Replication Origins in XenopusEgg Extract Are 5–15 Kilobases Apart and Are Activated in Clusters That Fire at Different Times

Abstract

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.

Defining replication origin efficiency using DNA fiber assays

“…DNA fiber analyses also revealed that initiation sites are irregularly distributed during early Xenopus development and are activated at different times throughout S phase (Blow et al 2001;Herrick et al 2000). Interestingly, replication dynamics in Xenopus egg extracts is modulated by the ATR checkpoint kinase even in the absence of exogenous replication stress (Marheineke and Hyrien 2004;Shechter et al 2004).…”

Asymmetry of DNA replication fork progression in Werner's syndrome

“…It is possible that stalled forks are resolved in the absence of WRN by an alternative mechanism that may involve recombination at the Holliday junctions formed, possibly accounting for the high rates of recombination in cells lacking functional WRN (Fukuchi et al ., 1989). A third possibility is that persistence of stalled forks in Werner's cells may influence downstream activation of later-firing replication origins (Blow et al ., 2001), perhaps by activating intra-S checkpoints. Any or all of these processes could account for the extension of S phase observed.…”

“…Cells were then washed in PBS, lysed in 1% SDS and spread over a glass surface as described previously (Para & Windle, 1993;Jackson & Pombo, 1998). BrdU-containing sites were visualized following acid denaturation and immunostaining, as described above and by Blow et al (2001). Images were recorded on film and measurements of labelled patches made from 10× projections of negatives.…”

Persistence Length of Chromatin Determines Origin Spacing in Xenopus Early-Embryo DNA Replication: Quantitative Comparisons between Theory and Experiment

“…7,8 In the second scenario ("fixed spacing"), one postulates a mechanism that imposes regularity in the distribution of potential origins, thus preventing the formation of problematic large gaps between origins. 9 In this article, we shall show that consideration of recent experimental results on early embryo Xenopus replication leads to a more nuanced, "intermediate" view that incorporates elements of both scenarios and, more important, suggests a biological picture in which the secondary structure of chromatinlooping in particular-plays an important biological role in DNA replication.…”

“…For example, molecular-combing 10 and direct visual hybridization (DIRVISH) 9,11 techniques can give detailed statistics about numbers and sizes of replicated domains as averaged over the genome, as well as many other related quantities. 11,12 Alternatively, gene-chips have given information about how replication proceeds at specific locations of the genome.…”