Topoisomerase 2 Beta Induces Dna Breaks To Regulate Human Papillomavirus Replication

Topoisomerases regulate higher-order chromatin structures through the transient breaking and religating 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 topologically associated domains (TADS) to relieve torsional stress arising during transcription or replication. TADS are anchored by CCCTC-binding factor (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 human papillomavirus (HPV) life cycle, we investigated whether 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 large amounts of DNA breaks. Knockdown of TOP2 beta with short hairpin RNAs (shRNAs) reduced DNA breaks by over 50% as determined through COMET assays. Furthermore, this correlated with substantially reduced formation of repair foci such as phosphorylated H2AX (gamma H2AX), phosphorylated CHK1 (pCHK1), and phosphorylated SMC1 (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.IMPORTANCE High-risk human papillomaviruses (HPVs) infect epithelial cells and induce viral genome amplification upon differentiation. HPV proteins activate DNA damage repair pathways by inducing high numbers of DNA breaks in both viral and cellular DNAs. This activation is required for HPV genome replication. TOP2 beta is a type II topoisomerase that induces double-strand DNA breaks at topologically associated domains (TADS) to relieve torsional stress arising during transcription or replication. Our studies demonstrate that TOP2 beta levels are increased in HPV-positive cells and that this is required for HPV replication. Importantly, our studies further show that knockdown of TOP2 beta reduces the number of breaks by over 50% in HPV-positive cells and that this correlates with substantially impaired activation of DNA repair pathways. This study identifies a critical mechanism by which HPV replication is regulated by the topoisomerase TOP2 beta through DNA break formation.