Chromosome replication in cell-free systems from Xenopus eggs

Abstract

Cell-free systems from eggs of the frog Xenopus laevis are able to perform most of the acts of eukaryotic chromosome replication in vitro . This now includes the crucial regulatory step of initiation, which had only been achieved for viral systems previously. Purified DNA or nuclei are able to initiate and complete semiconservation replication in egg extracts in vitro (Blow & Laskey, Cell 47, 557-587 (1986)). Replication does not require specialized DNA sequences either in vitro or in microinjected eggs, but in both systems large templates replicate more efficiently than small templates. In some cases replication can re-initiate, excluding the possibility that replication is primed by preexisting primers in the template preparations. When nuclei are replicated in vitro , only one round of replication is observed in a single incubation resembling the single round of replication observed for purified DNA after micro-injection. The mechanism that prevents re-initiation of replication within a single cell cycle is discussed and certain models are eliminated. Nucleosome assembly from histones and DNA has also been studied in cell-free systems from Xenopus eggs. Fractionation has led to the identification of two acidic proteins called nucleoplasmin and N1, which bind histones and transfer them to DNA. The sequences of both proteins have been determined by cDNA cloning and sequencing. Both proteins are found as complexes with histones in eggs.

Dynamic interaction of Y RNAs with chromatin and initiation proteins during human DNA replication

“…We have found that hY RNAs associate with chromatin before the initiation of DNA replication, interact with proteins of the pre-replication complex, and dissociate from sites of replicated DNA. These data suggest a molecular mechanism for Y RNA function during DNA replication, which is consistent with the original licensing factor model for once-per-cell-cycle control of eukaryotic DNA replication (Laskey et al, 1981;Blow et al, 1987).…”

“…Their binding to unreplicated chromatin during G1 and S phase discriminates replicated from unreplicated sections of the genome and ensures that chromosomal DNA is replicated once and only once during each cell cycle (Blow and Dutta, 2005;DePamphilis et al, 2006;Aladjem, 2007). Y RNAs are thus consistent with the 'activator' factor as proposed in the original model of replication licensing for once-per-cell-cycle control of eukaryotic DNA replication (Laskey et al, 1981;Blow et al, 1987).…”

An ATM- and ATR-dependent checkpoint inactivates spindle assembly by targeting CEP63

“…A particularly useful quality of Xenopus egg extract is the synchronous progression through multiple rounds of the cell cycle, driven by oscillations in Cdk activities (Murray, 1991). In addition, Xenopus egg extract has been shown to recapitulate cellular processes including semiconservative DNA replication and mitotic spindle assembly (Blow and Laskey 1986;Blow et al, 1987;Lohka and Maller 1985). This extract system is also enhanced by the ease of protein depletions and reconstitutions.…”

Cell‐cycle‐dependent regulation of DNA replication and its relevance to cancer pathology

“…Their presence on chromatin during S phase is indispensable for the process of DNA replication and is referred to as chromatin 'licensing' [2,3]. In the licensing process, all the chromatin that needs to be replicated during S phase is marked by bound MCM proteins following cell cycle entry in early G1 phase [1].…”