Pre-Steady-State Kinetic Studies of the Fidelity of Sulfolobus solfataricus P2 DNA Polymerase IV

Abstract

Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) is a thermostable archaeal enzyme and a member of the error-prone and lesion-bypass Y-family. In this paper, for the first time, the fidelity of a Y-family polymerase, Dpo4, was determined using pre-steady-state kinetic analysis of the incorporation of a single nucleotide into an undamaged DNA substrate 21/41-mer at 37 degrees C. We assessed single-turnover (with Dpo4 in molar excess over DNA) saturation kinetics for all 16 possible nucleotide incorporations. The fidelity of Dpo4 was estimated to be in the range of 10(-3)-10(-4). Interestingly, the ground-state binding affinity of correct nucleotides (70-230 microM) is 10-50-fold weaker than those of replicative DNA polymerases. Such a low affinity is consistent with the lack of interactions between Dpo4 and the bound nucleotides as revealed in the crystal structure of Dpo4, DNA, and a matched nucleotide. The affinity of incorrect nucleotides for Dpo4 is approximately 2-10-fold weaker than that of correct nucleotides. Intriguingly, the mismatched dCTP has an affinity similar to that of the matched nucleotides when it is incorporated against a pyrimidine template base flanked by a 5'-template guanine. The incoming dCTP likely skips the first available template base and base pairs with the 5'-template guanine, as observed in the crystal structure of Dpo4, DNA, and a mismatched nucleotide. The mismatch incorporation rates, regardless of the 5'-template base, were approximately 2-3 orders of magnitude slower than the incorporation rates for matched nucleotides, which is the predominant contribution to the fidelity of Dpo4.

DNA Adduct Bypass Polymerization by Sulfolobus solfataricus DNA Polymerase Dpo4

“…13D). This behavior of Dpo4 has been suggested previously in kinetic (35) and structural (31) studies with mispaired bases, without DNA adducts. The minor product has the A inserted opposite 1,N 2 -⑀-G, followed by normal incorporation.…”

“…When the addition of C or A to the primer was examined using rapid quench methods, no burst phase was observed (results not presented), indicating that the addition is an inherently slow process, and the k off rate (for the oligonucleotide) is not rate-limiting, in contrast to results reported for normal base incorporation (35) and confirmed here.…”

“…Several crystal structures have been reported, including some with damaged DNA (31)(32)(33)(34), and pre-steady-state kinetic analysis has helped define a general catalytic mechanism (for undamaged DNA) (35,36). A number of questions still exist about the interactions of 1,N 2 -⑀-G with DNA polymerases, in that different misincorporations and frameshifts have been observed with individual polymerases, and in some cases undefined mixtures of products are obtained with a single polymerase (15,16).…”

DNA Lesion Alters Global Conformational Dynamics of Y-family DNA Polymerase during Catalysis

“…Preparation of Fluorescently Labeled Dpo4 and DNA Substrates-Unlabeled wild-type S. solfataricus Dpo4 was purified as described previously (18). The creation of the Dpo4 enzymes containing the C31S substitution and site-specific labeling with Alexa Fluor 594 (Invitrogen) at another engineered Cys substitution (i.e.…”

Versatility of Y-family Sulfolobus solfataricus DNA Polymerase Dpo4 in Translesion Synthesis Past Bulky N2-Alkylguanine Adducts

“…The DNA polymerase Dpo4 from Sulfolobus solfataricus P2 (polymerase IV) (30) has been investigated in considerable detail both in terms of its function (31)(32)(33)(34)(35) and structure (36 -38). Dpo4 can bypass various DNA adducts, including 8-oxoG (39,40), O 6 -MeG (41), O 6 -BzG (42), UV-cross-linked products (43), BPDE adducts (44,45), and abasic lesions (38).…”