Similarly, eukaryotic topoisomerase I incision, in the presence of specific inhibitors like camptothecin and after enzymatic removal of the dead-end covalent complex, produces nicks with this configuration of termini 8. It has been suggested several times, most recently by Yang et al.
Several PNK enzymes have been purified from a variety of different mammalian tissues 10— Composition of oligonucleotides and model substrates. Model duplexes were prepared as described in Materials and Methods by annealing three oligonucleotides to generate either a nick duplex A or a single-nucleotide gap duplexes B and C. The pertinent termini for this study are indicated above or below their respective oligonucleotides. Polynucleotide kinase was isolated from calf thymus as previously described Recombinant human DNA ligase I was purified as described previously Oligonucleotides Fig.
Small aliquots were examined by non-denaturing polyacrylamide gel electrophoresis to ensure that all the labelled oligonucleotide was annealed with the complementary strand. Conditions for each reaction are given in the respective figure legends.
The results show very similar enzyme concentration dependence regardless of the structure of the substrate Fig. In each reaction, 2. Figure 3a shows the results obtained with duplex A, which contains a nick. However, the combination of the two enzymes lane 5 sealed the nick to produce the labelled 45mer p The results of enzyme treatment of the duplex containing a nucleotide gap duplex B are shown in Figure 3b.
Incubation with PNK alone dephosphorylated p20p to give p20 lane 3. Surprisingly a small quantity of p21 was detected. Lane 7 shows that the combination of all three enzymes effectively filled in and sealed the nucleotide gap to produce p In order to carry out a more detailed analysis of these reactions, we first determined enzyme conditions under which the action s of PNK was rate limiting. Because of the acidic pH optimum pH 5.
Results of 5 min reactions using increasing concentrations of PNK with constant levels of polymerase and ligase indicated that the rate of formation of p45 was comparable at both pHs and that 0. Duplex A containing 0. Duplex B was incubated under similar conditions with the inclusion of dCTP 0.
In a , lanes 1 and 2 show markers for p21p and p21, and in b , lanes 1 and 8 show markers for p21 and p45, respectively. Plots of the reactions are displayed in Figure 4b B-E. For the reactions with duplex B, very little, if any, p21 was seen in the autoradiograms, therefore, the plotted products are p20p, p20 and p This would suggest that the p21 produced by polymerase b was rapidly acted upon by DNA ligase.
This may not be surprising given that the two enzymes can form a complex As is evident from Figure 4 , the pH of the reaction did not have a dramatic influence on the rates of reaction, although the overall rate appears to be a little slower at the higher pH. Time course of repair of nicked and gapped duplexes. Duplex B containing 0. D and E Plots of the nick-sealing reactions at pH 6. Autoradiogram showing the results of incubating duplex C Fig. Duplex C containing 0.
Lane 1 shows a marker for p In the preceding experiments, which show that a combination of the three enzymes can repair the gapped substrate, both functions of PNK were required. However, increasing the concentration of the polymerase fold did result in gap-filling lanes 7 and 8 in the absence of PNK.
Interestingly, when DNA ligase I was included in the reaction mixture, gap-filling and ligation went almost to completion, implying that DNA ligase I also stimulated the steps prior to strand rejoining. To test the possibility that the presence of DNA ligase I enhanced the kinase activity of PNK, a similar set of reactions were carried out as described above but dCTP was omitted to prevent gap-filling and ligation of the two shorter oligonucleotides.
PhosphorImager analysis of the gel shown in Figure 6 indicated a 1. A similar level of enhancement, i. Even more marked enhancement of phosphorylation was observed with the complementary 45mer oligonucleotide p45 comp.
The control, in which PNK was omitted lane 6 , confirmed that the other two enzyme preparations were free of kinase activity. It was thus possible to monitor the phosphorylation of the 24mer and the complementary 45mer p45 comp. The radioactivity in p20 in lanes 3—7 indicated that there was no artefact due to unequal sample loading onto the gel. A role for the enzyme in DNA repair has been espoused ever since the existence of PNK in mammalian cells was first reported 28 , Clearly, as a prerequisite for such an enzyme to be involved in repair of single-strand breaks induced by topoisomerase or ionizing radiation it would have to act at the relatively sterically-hindered termini of nicks and 1 nt gaps, as opposed to more accessible ends of single-stranded DNA or of double-strand breaks.
This enzyme acts to restore tRNA, cleaved by a phage anticodon nuclease, to a ligatable form 31 , and is not considered to be a DNA repair enzyme since PNK mutants of phage T4 display no phenotypic defect in repair 32 , However, the latter is a relatively ill-defined substrate because such treatment can generate gaps as well as nicks But even this substrate has the potential for gap formation by slippage of the oligo dT on the complementary strand.
For this reason we employed oligonucleotide models with a well-defined nick or 1 nt gap. The latter two enzymes were chosen because they are believed to form a complex in cells which is involved in one of the two known pathways for base excision repair Figure 3 establishes that the combination of these three mammalian enzymes rapidly restored the damaged sites. The repair of the substrates required both functions of PNK.
The gap does not close completely. Using distance constraints, MD calculations show that the nick conformation is very close to a duplex with no lesion but is indeed more flexible in the central part. The gapped structure shows two families of conformations. One is close to B-DNA, the other is significantly kinked at the gap which reduces the size of the cavity.
We observe a spine of hydration within the cavities, similar, but of different geometry in the two cases. Abstract We have analyzed and compared the molecular structures and dynamics of DNA duplexes containing a nick or a gap of one nucleotide where the base in front of the gap is a guanine.
Publication types Research Support, Non-U.
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