The primary focus of my Section is on the role of DNA damage in human diseases, including both neurological diseases and cancer. Specifically, the major focus is on two different DNA lesions. The first DNA lesion is 8, 5-cyclo-deoxyadenosine (cyclo-dA). We have presented several lines of evidence indicating that this lesion is responsible, at least in part, for the neurodegeneration in patients with the hereditary DNA repair diseases xeroderma pigmentosum (see Brooks, 2008). Another adduct of interest is N2-ethyl-dG, which results from the reaction of acetaldehyde, the first metabolite of ethanol, with DNA. ? ? In the past year, our effort with both of these lesions has shifted towards a more mechanistic and structural biology approach. This shift was prompted by our discovery in 2007 that the oxidative DNA lesion 8, 5-cyclo-dA could produce different types of mutant RNA transcripts in vivo in human cells (Marietta et al, 2007). This observation led us to begin focusing on the mechanistic basis of these observations, as well as more generally how DNA lesions impact the transcription process. Towards this goal, we have taken a reductionist approach to the question by using a minimal transcription system composed of an RNA primer, a DNA template, and purified polymerase. The polymerases studied included multisubunit enzymes bacterial RNA polymerase, eukaryotic RNA polymerase II and yeast polymerase II, as well as the single subunit T7 RNA polymerase, which serves as a model for eukaryotic mitochondrial RNA polymerase. ? Using the minimal system, we found that all multisubunit polymerases incorporated UMP opposite the lesion, consistent with our results in vivo. Interestingly, molecular modeling predicted that T7 RNAP would be able to insert UTP opposite cyclo-dA with essential the same kinetics as opposite a natural template dA, a prediction that was experimentally confirmed. The manuscript describing these results will be submitted shortly, following the completion of the analysis of the comparative kinetic determinations.? We have also used this comparative minimal system to examine the effects of N2-ethyl-dG, on transcription. In our paper published in JBC (Cheng et al, 2008), we showed that this lesion blocks transcription by both multisubunit and single subunit polymerases, but at different steps. As with cyclo-dA, eukaryotic RNA Pol II inserted the correct nucleotide, CTP, opposite the N2-ethyl-dG, prior to stalling. However, kinetic studies showed that the addition ethyl group showed incorporation by a factor of approx 1500 fold. Using molecular modeling we were able to identify two mutually exclusive configurations for the ethyl group within the active site of the polymerase. It should be possible to distinguish between these two models using X-ray crystallography of the polymerase transcribing DNA containing the lesion, and efforts towards that goal are ongoing. ? In contrast to the effect of N2-ethyl-dG on transcription by multisubunit RNA polymerases, we found that T7 RNA polymerase was unable to incorporating any rNTP opposite the lesion (Cheng et al, 2008). Thus, the lesion blocks the two different types of polymerase at different mechanistic steps. ? Subsequent molecular modeling studies have indicated that the blocking effect of the lesion on T7 RNAP is due in large part to a steric clash between the ethyl group of the adduct and a specific His residue located within the active site of the enzyme. Using site-directed mutagenesis, TF Cheng in my lab has now directly verified this prediction, and is now mutating the same site in yeast mt RNA polymerase.? ? Other work ? ? In collaboration with the laboratory of Kiyoji Tanaka, in Japan, we found that Xpa knockout mice have an elevated rate of spontaneous mutagenesis, as well as a severe defect in spermatogenesis (Nakane et al, DNA Repair 2008 in press). Both effects may be due to the cyclo-dA lesion we have studies, or other endogenous DNA lesions that are repaired by the NER pathway. ? ? Based on a review of the literature regarding the brain pathology in patients with Cockayne syndrome, we proposed that the cause of the unique forms of neuropathology in this disease may not be due to DNA repair defects, as generally believed, but to defects in the transcription of certain cell-type specific genes (Brooks et al, 2008). We are now pursuing this idea by analyzing postmortem brain tissue samples from CS patients. ? ? Finally, we had earlier proposed a key role for the Fanconi anemia BRCA1 pathway in protecting against the genotoxic effects of acetaldehyde, the first metabolite of ethanol (Brooks and Theruvathu, 2005). This hypothesis was based on the characteristics of a different alcohol related lesion called a Cr-PdG adduct. At the time we proposed that hypothesis, we were focusing on the measurement of the adduct, rather than its biological effects. Subsequently, due to personnel changes, our work on this adduct has evolved to more cellular studies of its biological effects. In work completed this year, we have obtained evidence to support this hypothesis, as well a mechanistic insight into how acetaldehyde triggers activation of the FA-BRCA pathway. This manuscript will also be submitted in the near future.
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