Mechanisms of Topoisomerases and Topoisomerase Poisons During Vertebrate DNA Replication

Abstract

Daughter DNA molecules must be unlinked to ensure complete and accurate duplication of the genome, which is critical for cell survival. We have previously found that during vertebrate DNA replication topoisomerase IIα (TOP2) is crucial for resolving catenanes, or interwound daughter DNA molecules, that form throughout replication. This suggested an expanded role for TOP2 during DNA replication, yet it was unclear how replication was affected in the absence of TOP2 activity. My thesis work uses Xenopus egg extracts to explore the consequences of impaired catenane resolution during DNA replication. First, I found that the TOP2 poisons etoposide and doxorubicin both inhibit DNA replication through different mechanisms. Etoposide induces TOP2-dependent DNA breaks and TOP2-dependent fork stalling by trapping TOP2 behind replication forks. In contrast, doxorubicin does not lead to appreciable break formation and instead intercalates into parental DNA to stall replication forks independently of TOP2. In mammalian cells, etoposide stalls forks in a TOP2-dependent manner, while doxorubicin stalls forks independently of TOP2. However, both drugs exhibit TOP2-dependent cytotoxicity. Thus, etoposide and doxorubicin inhibit DNA replication through distinct mechanisms despite shared genetic requirements for cytotoxicity. Second, I investigated whether other topoisomerases could compensate for loss of TOP2 and discovered that topoisomerase IIIα (TOP3α) normally has a limited role during DNA replication but becomes crucial when TOP2 cannot function. TOP3α carries out this role as part of the TOP3α-RMI1-RMI2 complex, independent of the BLM helicase. Moreover, TOP3α aids replication fork progression when extensive lagging strand gaps limit the amount of double-stranded DNA that is the substrate for TOP2. Finally, extensive lagging strand single-stranded DNA generates a novel DNA structure that contains intramolecular single-stranded DNA intertwines that are resolved by TOP3α. These data suggest that the TOP3α-RMI1-RMI2 complex is crucial to resolve catenanes in the absence of TOP2 during eukaryotic DNA replication. Ultimately, my work highlights the consequences of inhibited catenane resolution during vertebrate DNA replication and suggests that targeting TOP3α may help overcome resistance to etoposide, but not doxorubicin.