Characteristics of DNA replication in isolated nuclei initiated by an aprotinin‐binding protein

Abstract

Isolated cell nuclei were used as the source of template DNA to investigate the role of a cytosolic aprotinin-binding protein (ADR) in the initiation of eukaryotic DNA replication. Computerized image cytometry demonstrated that the DNA content of individual nuclei increased significantly following incubation with ADR-containing preparations, and the extent of DNA synthesis is consistent with that allowed by the limiting concentration of dTTP. Thus, dTTP incorporation into isolated nuclei represents DNA synthesis and not parent strand repair. We found that dTTP incorporation into the isolated nuclei is dependent on DNA polymerase alpha (a principal polymerase in DNA replication) but that DNA polymerase beta (a principal polymerase in DNA repair processes) does not play a significant role in this system. Finally, neither aprotinin nor a previously described cytosolic ADR inhibitor can block the replication of nuclease-treated calf thymus DNA, while both strongly inhibit replication of DNA in isolated nuclei. This result, coupled with the relative ineffectiveness of nuclease-treated DNA compared with nuclear DNA to serve as a replicative template in this assay, argues against a significant contribution from repair or synthesis which initiates at a site of DNA damage. These data indicate that ADR-mediated incorporation of 3H-dTTP into isolated nuclei results from DNA replicative processes that are directly relevant to in vivo S phase events.

oriGNAI3: A narrow zone of preferential replication initiation in mammalian cells identified by 2D gel and competitive PCR replicon mapping techniques

“…A remarkable conclusion of this study is that a narrow zone of active replication initiation, referred to as oriGNAI3, has been identified both by a 2D gel electrophoresis and a competitive PCR technique. This differs from the data obtained for the Chinese hamster DHFR and human rDNA loci, in which broad zones of initiation, at least 30 kb in length, were disclosed by 2D gels (13,14), whereas narrow regions were observed with other replicon mapping techniques (5,6,8,(16)(17)(18)(19).…”

“…While precise mapping of this preferential region was not attempted, Little et al observed that the strongest bubble arc signals were located in an ∼8 kb long region of the broad initiation zone (14). Interestingly, alternative techniques later mapped a 1.5 kb long (17) or a 4 kb long (18) initiation region within the preferential region identified by 2D gels (14). Thus oriGNAI3 and the origin at the rDNA locus appear to share similar properties, if one considers either that infrequent initiation events were below the threshold of detection in our 2D NA study or that the degree of preferential initiation is higher at the initation region described here.…”

“…Indeed, when 2D techniques were applied to analysis of the Chinese hamster dihydrofolate reductase gene (DHFR) origin region, replication was found to initiate anywhere within 30-60 kb (13,15), whereas several other techniques suggested the existence of two discrete sites of initiation (5,6,16), one of which was circumscribed to only 450 bp (8). Likewise, 2D gel analysis of initiation at the human rDNA locus identified a 30 kb long broad zone of replication initiation (14), but more restricted sites of initiation were found with several other techniques (17)(18)(19). The reasons for such contradictions are poorly understood: one tentative explanation for this is that 2D gels detect all initiation events, even those occuring in regions firing very rarely, whereas techniques such as quantitative PCR (3) or Okazaki fragment polarity (8) detect only the region(s) where most initiation events occur, because they measure signal-to-noise ratios.…”

Enhanced flexibility and aphidicolin-induced DNA breaks near mammalian replication origins: implications for replicon mapping and chromosome fragility

“…sequences encoding a 13-kb primary transcript and a 30 kb segment referred to as the non-transcribed spacer (NTS). Replication analyses suggest that replication initiation occurs in the NTS, preferentially in a 5 kb-long region upstream, and a 2 kb-long region downstream of the rRNA genes (51)(52)(53)(54)(55). Flexibility analysis shows that all peaks map exclusively within the NTS, with two and three peaks respectively located within the initiation region downstream or upstream the genes (Fig.…”