Investigating the Role of Transcription on DNA Damage at Early Replicating Fragile Sites

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Project Summary/Abstract Genome instability is a hallmark of cancer. This instability initiates with a DNA lesion that is unfaithfully repaired. Replication stress is a major source of these lesions and transcription greatly enhances replication stress. Early replicating fragile sites (ERFS) are specific locations in the genome that experience recurrent DNA damage in early S-phase in the presence of replication stress. When ERFS were mapped in mouse primary B-cells, it was found that they are found in in gene rich areas and largely overlap with recurrent sites of amplifications and deletions in diffuse large B-cell lymphoma. It is not known why these sites are prone to DNA damage at ERFS or in the cancer. It was shown at one site that a decrease in transcription decreased chromosome fragility. It has been previously shown that collisions between replication forks (RF) and RNA polymerase (RNAP) are mutagenic. Transcriptional dysregulation in early replicating regions of the genome may play a role in this chromosome fragility. The hypothesis is that an increase in transcriptional activity in early replicating regions will lead to elevated levels of DNA damage due to the spatial and temporal co-localization of RF and RNAP. These experiments will be performed in primary mouse B-cells, as their distinct transcriptional profile and normal karyotype will allow us to accurately document deviations from wild type. The first aim, to test the hypothesis, will be modulating transcription at early replicating regions by targeting a catalytically defective CRISPR-dCas9 tethered to either transcriptional activators or repressors. This will allow the modulation of transcription at specific loci. The DNA damage will be measured by observing chromosomal abnormalities at the same loci using fluorescence in situ hybridization (FISH). This approach will show how alterations in transcriptional activity influence the levels of DNA damage in early replicating regions. The second aim, to test the hypothesis, will be finding sites in the genome that have both RF and RNAP co-localized in early S-phase. To locate RF and RNAP spatially and temporally co- localizing, I will perform a sequential immunoprecipitation (IP) for RNAP and nascent DNA, labeled with 5-ethynyl-2'-deoxyuridine The DNA at these locations will be sequenced and the DNA damage will be quantified using FISH. The sequential IP experiment will provide us with genomic loci that have both RF and RNAP co-localized in early S-phase. Together the data will suggest that changes in transcriptional programming leads to conflicts between RF and RNAP generating recurrent DNA damage observed in cancer.