FIGURE S1. Anti-Tcpolß Is Specific. Immunoblot Using Anti-Tcpolßshows Reaction Only With

FIGURE S1. Anti-Tcpolß is specific.Immunoblot using anti-Tcpolßshows reaction only with T. cruzi protein extract (lane 1) and recombinant Tcpolß (lane 5). Other extracts and polymerases do not show reaction with the antibody. Lane 2: S.cerevisiae extract, Lane 3: S. pombe extract. Lane 4: HeLa cells extract. Lane 6: T4 DNA polymerase. Lane 7: Klenow fragment. The protein amount of each T. cruzi, S.cerevisiae, S. pombeand HeLa extracts was 50 g. The protein amount of recombinantTcpolß, T4 DNA polymerase and Klenow fragment was 25 ng.

FIGURE S2. Amino acid sequence alignment of DNA beta polymerases with their corresponding secondary structure described in the literature and putative phosphorylation sites. The cylinders marked with the letters A to O represent alpha helices described in the literature (Davies et al. ; Pelletier et al. 1994; Lindhl and Wood 1999;El-Sayed et al. 2005a). The gray rectangles numbered 1 through 9 represent beta sheets, also described previously (Davies et al. 1994; Pelletier et al. 1994; Sawaya et al. 1994). Motifs A and C correspond to segments containing the catalytic residues D190 and D191 (motif C) and R256 (motif A). The two largewhite vertical arrows ( ) indicate the S44 and S55 serine residues of rat polphosphorylated by in vitroassay with protein kinase C (Tokui et al. 1991). The large grey arrow indicatesthe lysine 72 (Lys72) responsible forthedRP-lyaseactivity(Deterding et al 2000) and the acetylation site (Hasan et al. 2002).

The hypothetical phosphorylation sites are indicated by thin vertical arrows on which there are letters that correspond to the following kinases: protein kinase A (A), protein kinase C (C), epidermal growth factor receptor (E), protein kinase G (G),caseine kinase I (K1), caseine kinase II (K2), RSK (R) and GSK3 (X).These hypothetical phosphorylation sites were identified by the NetPhosK 1.0 program.

The vertical arrows at the bottom of the alignments indicate the alignment column where there are four different amino acids of TrypanosomacruziMiranda pol. The access codes for the sequences are: Human (gi|4505931), rat (gi|206278), X-laevis (Xenopuslaevis, gi|2661842), L-brazil (Leishmaniabraziliensis, gi|134059577), C-fasci (Crithidia fasciculate,gi|33356563), Tc-Miranda (T. cruziMiranda clone, translated from gi 71666010), Tc-CLBr-Hap-N (T. cruziCLBrener clone haplotype non-Esmeraldo gi|70885292), Tc-CLBr-Hap-E (T. cruziCLBrener clone,haplotype Esmeraldo gi|71650066), Tc-Dm28c (T. cruziDm28c clone, gi|557863712), Tc-JRcl4 (T. cruzi JRcl4 clone translated from gb|AODP01000717.1), Tc-Tula (T. cruziTula clone, translated from gb|AQHO01018169.1|:123-1417), Tc-Esm (T. cruziEsmeraldo clone, translated from gb|ANOX01000503.1|:1035-2330), Tc-marink (Trypanosomacruzimarinkellei, gi|407395989), T-B-brucei (Trypanosomabruceibrucei strain 927/4, gi|72389310), S- pombe (Schizosaccharomycespombepol4. (SPAC2F7.06c.1) and S-cerv (Saccharomyces cerevisiaepol4, YCR014C).