Wapl is a new regulator of sister chromatid resolution and promotes release of cohesin from chromosomes by directly interacting with its regulatory subunits.

PrefaceCorrect regulation of the replication licensing system ensures that chromosomal DNA is precisely duplicated in each cell division cycle. Licensing proteins are inappropriately expressed at an early stage of tumorigenesis in a wide variety of cancers. Here we discuss evidence that misregulation of replication licensing is a consequence of oncogene-induced cell proliferation. This misregulation can cause either under-or over-replication of chromosomal DNA, and could explain the genetic instability commonly seen in cancer cells. Overview of the replication licensing systemTo prevent the occurrence of potentially cancer-causing alterations to the genome, it is crucial that chromosomal DNA is precisely duplicated during S phase of the cell division cycle. Because of the large size of animal chromosomes, it is necessary for them to be replicated by thousands of replication forks initiated at replication origins scattered throughout the genome. The activity of these replication origins must be carefully regulated to ensure precise chromosome duplication. If too few replication origins are active, there is a danger that the chromosomal DNA will not be completely replicated during S phase, which can potentially lead to DNA strand breaks and gross chromosomal rearrangements in surviving daughter cells. It is equally important to ensure that no replication origin initiates more than once in each cell cycle, as this would lead to re-duplication (amplification) of the DNA in the vicinity of the over-firing origin and other consequent chromosomal rearrangements.Correct regulation of the replication licensing system is responsible for ensuring the proper regulation of replication origins during cell cycle progression1-3. Origin licensing, which occurs prior to S phase in late mitosis and early G1 (Figure 1) involves the stable loading of the Mcm2-7 replication proteins onto DNA at replication origins. Mcm2-7 are essential replication fork proteins which probably provide the helicase activity to unwind the template DNA ahead of the fork4,5. Because of this behaviour, Mcm2-7 complexes move away from each origin as it initiates, thereby leaving the origin in an unlicensed state. In the absence of DNA-bound Mcm2-7, replication origins cannot initiate (Figure 1). Therefore to prevent replicated origins from being re-licensed (and hence re-replicated) after they have initiated, it is necessary for the replication licensing system to be shut down prior to entry into S phase.Replication licensing requires at least 3 proteins in addition to Mcm2-7: the origin recognition complex (ORC), Cdc6 and Cdt11-3,6. ORC first binds to DNA at replication origins and recruits Cdc6 and Cdt1. These proteins then act together to load Mcm2-7 onto DNA, plausibly by opening up the ring-shaped Mcm2-7 complex and clamping it around the DNA7. Consistent with this model, once Mcm2-7 have been loaded onto the DNA, ORC, Cdc6 and Cdt1 are no longer required for Mcm2-7 to remain bound and for the origin to * Author for correspondence: email j.j.blow@dunde...

When Xenopus eggs and egg extracts replicate DNA, replication origins are positioned randomly with respect to DNA sequence. However, a completely random distribution of origins would generate some unacceptably large interorigin distances. We have investigated the distribution of replication origins in Xenopus sperm nuclei replicating in Xenopus egg extract. Replicating DNA was labeled with [3H]thymidine or bromodeoxyuridine and the geometry of labeled sites on spread DNA was examined. Most origins were spaced 5–15 kb apart. This regular distribution provides an explanation for how complete chromosome replication can be ensured although origins are positioned randomly with respect to DNA sequence. Origins were grouped into small clusters (typically containing 5–10 replicons) that fired at approximately the same time, with different clusters being activated at different times in S phase. This suggests that a temporal program of origin firing similar to that seen in somatic cells also exists in the Xenopus embryo. When the quantity of origin recognition complexes (ORCs) on the chromatin was restricted, the average interorigin distance increased, and the number of origins in each cluster decreased. This suggests that the binding of ORCs to chromatin determines the regular spacing of origins in this system.

The cohesin complex is a central player in sister chromatid cohesion, a process that ensures the faithful segregation of chromosomes in mitosis and meiosis. Previous genetic studies in yeast show that Scc2/Mis4, a HEAT-repeat-containing protein, is required for the loading of cohesin onto chromatin. In this study, we have identified two isoforms of Scc2 in humans and Xenopus (termed Scc2A and Scc2B), which are encoded by a single gene but have different carboxyl termini created by alternative splicing. Both Scc2A and Scc2B bind to chromatin concomitant with cohesin during DNA replication in Xenopus egg extracts. Simultaneous immunodepletion of Scc2A and Scc2B from the extracts impairs the association of cohesin with chromatin, leading to severe defects in sister chromatid pairing in the subsequent mitosis. The loading of Scc2 onto chromatin is inhibited in extracts treated with geminin but not with p21(CIP1), suggesting that this step depends on replication licensing but not on the initiation of DNA replication. Upon mitotic entry, Scc2 is removed from chromatin through a mechanism that requires cdc2 but not aurora B or polo-like kinase. Our results suggest that vertebrate Scc2 couples replication licensing to sister chromatid cohesion by facilitating the loading of cohesin onto chromatin.