opt rdg 1 - Introduction In mammalian cell nuclei, DNA...

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Unformatted text preview: Introduction In mammalian cell nuclei, DNA replication takes place at so-called replication foci. Here, proteins involved in DNA replication are assembled into microscopically visible functional complexes (Leonhardt et al., 2000b). The proliferating cell nuclear antigen (PCNA) is a central component of these complexes (Wyman and Botchan, 1995). At replication foci, nucleotide analogues like bromodeoxyuridine (Berezney et al., 1995b; Gratzner, 1982) or fluorescently labelled nucleotides (Manders et al., 1999; Pepperkok and Ansorge, 1995; Zink et al., 1998) are incorporated into nascent DNA, which gives rise to typical focal DNA labelling patterns in pulse-labelling experiments. A large percentage of individually labelled DNA foci correspond to replicon clusters rather than to single replicons (Berezney et al., 2000; Jackson and Pombo, 1998). The average DNA content of a replicon cluster organized into a microscopically visible focus is about 1 megabase pair (Mb), although the DNA content of individual foci may vary considerably (Berezney et al., 2000). It has been observed that focal DNA labelling patterns obtained after pulse-labelling with nucleotide analogues were maintained at subsequent cell cycle stages and cell cycles (Berezney et al., 1995b; Jackson and Pombo, 1998; Ma et al., 1998; Sparvoli et al., 1994; Zink et al., 1999; Zink et al., 1998). Based on this observation, it has been suggested that DNA might be organized into stable aggregates equivalent to replication foci. One way to prove that microscopically visible foci represent such stable DNA aggregates is to show that individual DNA foci labelled during the first S phase convert into replication foci again at subsequent S phases. Corresponding experiments have been performed with cells synchronized at the G1/S border, which have been pulse- labelled with two different nucleotide analogues at the very beginning of two consecutive S phases (Jackson and Pombo, 1998; Ma et al., 1998). The results reveal a high degree of colocalization at nuclear sites first initiated during S phase (Jackson and Pombo, 1998; Ma et al., 1998). These findings indicate the presence of stable DNA aggregates corresponding to the earliest replicating foci, which stably maintain their replication timing. Nevertheless, it has been difficult to show exact colocalization at the level of individual foci and the question remains whether replication timing is stably maintained for the chromosomal regions replicating later during S phase. Given these uncertainties, the question whether chromosomes are organized into stable subunits equivalent to replication foci, that stably maintain their replication timing, remains controversially discussed....
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This note was uploaded on 10/07/2009 for the course GEN 409 taught by Professor Linda during the Fall '09 term at Iowa State.

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opt rdg 1 - Introduction In mammalian cell nuclei, DNA...

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