Abstract
Sperm chromatin incubated in Xenopus egg extracts undergoes origin licensing and nuclear assembly before DNA replication. We found that depletion of DNA topoisomerase IIα (topo IIα), the sole topo II isozyme of eggs and its inhibition by ICRF-193, which clamps topo IIα around DNA have opposite effects on these processes. ICRF-193 slowed down replication origin cluster activation and fork progression in a checkpoint-independent manner, without altering replicon size. In contrast, topo IIα depletion accelerated origin cluster activation, and topo IIα add-back negated overinitiation. Therefore, topo IIα is not required for DNA replication, but topo IIα clamps slow replication, probably by forming roadblocks. ICRF-193 had no effect on DNA synthesis when added after nuclear assembly, confirming that topo IIα activity is dispensable for replication and revealing that topo IIα clamps formed on replicating DNA do not block replication, presumably because topo IIα acts behind and not in front of forks. Topo IIα depletion increased, and topo IIα addition reduced, chromatin loading of MCM2-7 replicative helicase, whereas ICRF-193 did not affect MCM2-7 loading. Therefore, topo IIα restrains MCM2-7 loading in an ICRF-193-resistant manner during origin licensing, suggesting a model for establishing the sequential firing of origin clusters.
Highlights
Eukaryotic DNA replication starts at multiple sites called replication origins [1]
Cdc45 binding was not detected here, we found in other experiments (Supplementary Figure S6) that when ICRF-193 was added at 0 min, the chromatin binding of Cdc45 was not affected, but its dissociation during the progression of DNA replication was slower, consistent with the prolonged S phase observed in these conditions
The results reported here demonstrate that (i) no topoisomerase II (topo II) activity is required during S phase to promote a normal rate of DNA replication; (ii) trapping topo IIaDNA clamps by ICRF-193 during nuclear assembly, but not later, slows down the activation of origin clusters and the progression of replication forks in a checkpoint-independent manner; (iii) topo IIa acts in an ICRF-193-resistant manner during nuclear assembly to negatively regulate MCM loading and to establish a temporal program of origin cluster activation during the subsequent S phase
Summary
Eukaryotic DNA replication starts at multiple sites called replication origins [1]. Origins tend to fire coordinately in clusters of 5–10 origins that are activated at different times through S phase [2]. A replication timing program exists in Xenopus egg extracts where no transcription is taking place [5] Despite their importance for embryonic development and genome stability, the mechanisms controlling the temporal programme of genome replication and the length of S phase have remained elusive. Recent experiments suggest that competition for limiting replication factors establishes the timing and efficiency of origin firing in fission yeast [6,7], budding yeast [8,9] and mammalian cells [10]. The Rif protein in fission yeast [12] and the Forkhead transcription factors Fkh1/ 2 in budding yeast [13] act as global regulators of origin firing time by affecting not pre-RC assembly but the loading of Cdc, a cofactor of the MCM replicative helicase. Rif regulates replication timing domains in human [14] and mouse [15] cells
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