Abstract
The Dbh polymerase of Sulfolobus solfataricus is a member of the recently described family of low fidelity DNA polymerases involved in bypass of DNA lesions. To investigate the enzymatic properties of Dbh, we characterized the errors made by this polymerase in vitro. Not only is Dbh much less accurate than the "classical" polymerases, but it showed a remarkable tendency to skip over a template pyrimidine positioned immediately 3' to a G residue, generating a single-base deletion. Single-turnover kinetic measurements suggest possible mechanisms. First, Dbh shows a bias in favor of dCTP, such that the rate of incorporation of dCTP opposite a template G is about 10-fold faster than for the other three dNTPs opposite their complementary partners. On a DNA substrate corresponding to a frameshift hotspot, the rate of frameshift insertion of dCTP opposite a template G that is one residue 5' to the expected templating position is approximately equal to the rate of the non-frameshifted C-dGTP insertion. We suspect that the unusual mutational specificity of Dbh (which is shared with other polymerases from the DinB branch of the bypass polymerase family) may be related to the type of DNA lesion(s) that it serves to bypass in vivo.
Highlights
At a molecular level, an obvious question is what modifications to the classical polymerase active site are responsible for the novel properties of the lesion bypass polymerases
We suspect that the unusual mutational specificity of Dbh may be related to the type of DNA lesion(s) that it serves to bypass in vivo
0.0084 0.053 Յ0.011 Յ0.0063 0.0092 0.053 0.040 0.071 0.0063 0.026 a The number of occurrences of each template base within the 167-nucleotide region of the tk target for which reliable sequence data were obtained. b The number of mutations that could have resulted from misalignment-mediated misinsertion is shown in parentheses. c The calculation is analogous to that described in Table II. d Error rates for exonuclease-deficient Klenow fragment (KF) are taken from Ref. 33. e The large number of C-to-T transition mutations may have resulted from deamination of C at the temperature of 65 °C used for the Dbh-primed synthesis reaction
Summary
Materials—DNA oligonucleotides were synthesized by the Keck Biotechnology Resource Laboratory at Yale Medical School and were purified as described previously [28]. To increase the probability that the plasmids sequenced would contain independent mutations, the FT334 cells after electroporation were divided into multiple aliquots for outgrowth and plating, and no more than five tkϪ colonies from each plate were used to isolate DNA for sequencing (at the Keck Biotechnology Resource Laboratory at Yale Medical School). For Klenow fragment, the error rate for a particular class of errors was determined by calculating the fraction of the total errors contributed by that class, multiplying by the mutant frequency, and dividing by the number of detectable sites for that class of errors Kinetic Measurements—Single-turnover kinetic measurements of the polymerase reaction catalyzed by Dbh were carried out in a mixture containing 0.1 M duplex DNA oligonucleotide, 5Ј-32P-labeled on the primer strand, and variable concentrations of one or more dNTPs in 10 mM HEPES-NaOH, pH 8.5, 10 mM MgCl2, and 5 mM DTT. By showing that the reaction rate was unaffected by an increase in Dbh concentration to 20 M, we established that all the DNA substrate was enzyme-bound under our reaction conditions
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
