Nucleic Acids Research, 2005, Vol. 33, No. 15 4769 Figure 4. Lack of sugar discrimination by SpPol4. (A) Multiple amino acid alignment of the amino acid region (connecting subdomains palm and thumb) probably involved in sugar discrimination in the Pol X family. Numbers between slashes indicate the amino acid position relative to the N-terminus of each polymerase. Invariant (in white letters over a black background) and conservative substitutions referred to SpPol4 residues are boxed in dark gray. The two amino acid residues most probably involved in sugar discrimination are inside a box, and the Gly residue critical for rNTP incorporation is indicated with an asterisk. Abbreviations used are: Hom.sa. (Homo sapiens), Sch.po. (S.pombe), and Sac.ce. (S.cerevisiae). (B) SpPol4 inserts rNTPs efficiently. The assay, essentially as described in Figure 2, evaluates complementary rNMP incorporation into the four 1 nt gapped molecules, at different rNTP concentrations. The plotted values represent the ratio between the amount of extended versus total primers, and are the mean of four independent experiments. (C) Lack of sugar discrimination by SpPol4. The four 1 nt gapped DNA substrates described in Figure 2, differing in the templating base (X) (a scheme is shown), were used. In this competition assay, each complementary duo of nucleotides (rNTP + dNTP) was simultaneously provided at 100 nM. Reactions were carried out as described in Materials and Methods using 125 nM SpPol4. Mobility of the primer (P) and the rNTP (rN) or dNTP (dN) extended primers was analyzed by 8 M urea–20% PAGE and autoradiograph. The sugar selectivity factor is given as the ratio between the amount of rNTP- versus dNTP-extended primers. the mating type appear yellowish. As expected, iodine vapors stained the smt-0 mutant colonies yellowish, whereas the h90 wild-type and the pol4D colonies were stained black (data not shown). Therefore, the imprint at mat1 remains unaffected in the absence of SpPol4 and if there were one or two RNA residues in the DNA, the incorporation would be SpPol4- independent. SpPol4 has no TdT activity Polm, as TdT, displays an intrinsic deoxynucleotidyltrans- ferase activity, which is stronger in the presence of Mn2+ as cofactor (33). This enzymatic activity requires a region of the palm subdomain called loop1, which is absent in SpPol4, Polb and Poll (Supplementary Figure 3A). It has been demon- strated that the deletion of this loop abolishes the TdT-like activity of human Polm (29) (R. Jua´rez, J. F. Ruiz, S. A. Nick McElhinny, D. A. Ramsden and L. Blanco, manuscript sub- mitted). TdT activity can only be unambiguously determined by using single-stranded homopolymeric DNA as primer and any of the three dNTPs not included in the primer sequence. Thus, using a 32P-labeled 15mer dT oligonucleotide, in the presence of either Mg2+ or Mn2+, SpPol4 displayed no TdT activity (Supplementary Figure 3B). SpPol4 has an intrinsic dRP lyase activity most probably involved in BER The amino acid residues that are critical for dRPase activity are conserved in the 8 kDa domain of Polb and Poll (18,24) and are indicated with dots in Figure 5A. Among them, a speciﬁc lysine (Lys72 in Polb and Lys312 in Poll) is the cat- alytic residue acting as a Schiff-base during b-elimination of the dRP moiety. As shown in Figure 5A, Polm and SpPol4 lack the catalytic lysine residue, and as it has been demonstrated for Polm (24), it was probable that SpPol4 was devoid of dRP lyase activity. However, by using standard BER assays (18,24), here we show that SpPol4 is able to remove a dRP group and