The Cockayne syndrome B protein (CSB) has long been known to be involved in the repair of DNA modifications that block the RNA polymerase in transcribed DNA sequences (transcription-coupled repair). Recent evidence suggests that it also has a more general role in the repair of oxidative DNA base modifications such as 7,8-dihydro-8-oxo-2 0 -deoxyguanosine (8-oxoG). In mammalian cells, 8-oxoG is a substrate of the repair glycosylase OGG1. Mice without this enzyme accumulate 8-oxoG in the genome and have elevated spontaneous mutation rates. To elucidate the role of CSB in the prevention of mutations by oxidative DNA base damage, we have generated mice that are deficient in Csb or Ogg1 or both genes and carry a non-transcribed bacterial lacI gene for mutation analysis (Big Blue mice). Our results indicate that the overall spontaneous mutation frequencies in the livers of Csb m/m / Ogg1 À/À -mice are elevated not only compared with heterozygous control mice (factor 3.3), but also with Ogg1 À/À -animals (factor 1.6). Sequence analysis revealed that the additional mutations caused by CSB deficiency in an Ogg1 À/À background are mostly G:C to T:A transversions and small deletions. For all mouse strains, the background levels of oxidative purine modifications in the livers correlate linearly with the numbers of G:C to T:A transversions observed. The data indicate that CSB is involved in the inhibition of mutations caused by spontaneous oxidative DNA base damage in a nontranscribed gene.

No abstract

BackgroundDNA is a carrier of biological information. The hybridization process, the formation of the DNA double-helix from single-strands with complementary sequences, is important for all living cells. DNA microarrays, among other biotechnologies such as PCR, rely on DNA hybridization. However, to date the thermodynamics of hybridization is only partly understood. Here we address, experimentally and theoretically, the hybridization of oligonucleotide strands of unequal lengths, which form a bulged loop upon hybridization. For our study we use in-house synthesized DNA microarrays.ResultsWe synthesize a microarray with additional thymine bases in the probe sequence motifs so that bulged loops occur upon target hybridization. We observe a monotonic decrease of the fluorescence signal of the hybridized strands with increasing length of the bulged loop. This corresponds to a decrease in duplex binding affinity within the considered loop lengths of one to thirteen bases. By varying the position of the bulged loop along the DNA duplex, we observe a symmetric signal variation with respect to the center of the strand. We reproduce the experimental results well using a molecular zipper model at thermal equilibrium. However, binding states between both strands, which emerge through duplex opening at the position of the bulged loop, need to be taken into account.ConclusionsWe show that stable DNA duplexes with a bulged loop can form from short strands of unequal length and they contribute substantially to the fluorescence intensity from the hybridized strands on a microarray. In order to reproduce the result with the help of equilibrium thermodynamics, it is essential (and to a good approximation sufficient) to consider duplex opening not only at the ends but also at the position of the bulged loop. Although the thermodynamic parameters used in this study are taken from hybridization experiments in solution, these parameters fit our DNA microarray data well.

Basal levels of endogenously generated oxidative DNA modifications such as 7,8-dihydro-8-oxoguanine (8-oxoG) mice, which were f3-fold higher than in the repair-proficient mice, declined by 39% under the treatment, whereas the frequencies in the livers of the repair-proficient animals remained unchanged. Preneoplastic lesions (staining positive or negative for glucose-6-phoshatase) developed in the livers of both wild-type and Csb m/m /Ogg1 À/À mice after 30 weeks.Both the numbers and the total volumes of the lesions were f6-fold higher in the repair-deficient mice than in the wildtype mice. The results indicate that spontaneous mutations generated from endogenous oxidative DNA base damage efficiently translate into increased tumorigenesis when cell proliferation is stimulated. [Cancer Res 2007;67(11):5156-61]

The specificity of molecular recognition is important for molecular self-organization. A prominent example is the biological cell where a myriad of different molecular receptor pairs recognize their binding partners with astonishing accuracy within a highly crowded molecular environment. In thermal equilibrium it is usually admitted that the affinity of recognizer pairs only depends on the nature of the two binding molecules. Accordingly, Boltzmann factors of binding energy differences relate the molecular affinities among different target molecules that compete for the same probe. Here, we consider the molecular recognition of short DNA oligonucleotide single strands. We show that a better matching oligonucleotide can prevail against a disproportionally more concentrated competitor with reduced affinity due to a mismatch. We investigate the situation using fluorescence-based techniques, among them Förster resonance energy transfer and total internal reflection fluorescence excitation. We find that the affinity of certain strands appears considerably reduced only as long as a better matching competitor is present. Compared to the simple Boltzmann picture above we observe increased specificity, up to several orders of magnitude. We interpret our observations based on an energy-barrier of entropic origin that occurs if two competing oligonucleotide strands occupy the same probe simultaneously. Due to their differences in binding microstate distributions, the barrier affects the binding affinities of the competitors differently. Based on a mean field description, we derive a resulting expression for the free energy landscape, a formal analogue to a Landau description of phase transitions reproducing the observations in quantitative agreement as a result of a cooperative transition. The advantage of improved molecular recognition comes at no energetic cost other than the design of the molecular ensemble and the presence of the competitor. As a possible application, binding assays for the detection of single nucleotide polymorphisms in DNA strands could be improved by adding competing strands. It will be interesting to see if mechanisms along similar lines as exposed here contribute to the molecular synergy that occurs in biological systems.