Processing of DNA lesions by archaeal DNA polymerases from Sulfolobus solfataricus

Abstract

Spontaneous damage to DNA as a result of deamination, oxidation and depurination is greatly accelerated at high temperatures. Hyperthermophilic microorganisms constantly exposed to temperatures exceeding 80 degrees C are endowed with powerful DNA repair mechanisms to maintain genome stability. Of particular interest is the processing of DNA lesions during replication, which can result in fixed mutations. The hyperthermophilic crenarchaeon Sulfolobus solfataricus has two functional DNA polymerases, PolB1 and PolY1. We have found that the replicative DNA polymerase PolB1 specifically recognizes the presence of the deaminated bases hypoxanthine and uracil in the template by stalling DNA polymerization 3-4 bases upstream of these lesions and strongly associates with oligonucleotides containing them. PolB1 also stops at 8-oxoguanine and is unable to bypass an abasic site in the template. PolY1 belongs to the family of lesion bypass DNA polymerases and readily bypasses hypoxanthine, uracil and 8-oxoguanine, but not an abasic site, in the template. The specific recognition of deaminated bases by PolB1 may represent an initial step in their repair while PolY1 may be involved in damage tolerance at the replication fork. Additionally, we reveal that the deaminated bases can be introduced into DNA enzymatically, since both PolB1 and PolY1 are able to incorporate the aberrant DNA precursors dUTP and dITP.

Figure 1

Expression of DNA polymerases B1 and Y1 in S.solfataricus cells. Quantitative western blots showing detection of the polymerases in cell lysates including standards with different amounts of purified proteins. The S.solfataricus cell extract was prepared as described in Materials and Methods. Lanes 1–5, purified proteins applied at 15, 30, 60, 120 and 240 ng/lane; lanes 6–8, S.solfataricus total cell extract applied at 5, 10 and 15 µl/lane. The chemiluminescence values shown beneath the lanes were expressed as the sum of the intensities of the pixels inside the volume boundary manually selected around each band per area of a single pixel, as described in Materials and Methods. The intensity values obtained for the amounts of 15, 30 and 60 ng (lanes 1–3) were used to construct a linear regression curve for each protein. From these titration curves, the protein concentration values were extrapolated for the lanes loaded with 15 µl of total cell extract.

Figure 2

Comparison of DNA lesion bypass by thermostable DNA polymerases from the A, B and Y families. The side-by-side comparison of DNA polymerase abilities to bypass several heat-induced DNA lesions. The enzymes used were PolY1 and PolB1 from S.solfataricus and Taq DNA polymerase from T.aquaticus, shown schematically from left to right in the picture. The types of DNA lesions tested and associated controls (no lesion) are shown below the gel lines. The position of the lesions (or G in the control) is underlined within the full sequence context at the top. dNTPs were used at a concentration of 100 µM and the reactions were carried out at 55°C for 10 min as described in Materials and Methods.

Figure 3

Recognition of deaminated bases in DNA by archaeal B family DNA polymerases by the read-ahead mechanism. The deaminated base products uracil (U) and hypoxanthine (X) were positioned further downstream from the 3′-end of the primer to determine the exact position of the replication block. Sequence details of the relevant parts of oligonucleotide substrates as well as the exact positions of the lesions on the gels are shown in the picture. The order of experimental lanes on the gels is schematically shown as well. The enzymes used were S.solfataricus PolY1 and PolB1 as well as the control DNA polymerases Taq from T.aquaticus and Pfu from Pyrococcus furiosus.

Figure 4

Incorporation of deaminated dNTP derivatives into DNA by thermostable DNA polymerases. (A) Side-by-side comparison of incorporation of the normal base thymine (dTTP) and the erroneous uracil (dUTP) into DNA by the Y, B and A family DNA polymerases. The relevant substrate sequence is shown on the right next to the gel. (B) Incorporation and insertion specificity of the erroneous base hypoxanthine (dITP) into DNA by the Y, B and A family DNA polymerases. The relevant primer–template sequence is shown on the left next to the gel, where N stands for the variable base. Each of the four possible bases was tested at the variable base position as template for each enzyme in separate lanes as marked under the gel picture. The enzymes used were S.solfataricus PolY1 and PolB1 as well as the control DNA polymerase Taq from T.aquaticus.

Figure 5

Tight binding of PolB1 to ssDNA oligonucleotides containing deaminated bases. BIAcore sensograms showing the association and dissociation kinetics for binding of S.solfataricus DNA polymerases to immobilized oligonucleotides. (A) Demonstration of tight binding of PolB1 to oligonucleotides containing deaminated bases (either single uracil or single hypoxanthine as shown) compared to control oligonucleotide without lesions. (B) Comparison of PolY1 and PolB1 binding to control oligonucleotide. Binding to substrates was tested by injecting the appropriate protein independently five times at concentrations of 10, 50, 100, 250 and 500 nM.

Figure 5

Tight binding of PolB1 to ssDNA oligonucleotides containing deaminated bases. BIAcore sensograms showing the association and dissociation kinetics for binding of S.solfataricus DNA polymerases to immobilized oligonucleotides. (A) Demonstration of tight binding of PolB1 to oligonucleotides containing deaminated bases (either single uracil or single hypoxanthine as shown) compared to control oligonucleotide without lesions. (B) Comparison of PolY1 and PolB1 binding to control oligonucleotide. Binding to substrates was tested by injecting the appropriate protein independently five times at concentrations of 10, 50, 100, 250 and 500 nM.