A human DNA replication origin: localization and transcriptional characterization

Biamonti, Giuseppe; Perini, Giovanni; Weighardt, Florian; Riva, Silvano; Giacca, Mauro; Norio, Paolo; Zentilin, Lorena; Diviacco, Silvia; Dimitrova, Daniela S.; Falaschi, Arturo

doi:10.1007/bf02451782

Cited by 43 publications

(40 citation statements)

References 38 publications

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“…3 A and B and fit with the presence of a replication start site near the original isolate (B48 marker) for both pulse times and for all different size samples. These data confirm a preliminary mapping performed with a 10-min pulse with the same technique (15). As a control, we quantified in the same sample an unrelated DNA sequence [located within the 3-globin gene (10)] that, as shown in Fig.…”

Section: ~-F )W Lsupporting

confidence: 83%

Fine mapping of a replication origin of human DNA.

Giacca

Zentilin

Norio

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

179

202

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A highly sensitive procedure was developed for the identification of the origin of bidirectional DNA synthesis in single-copy replicons of ammalian cells. The method, which does not require cell synchronization or permeabilization, entails the absolute quantification, by a competitive PCR procedure in newly synthesized DNA samples, of the abundance of neighboring DNA framents distributed along a given genomic region. Terminal differentiation of HL-60 was achieved with retinoic acid and dimethylformamide, as described (17).Transfection. Plasmid pAWTSV (=9 kb), a kind gift of Cesare Vesco (Institute of Cell Biology, Rome), carries the whole simian virus 40 (SV40) genome inserted in the BamHI site of pAT153 (18). Six 10-cm tissue culture plates, containing about 106 COS-1 cells each, were transfected with 10 pg of pAWTSV by the calcium phosphate precipitation technique. After 10 hr of incubation in calcium phosphate solution, cells were extensively washed and fresh medium was added, containing 10 nCi of [14C]thymidine per ml. After 18 hr of incubation, BrdUrd (100 uM final concentration) and[3H]deoxycytidine (1 jAM final concentration) were added.After 1 min of incubation, cells were killed by addition of sodium azide and DNA was extracted as described below.Extrction and Purification of Newly Syntez DNA.Total DNA was extracted, denatured, and size-fractionated by sedimentation through neutral sucrose gradients as described (15).In the experiment involving transfection of plasmid pAW-TSV, DNA (700 /4 final volume) was fractionated on four 5-20%6 (wt/vol) linear sucrose gradients (5 ml each) for 210 min at 200C in a Beckman SW55Ti rotor at 55 krpm; 24 fractions of 200 j4 were collected.In the experiment with synchronized HL-60 cells, DNA (2 ml final volume) was fractionated on eight 5-30% sucrose Abbreviations: DHFR, dihydrofolate reductase; SV40, simian virus 40. tTo whom reprint requests should be addressed. 7119The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Section: ~-F )W Lsupporting

confidence: 83%

Fine mapping of a replication origin of human DNA.

Giacca

Zentilin

Norio

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

179

202

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“…ceding another still uncharacterized gene, provisionally named ppvl (13)(14)(15). The same origin, originally identified in HL-60 cells, was found to be active also in several other human cell types, including activated peripheral lymphocytes (S. Kumar, M.G., G.B., S.R., and A.F., unpublished results).…”

mentioning

confidence: 55%

In vivo protein-DNA interactions at human DNA replication origin.

