Variation_of_DNA_damage_and_repair_along_a_bacterial_genome-sc-2013-04-17T10:48:37Z-2600
Variation_of_DNA_damage_and_repair_along_a_bacterial_genome
Recent evidence from cancer and bacterial genome sequencing studies evidences that the mutation rate varies largely along a genome, suggesting that the activity of DNA mutagenesis and repair is different in different regions of a genome.  Unfortunately, while decades of research on the field of DNA mutagenesis and repair have yielded a detailed molecular understanding of these processes, we know virtually nothing about their variable activity along a genome. A major reason for this is the lack of functional genomic techniques to study DNA mutagenesis and DNA repair along the genome, which contrasts with the impact of genomics in other fields such as transcriptional regulation and epigenetics.   We have now successfully used DNA immunoprecipitation to isolate DNA fragments carrying 8-oxoguanine sites (one of the major oxidative lesions) using a protocol similar to certain MEDIP-seq protocols used to identify methylated DNA. These DNA fragments will then be sequenced and mapped to the reference genome, obtaining a map of the relative rate of DNA oxidative damage along the E.coli genome.  This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/
We have developed a protocol similar to certain MEDIP-seq protocols to identify genomic regions containing 8-oxoguanine bases. To develop this protocol, we are using a simple experimental design on E.coli. Briefly, cells are exposed to high but sublethal doses of oxidative damage (5mM H2O2 for 20 min). Then DNA is extracted immediately after treatment to minimise the action of DNA repair (condition 1) or cells are incubated for 1h to allow substantial DNA repair (condition 2). The comparison of these two conditions with an unexposed control population will allow us to quantify the extent of DNA damage at different parts of a genome, before and after substantial DNA repair. Three biological replicates have been produced for each of the three conditions (9 samples in total).   The use of E.coli is ideal to develop this technology as it allows us to obtain high coverage data at a dramatically lower sequencing cost. Further, we have extensive RNA-sequencing and methylation data for the same strain under comparable conditions, which will allow us to demonstrate the potential of this technology to shed light on the interplay of DNA mutagenesis and DNA repair with genome organisation, transcription and epigenetics.