Transient dsDNA breaks during pre-replication complex assembly.
Initiation of DNA replication involves the ordered assembly of the multi-protein pre-replicative complex (pre-RC) during G(1) phase. Previously, DNA topoisomerase II (topo II) was shown to associate with the DNA replication origin located in the lamin B2 gene locus in a cell-cycle-modulated manner. Here we report that activation of both the early-firing lamin B2 and the late-firing hOrs8 human replication origins involves DNA topo II-dependent, transient, site-specific dsDNA-break formation. Topo IIbeta in complex with the DNA repair protein Ku associates in vivo and in vitro with the pre-RC region, introducing dsDNA breaks in a biphasic manner, during early and mid-G(1) phase. Inhibition of topo II activity interferes with the pre-RC assembly resulting in prolonged G(1) phase. The data mechanistically link DNA topo IIbeta-dependent dsDNA breaks and the components of the DNA repair machinery with the initiation of DNA replication and suggest an important role for DNA topology in origin activation.
Rampakakis
Emmanouil
E
Goodman Cancer Center and Department of Biochemistry, McGill University, Montreal, Quebec, Canada H3A 1A3.
Zannis-Hadjopoulos
Maria
M
D034961:Antigens, Nuclear / Q000378:metabolism*; D053903:DNA Breaks, Double-Stranded; D004261:DNA Replication; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D016193:G1 Phase / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D000072200:Ku Autoantigen; D018741:Replication Origin
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen; D004250:DNA Topoisomerases, Type II