+dRP) is obtained. As shown in the autoradiogram, the dRP moiety can be cleaved by incubation with either human Poll (60 nM), wild-type SpPol4 or SpPol4D355A/D357A (20, 60 and 120 nM), as the labeled strand is detected as an 18mer product after denaturing electrophoresis. Human Polm (70 nM) was included as a negative control, lacking dRP lyase activity. (C) In vitro reconstitution of a BER reaction with SpPol4. A 34mer double-stranded oligonucleotide containing an uracil residue at position 16 in one strand is treated with UDG (100 nM) and hAPE (40 nM) to release a dRP-containing nicked substrate. By adding a labeled dNTP (a-dCTP) and either purified human Poll (245 nM) or SpPol4 (125 nM), two labeled products can be observed after denaturing electrophoresis and autoradiography: (i) a 16mer product generated by a single nucleotide insertion at the 30-hydroxyl end of the 50-incised AP site; (ii) a 34mer product that corresponds to the complete repair of the DNA strand upon T4 DNA ligase action. promote single-patch BER in vitro. By adding UDG and hAPE to a uracil-containing substrate, a nicked strand with a 3-hydroxyl and a 50-dRP is produced. The strand containing the 50-dRP moiety, which is 30 end labeled, migrates at the expected position of an 18mer + dRP (Figure 5B). By using increasing amounts of SpPol4, this product was converted to a shorter product (18mer), indicating that SpPol4 as Poll, and unlike Polm, has an intrinsic dRP lyase activity (Figure 5B). Similarly, the SpPol4D355A/D357A polymerization-deﬁcient mutant was proﬁcient in dRP lyase activity similar to the wild-type SpPol4 (Figure 5B). Unexpectedly from the alignment shown in Figure 5A, SpPol4 has dRP lyase activity though it lacks the lysine residue conserved in Polb (Lys72) and Poll (Lys312) responsible for their dRP lyase activities (24,58). Removal of a dRP residue is an essential step for the com- pletion of single nucleotide BER. Polb and Poll are able to efﬁciently promote in vitro BER of a uracil-containing duplex