Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4): an archaeal DinB-like DNA polymerase with lesion-bypass properties akin to eukaryotic poleta

Abstract

Phylogenetic analysis of Y-family DNA polymerases suggests that it can be subdivided into several discrete branches consisting of UmuC/DinB/Rev1/Rad30/Rad30A and Rad30B. The most diverse is the DinB family that is found in all three kingdoms of life. Searches of the complete genome of the crenarchaeon Sulfolobus solfataricus P2 reveal that it possesses a DinB homolog that has been termed DNA polymerase IV (Dpo4). We have overproduced and purified native Dpo4 protein and report here its enzymatic characterization. Dpo4 is thermostable, but can also synthesize DNA at 37 degrees C. Under these conditions, the enzyme exhibits misinsertion fidelities in the range of 8 x 10(-3) to 3 x 10(-4). Dpo4 is distributive but at high enzyme to template ratios can synthesize long stretches of DNA and can substitute for Taq polymerase in PCR. On damaged DNA templates, Dpo4 can facilitate translesion replication of an abasic site, a cis-syn thymine-thymine dimer, as well as acetyl aminofluorene adducted- and cisplatinated-guanine residues. Thus, although phylogenetically related to DinB polymerases, our studies suggest that the archaeal Dpo4 enzyme exhibits lesion-bypass properties that are, in fact, more akin to those of eukaryotic poleta.

Figure 1

Purification of S.solfataricus Dpo4 protein. Aliquots from various stages of the purification were separated on a 12% polyacrylamide–SDS gel and proteins visualized after staining with Coomassie Brilliant Blue R-250. –, Whole cell extract of RW382; +, whole extract from RW382 harboring p1914 (P2 Dpo4); S, clarified supernatant after heating the soluble cell extract at 85°C for 5 min; Q, pooled HiQ column fractions; H, pooled hydroxylapatite column fractions; M, Dpo4 fractions after Superdex 75 gel-filtration and MonoS columns. The molecular weight of marker proteins (BSA 66 kDa and E.coli RecA 38 kDa) are indicated on the left of the gel.

Figure 2

Ability of S.solfataricus Dpo4 to incorporate nucleotides at various template sites. The extent of (mis)incorporation was measured at each template site in the absence (0) or presence of all four dNTPs (4) or presence of each individual dNTP (100 µM) (G, A, T, C). Reactions were for 5 min at 37°C with 10 nM of Dpo4. The sequence context of each template is given above each panel and the target template nucleotide is indicated in bold.

Figure 3

Sulfolobus solfataricus P2 Dpo4 is a distributive polymerase. The ability of Dpo4 to extend a primer annealed to a long (∼7.2 kb) single-stranded DNA template was assayed over a range of enzyme concentrations. The P/T was kept fixed at 10 nM and the enzyme concentration varied from 200 to 0.5 nM as indicated. Reactions contained all four dNTPs (100 µM each) and were performed for 5 min at 37°C. This experiment shows that in the absence of additional cofactors, P2 Dpo4 is a distributive enzyme, primarily synthesizing 1–2 nt per binding event.

Figure 4

Thermostability of S.solfataricus P2 Dpo4. Aliquots of P2 Dpo4 (triangle), Taq polymerase (circle) and E.coli pol I Klenow fragment (diamond) were heated at the temperature indicated for 5 min. The effect of such treatment on polymerase activity was subsequently determined by assaying the respective enzyme’s ability to extend a radiolabeled primer via the correct incorporation of dCMP opposite template G. Replication assays were performed at 37°C and 100% incorporation occurred when all of the primer was extended by 1 nt.

Figure 5

PCR amplification DNA fragments by S. solfataricus P2 Dpo4. (Upper) Amplification of an ∼180 bp fragment by P2 Dpo4 or Taq polymerase. The exact amplification conditions are given in the Materials and Methods. The P2 enzyme concentration was varied from 20 to 400 ng. Track MW represents 100 bp molecular weight markers (Life Technologies). (Lower) Amplification of a ∼1300 bp DNA fragment. Enzyme concentrations are identical to those indicated above and amplification conditions are also given in the Materials and Methods. Track MW represents 1 kb molecular weight markers (Life Technologies).

Figure 6

Time-course experiments showing the ability of S.solfataricus P2 Dpo4 to bypass various DNA lesions. CPD, cis-syn cyclobutane pyrimidine thymine–thymine dimer: 6-4, 6-4 pyrimidine–pyrimidone thymine–thymine dimer; Abasic, synthetic abasic site; Cis-Pt, 1.2-cisplatinated guanine; AAF, acetyl aminofluorene guanine. The T-template is an undamaged control for the CPD, 6-4 and Abasic lesions. The G-template is the undamaged control for the Cis-Pt and AAF templates. Replication reactions were performed in the presence of 10 nM Dpo4 and 100 µM dNTPs for the times noted.

Figure 7

Quantitation of primer elongation shown in Figure 6. Filled circle, undamaged T; filled diamond, undamaged G: open triangle, abasic site; filled square, cisplatin-G; open circle, cis-syn TT; filled triangle, 6-4 TT; open square, AAF-G.

Figure 8

Ability of S.solfataricus P2 Dpo4 to incorporate nucleotides at various damaged template sites. The extent of (mis)incorporation was measured at each template site in the absence (0) or presence of all four dNTPs (4) or presence of each individual dNTP (100 µM) (G, A, T, C). Reactions were for 5 min at 37°C with 10 nM of Dpo4. The sequence context of each template is given above each panel. The extent of primer utilization (expressed as a percentage of the 0 dNTP control) is given below each track. From this qualitative assay, one observes that Dpo4 prefers to incorporate the correct nucleotide at each lesion. These observations have been confirmed by steady-state kinetic analysis (Table 2).