Dissecting the Roles of Divergent and Convergent Transcription in Chromosome Instability

Abstract

SUMMARY The interplay of transcription, topological tension and chromosome breakage is a subject of intense interest, but, with so many facets to the problem, difficult to test. Here, we vary the orientation of promoters relative to one another in a yeast system that permits sensitive detection of chromosome breaks. Interestingly, convergent transcription that would direct RNA polymerases into one another does not increase chromosome breakage. In contrast, divergent transcription that would create underwound (potentially single-stranded) DNA does cause a marked increase in chromosome breakage. Furthermore, we examine the role that topoisomerases are playing in preventing genome instability at these promoters and find that Top2 is required to prevent instability at converging promoters.

AID and Reactive Oxygen Species Can Induce DNA Breaks within Human Chromosomal Translocation Fragile Zones

“…In order to construct the unique ZBE assay (Figure 1B), we have refined the haploid genetic assay from our previous study (Pannunzio and Lieber, 2016). The assay relies on the loss of the URA3 , CAN1 , and KAN-MX4 markers from the left arm of S. cerevisiae chromosome I, and retention of a LYS2 marker to ensure breaks detected are occurring on the telomeric-side of LYS2 and not in the 25 kb region between LYS2 and the first essential gene on chromosome I, PTA1 .…”

“…The effect of transcription was measured by inserting the GAL1 promoter into this region upstream of the inserted mammalian sequences. We have previously shown that the ectopic promoter leads to stable transcription upon galactose induction (Pannunzio and Lieber, 2016). For consistency, all strains, both those with and without the GAL1 promoter, were grown in YPG (1% yeast extract, 2% peptone, and 2% galactose).…”

“…Standard yeast genetic and molecular techniques were employed (Sambrook et al, 1989; Sherman F., 1986). As before (Pannunzio and Lieber, 2016), the assay relies on the loss of a URA3 and CAN1 cassette from the left arm of S. cerevisiae chromosome I, conferring resistance to 5-fluoroorotic acid (5-FOA R ) and canavanine (Can R ), respectively. Previously, a DSB anywhere in the 25 kb region between the CAN1 gene and the first essential gene on chromosome I, PTA1 , could result in loss of the non-essential chromosome arm.…”

“…For construction of the assay, as before, the various substrates were first assembled on a plasmid (Pannunzio and Lieber, 2016). A 1059 bp fragment carrying the URA3 CDS plus 177 bp of upstream and 78 bp of downstream sequence was cloned into pUC18, followed by a 2475 bp fragment carrying the CAN1 CDS plus 486 bp of upstream and 216 bp of downstream sequence.…”

“…Fragment insertion replaces a 690 bp fragment of the BDH1 CDS with the LYS2 gene. Additional alleles common to all of the assay strains are ura3::TRP1 and can1::LEU2 generated by fragment insertion (Pannunzio and Lieber, 2016) and the lys2-Δ4443 allele generated by the delitto perfetto method (Storici et al, 2001). All chromosomal insertions and disruptions were confirmed by PCR and meiotic mapping.…”