Functional interactions of DNA topoisomerases with a human replication origin

The mechanism by which certain DNA sequences are chosen to function as DNA replication origins, in metazoan genomes, is currently not understood. However, a comparison of the DNA loci identified as replicators so far points towards a role for DNA topology in this process. DNA topoisomerases are the modulators of DNA topology inside the cell, their activity being essential in all living organisms, nevertheless, the involvement of topoisomerases in metazoan DNA replication is poorly characterized. In this study, the role of topological modulation of the origin DNA was investigated by mapping the interaction of human topoisomerase I and II with a human origin. The lamin B2 DNA replication origin, located on human chromosome 19, interacts with the DNA topoisomerases I and II in a cell cycle modulated fashion. The topoisomerases interact in vivo with precise bonds ahead of the start sites of bidirectional replication, within the pre-replicative complex region. Topoisomerase I introduces two single stranded cleavages, on the origin upper and lower strand respectively, in M, early G1 and at late G1 - G1/S border, with topoisomerase II introducing also two single stranded cleavages, on the origin upper and lower strand respectively, in M and middle of G1 phase of the cell cycle. At the origin, topoisomerase II interacts with Orc2p during the assembly of the pre-replicative complex in the middle of G1 phase of the cell cycle. Furthermore, topoisomerase I interacts with Orc2p in late G1 - G1/S border as part of the origin initiation complex and inhibition of topoisomerase I activity abolishes origin firing. The two topoisomerases also compete for the same sites bound by the Orc2 protein, in different moments of the cell cycle. In vitro, human recombinant DNA topoisomerase I is able to distinguish the same sites on origin DNA as the in vivo cleaved ones, with some additional cuts on the upper strand. In contrast, human recombinant DNA topoisomerase II, alone, cannot introduce the same precise origin cleavages, but as part of an in vitro origin specific multi-protein complex it can recognize and cut exactly the same sites as in vivo. Thus, the two topoisomerases are members of the replicative complexes with DNA topology playing an important functional role for origin activation.