Topoisomerase 1-catalyzed genomic ribonucleotide excision, its regulation, and its implication in transcription.

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PROJECT SUMMARY In eukaryotes, ribonucleotides are frequently incorporated into DNA during replication (1 ribonucleotide per every 1000-5000 deoxyribonucleotides). Canonically, RNase H2 is the protein responsible for the removal of these embedded ribonucleotides. However, it has been recently shown that topoisomerase 1 (Top1) also has its own genomic ribonucleotide processing activity. When this processing occurs in specific short repeat sequences, it can lead to 2-7 bp deletions. These deletions are the result of two sequential nicks by Top1 that releases a small single-stranded DNA segment and is then followed by a strand slippage and ligation across the formed gap. These deletions have been shown to be biased towards the non-transcribed strand (NTS) of highly transcribed genes, but the reasoning for this strand specificity is yet to be elucidated. With the help of our recent preliminary data, we propose that this strand specificity for Top1 activity is due to the formation of DNA topological structures, specifically negative supercoils, behind the RNA polymerase that bias the initial cleavage by Top1. This would re-define the way we think about Top1-mediated DNA relaxation by limiting the cleavage of Top1 mainly towards the NTS during transcription. This limitation also allowed us to hypothesize about a possible biological implication for Top1-catalyzed excision of ribonucleotides. When Top1 cleaves a ribonucleotide, it can generate a unique nick lesion known as 2’,3’-cyclic phosphate (CP). The bias of Top1 cleavage at the NTS and the formation of CPs lead us to hypothesize that this transient nick lesion could serve as a way of continually relieving transcriptional torsional stress, as CPs would be forming at a strand that would potentially not interfere with transcription, unlike nicks at the transcribing strand. We plan to investigate this by looking at mRNA expression after depleting genomic ribonucleotides in yeast. This investigation will provide us with potential roles of ribonucleotide incorporation in eukaryotes. Additionally, the processing of genomic ribonucleotides by Top1 produces PARP-trapping lesions, a chemotherapeutic target for BRCA1/BRCA2 deficient tumors. I will look at the interactions that could lead to the trapping of PARP1 after ribonucleotide cleavage by Top1 and its link to the ability of PARP1 to regulate CP formation (identified recently by us and shown in our preliminary data). The presence of either CPs or adducts between DNA and Top1 are likely candidates for the recruitment of PARP1. Overall, our findings will help clarify the relevance of ribonucleotide incorporation for transcriptional regulation by providing insight into a novel mechanism of DNA relaxation by Top1 through the processing of those genomic ribonucleotides. Our proposal will also aim to describe the regulation of CP formation by PARP1, and the physiological relevance of the interaction between Top1, PARP1, and genomic ribonucleotides.