Topoisomerase 2b induces DNA breaks to regulate human papillomavirus replication

Abstract

Topoisomerases regulate higher order chromatin structures through the transient breaking and re-ligating of one or both strands of the phosphodiester backbone of duplex DNA. TOP2{beta} is a type II topoisomerase that induces double strand DNA breaks at topological-associated domains (TADS) to relieve torsional stress arising during transcription or replication. TADS are anchored by CTCF and SMC1 cohesin proteins in complexes with TOP2{beta}. Upon DNA cleavage a covalent intermediate DNA-TOP2{beta} (TOP2{beta}cc) is transiently generated to allow for strand passage. The tyrosyl-DNA phosphodiesterase TDP2 can resolve TOP2{beta}cc but failure to do so quickly can lead to long-lasting DNA breaks. Given the role of CTCF/SMC1 proteins in the HPV life cycle we investigated if TOP2{beta} proteins contribute to HPV pathogenesis. Our studies demonstrated that levels of both TOP2{beta} and TDP2 were substantially increased in cells with high risk HPV genomes and this correlated with high amounts of DNA breaks. Knockdown of TOP2{beta} with shRNAs reduced DNA breaks by over 50% as determined through COMET assays. Furthermore this correlated with substantially reduced formation of repair foci such as {gamma}H2AX, pCHK1 and pSMC1 indicative of impaired activation of DNA damage repair pathways. Importantly, knockdown of TOP2{beta} also blocked HPV genome replication. Our previous studies demonstrated that CTCF /SMC1 factors associate with HPV genomes at sites in the late regions of HPV31 and these correspond to regions that also bind TOP2{beta}. This study identifies TOP2{beta} as responsible for enhanced levels of DNA breaks in HPV positive cells and as a regulator of viral replication.