Thursday, April 10, 2025
4/10/2025
Mechanisms of Chromosome Fragile Site Formation in the Human Genome - PROJECT SUMMARY/ABSTRACT Common fragile sites (CFSs) are recurrent “wounds” in every person's genome that predispose the chromosomes to DNA double strand breaks (DSBs) and rearrangements. Known features associated with CFSs include late replication timing, which is further enhanced upon replication stress, and large transcribed genes, which may cause replication-transcription conflict. CFS formation/breakage underlies a wide variety of human diseases, including cancer and neurological disorders. We recently mapped replication stress-induced DNA DSBs in a normal human lymphoblastoid cell line, using Break-seq, a powerful NextGen-sequencing based technique developed in my laboratory. DSBs, with or without replication stress, are associated with late replication timing. However, these DSBs were not enriched inside large transcribing genes, nor are they enriched inside cytologially defined CFS core sequences. We hypothesize that the differences between Break-seq signals and CFS core sequences are attributable to the inherent differences between technological platforms as each is biased toward a partial feature of the human CFS, with Break-seq detecting the DSBs whereas the cytological methods detecting ssDNA gaps. The main objective of our proposal is to directly test this hypothesis by creating an upgraded sequencing technology, Fragile Site (FS)-seq, to simultaneously map ssDNA gaps and DSBs (Aim 1). Moreover, preliminary evidence suggests that the ssDNA inside the CFS core sequences is a consequence of “rogue” DNA initiation events upon high levels of DNA replication stress. Therefore, we will test if alterations in replication timing gives rise to ssDNA at the CFS core regions (Aim 2). The proposed project will bridge the gap in our current understanding of the mechanisms of CFS formation and genome instability.
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