Dataset Information

Sequence-dependent thymine dimer formation and photoreversal rates in double-stranded DNA.

ABSTRACT: The kinetics of thymine-thymine cyclobutane pyrimidine dimer (TT-CPD) formation was studied at 23 thymine-thymine base steps in two 247-base pair DNA sequences irradiated at 254 nm. Damage was assayed site-specifically and simultaneously on both the forward and reverse strands by detecting emission from distinguishable fluorescent labels at the 5'-termini of fragments cleaved at CPD sites by T4 pyrimidine dimer glycosylase and separated by gel electrophoresis. The total DNA strand length of nearly 1000 bases made it possible to monitor damage at all 9 tetrads of the type XTTY, where X and Y are non-thymine bases. TT-CPD yields for different tetrads were found to vary by as much as an order of magnitude, but similar yields were observed at all instances of a given tetrad. Kinetic analysis of CPD formation at 23 distinct sites reveals that both the formation and reversal photoreactions depend sensitively on the identity of the nearest-neighbour bases on the 5' and the 3' side of a photoreactive TT base step. The lowest formation and reversal rates occur when two purine bases flank a TT step, while the highest formation and reversal rates are observed for tetrads with at least one flanking C. Overall, the results show that the probabilities of CPD formation and photoreversal depend principally on interactions with nearest-neighbour bases.

SUBMITTER: Law YK

PROVIDER: S-EPMC3815666 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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Publications

Sequence-dependent thymine dimer formation and photoreversal rates in double-stranded DNA.

Law Yu Kay YK Forties Robert A RA Liu Xin X Poirier Michael G MG Kohler Bern B

Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology 20130801 8

The kinetics of thymine-thymine cyclobutane pyrimidine dimer (TT-CPD) formation was studied at 23 thymine-thymine base steps in two 247-base pair DNA sequences irradiated at 254 nm. Damage was assayed site-specifically and simultaneously on both the forward and reverse strands by detecting emission from distinguishable fluorescent labels at the 5'-termini of fragments cleaved at CPD sites by T4 pyrimidine dimer glycosylase and separated by gel electrophoresis. The total DNA strand length of near ...[more]

PMID: 23727985

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Project description:We develop a robust coarse-grained model for single- and double-stranded DNA by representing each nucleotide by three interaction sites (TIS) located at the centers of mass of sugar, phosphate, and base. The resulting TIS model includes base-stacking, hydrogen bond, and electrostatic interactions as well as bond-stretching and bond angle potentials that account for the polymeric nature of DNA. The choices of force constants for stretching and the bending potentials were guided by a Boltzmann inversion procedure using a large representative set of DNA structures extracted from the Protein Data Bank. Some of the parameters in the stacking interactions were calculated using a learning procedure, which ensured that the experimentally measured melting temperatures of dimers are faithfully reproduced. Without any further adjustments, the calculations based on the TIS model reproduce the experimentally measured salt and sequence-dependence of the size of single-stranded DNA (ssDNA), as well as the persistence lengths of poly(dA) and poly(dT) chains. Interestingly, upon application of mechanical force, the extension of poly(dA) exhibits a plateau, which we trace to the formation of stacked helical domains. In contrast, the force-extension curve (FEC) of poly(dT) is entropic in origin and could be described by a standard polymer model. We also show that the persistence length of double-stranded DNA, formed from two complementary ssDNAs, is consistent with the prediction based on the worm-like chain. The persistence length, which decreases with increasing salt concentration, is in accord with the Odijk-Skolnick-Fixman theory intended for stiff polyelectrolyte chains near the rod limit. Our model predicts the melting temperatures of DNA hairpins with excellent accuracy, and we are able to recover the experimentally known sequence-specific trends. The range of applications, which did not require adjusting any parameter after the initial construction based solely on PDB structures and melting profiles of dimers, attests to the transferability and robustness of the TIS model for ssDNA and dsDNA.

Dataset Information

Sequence-dependent thymine dimer formation and photoreversal rates in double-stranded DNA.

Publications

Sequence-dependent thymine dimer formation and photoreversal rates in double-stranded DNA.

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