Abstract
DNA adducts formed by platinum-based anticancer drugs interfere with DNA replication. The carrier ligand of the platinum compound is likely to affect the conformation of the Pt-DNA adducts. In addition, the conformation of the adduct can also change upon binding of damaged DNA to the active site of DNA polymerase. From the crystal structures of pol beta ternary complexes it is evident that undamaged gapped and primed single-stranded (non-gapped) DNA templates exist in very different conformations when bound to pol beta. Therefore, one might expect that the constraints imposed on the damaged templates by binding to the polymerase active site should also affect the conformation of the Pt-DNA adducts and their ability to inhibit DNA replication. In support of this hypothesis we have found that the efficiency, carrier ligand specificity, site of discrimination (3'-G versus 5'-G of the Pt-GG adducts), and fidelity of translesion synthesis past Pt-DNA adducts by pol beta are strongly affected by the structure of the DNA template. Previous studies have suggested that the conformation of Pt-DNA adducts may be affected by the sequence context of the adduct. In support of this hypothesis, our data show that sequence context affects the efficiency, fidelity, and pattern of misincorporation by pol beta.
Highlights
The efficiency and accuracy of this bypass was different for the each polymerase. This suggested that translesion replication past Pt-DNA adducts was determined by both the structure of the adduct and the DNA polymerase involved in translesion synthesis
In the present study we show that the replication blocking potential and mutagenicity of Pt-DNA adduct is likely to be strongly influenced by constraints imposed on the template when it binds to the active site of the polymerase
In support of this hypothesis we have shown that for translesion synthesis past Pt-DNA adducts by pol  the specificity, the site of discrimination (3Ј-G versus 5Ј-G), and the pattern of misincorporation is strongly dependent on the DNA template structure
Summary
Construction of Platinum Adduct-containing Templates—Primertemplates were constructed from synthetic oligonucleotides as described previously [8]. To determine the efficiency of nucleotide incorporation opposite the 3Ј-G, dCTP concentrations ranged from 1 to 8 M for the undamaged template and from 16 to 256 M for the platinated templates. The kcat values we report were obtained by dividing Vmax (in nM primer extended per min) by the enzyme concentration (0.5 nM) To compare these data with the Vmax and Vmax/Km values reported in our previous kinetic analysis of translesion synthesis by pol  with different DNA templates [9], these kcat and kcat/Km values must be divided by 30. Kinetic Misincorporation Assays—To measure nucleotide misinsertion opposite Pt-GG adducts, 150 fmol of single nucleotide-gapped DNA substrates were incubated with 5 fmol of pol  and variable concentrations of dTTP or dATP. The relative misinsertion frequency was determined as fmis.rel ϭ (fmis-Pt)/(fmis-control)
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