Dimitrova

Giacca

Demarchi

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

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Protein-DNA interactions were studied in vivo at the region containing a human DNA replication origin, located at the 3' end of the lamin B2 gene and partially overlapping the promoter of another gene, located downstream. DNase I treatment of nuclei isolated from both exponentially growing and nonproliferating HL-60 cells showed that this region has an altered, highly accessible, chromatin structure. High-resolution analysis of protein-DNA interactions in a 600-bp area encompassing the origin was carried out by the in vivo footprinting technique based on the ligation-mediated polymerase chain reaction. In growing HL-60 cells, footprints at sequences homologous to binding sites for known transcription factors (members of the basichelix-loop-helix family, nuclear respiratory factor 1, transcription factor Spl, and upstream binding factor) were detected in the region corresponding to the promoter of the downstream gene. Upon conversion of cells to a nonproliferative state, a reduction in the intensity of these footprints was observed that paralleled the diminished transcriptional activity of the genomic area. In addition to these protections, in close correspondence to the replication initiation site, a prominent footprint was detected that extended over 70 nucleotides on one strand only. This footprint was absent from nonproliferating HL-60 cells, indicating that this specific protein-DNA interaction might be involved in the process of origin activation. ceding another still uncharacterized gene, provisionally named ppvl (13-15). The same origin, originally identified in HL-60 cells, was found to be active also in several other human cell types, including activated peripheral lymphocytes (S. Kumar, M.G., G.B., S.R., and A.F., unpublished results). A map of the 13.7-kb genomic fragment containing the replication origin is shown in Fig. 1A.The finding that the lamin B2 ori is located in a highly transcribed region is not surprising, since numerous reports have implicated transcriptional regulatory elements in the control of DNA replication (16)(17)(18)(19). Moreover, the domain is replicated at the very beginning of the S phase (12), consistent with the well-documented correlation between transcriptionally active genomic regions and early replication.We have now addressed the study of the specific protein-DNA interactions at the lamin B2 ori. To this purpose, we have exploited the in vivo footprinting technique based on the ligation-mediated polymerase chain reaction (LMPCR) (20).In the past few years we have extensively utilized this technique (21), which, in comparison with in vitro binding studies, is particularly suitable for the detection of the protein-DNA interactions actually occurring in the nucleus, since it is sensitive to the accessibility of sites to protein factors and possible epigenetic modification of DNA such as methylation. Furthermore, we have taken advantage of the property of HL-60 myeloid cells to undergo terminal differentiation upon chemical stimulation (22), a process which is accom...

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“…(Note that this is a representative graph; the copy number of all the primer sets used in this study was measured; see Table 3 To assess the quality of nascent DNA samples, the distribution of nascent DNA from an origin region associated with the lamin B2 locus was quantified. This origin was shown to be active in a number of different proliferating human cells (26,27,41,43,44). Nascent DNA was measured at two reference points, one located at the center of the origin (LB2 primer set = peak activity), the other located f4 kb away (LB2C1 primer set = background activity), using the primer sets described by Ladenburger et al (45).…”

Section: Resultsmentioning

confidence: 99%

Differentially Active Origins of DNA Replication in Tumor versus Normal Cells

Paola

Price²,

Zannis‐Hadjopoulos

2006

Cancer Research

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Previously, a degenerate 36 bp human consensus sequence was identified as a determinant of autonomous replication in eukaryotic cells. Random mutagenesis analyses further identified an internal 20 bp of the 36 bp consensus sequence as sufficient for acting as a core origin element. Here, we have located six versions of the 20 bp consensus sequence (20mer) on human chromosome 19q13 over a region spanning f211 kb and tested them for ectopic and in situ replication activity by transient episomal replication assays and nascent DNA strand abundance analyses, respectively. The six versions of the 20mer alone were capable of supporting autonomous replication of their respective plasmids, unlike random genomic sequence of the same length. Furthermore, comparative analyses of the endogenous replication activity of these 20mers at their respective chromosomal sites, in five tumor/ transformed and two normal cell lines, done by in situ chromosomal DNA replication assays, involving preparation of nascent DNA by the L exonuclease method and quantification by real-time PCR, showed that these sites coincided with chromosomal origins of DNA replication in all cell lines. Moreover, a 2-to 3-fold higher origin activity in the tumor/ transformed cells by comparison to the normal cells was observed, suggesting a higher activation of these origins in tumor/transformed cell lines. (Cancer Res 2006; 66(10): 5094-103)

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A human DNA replication origin: localization and transcriptional characterization

Cited by 43 publications

References 38 publications

Fine mapping of a replication origin of human DNA.

Fine mapping of a replication origin of human DNA.

In vivo protein-DNA interactions at human DNA replication origin.

Differentially Active Origins of DNA Replication in Tumor versus Normal Cells

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