Genome-wide study to investigate the organisation of global genome nucleotide excision repair in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae efficient global genome nucleotide excision repair (GGR) requires a heterotrimeric protein complex of Abfl, Rad7 and Radl6 termed the GGR complex. Abfl is a site specific DNA binding protein with known roles in DNA replication, transcription and repair. Radl6 has a DNA translocase activity and is a functional component of an E3 ubiquitin ligase. Radl6 has recently been shown to regulate the occupancy of Gcn5 and histone H3 K9K14 acetylation in response to UV damage. The current study investigates how GGR is organised throughout the genome using chromatin-immunoprecipitation coupled to microarrays. Abfl is observed to bind the genome at a high frequency and is preferentially localised to promoters. By analysing other genome-wide datascts in relation to Abfl binding, Radl6 dependent histone H3 K.9K14 acctylation and efficient GGR are observed to colocalise with Abfl binding sites at promoters. Radl6 binding is also mapped and is found to colocalise with Abfl binding sites at many promoters. Peaks of Radl6 binding are lost in a UV dependent manner and based on previous studies, this is suggested to occur by DNA translocation of Radl6. The differences in Radl6 binding levels are found to correlate with Radl6 dependent acetylation and efficient GGR. In addition to studying the occupancy of Abfl, novel tools are built for the genome-wide analysis of Abfl DNA binding kinetics. A recombinant protein termed a competitor is designed for this purpose. The competitor consists of an Abfl DNA binding domain fused to a hormone dependent regulatory cassette. Following activation, the rate at which the competitor replaces Abfl at a DNA binding site is monitored by chromatin immunoprecipitation to qualitatively measure Abfl DNA binding kinetics. Preliminary results are shown that might suggest changes in Abfl DNA binding kinetics following UV are mechanistically linked to GGR