Interactions of USF and Ku antigen with a human DNA region containing a replication origin

Tóth, Éva Csordés; Marusic, Lidija; Ochem, Alexander; Patthy, Andrés; Pongor, Séndor; Giacca, Mauro; Falaschi, Arturo

doi:10.1093/nar/21.14.3257

Cited by 39 publications

(38 citation statements)

References 43 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…These reactions were repeated for 30 One-third of the PCR products was blotted to nitrocellulose filters with a Minifold II slot-blot set (Schleicher & Schuell) and hybridized with 32P-end-labeled oligonucleotide probes at 52°C for 16 h in a buffer containing 6x SSC (1 x SSC contains 0.3 M NaCl and 0.03 M sodium citrate), 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, and 0.1% SDS. The filters were washed at room temperature twice with 6x SSC-0.1% SDS for 10 min and twice with 0.1x SSC-0.1% SDS for 10 min and subjected to autoradiography.…”

Section: Materuils and Methodsmentioning

confidence: 99%

A novel DNA replication origin identified in the human heat shock protein 70 gene promoter.

Taira¹,

Iguchi-Ariga²,

Ariga³

1994

Mol. Cell. Biol.

View full text Add to dashboard Cite

A general and sensitive method for the mapping of initiation sites of DNA replication in vivo, developed by Vassilev and Johnson, has revealed replication origins in the region of simian virus 40 oyi, in the regions upstream from the human c-myc gene and downstream from the Chinese hamster dihydrofolate reductase gene, and in the enhancer region of the mouse immunoglobulin heavy-chain gene. Here we report that the region containing the promoter of the human heat shock protein 70 (hsp7O) gene was identified as a DNA replication origin in HeLa cells by this method. Several segments of the region were cloned into pUC19 and examined for autonomously replicating sequence (ARS) activity. The plasmids carrying the segments replicated episomally and semiconservatively when transfected into HeLa cells. In order to study DNA replication in higher eukaryotes, identification and mapping of DNA replication origins in chromosomes are required. Although origins in mammalian chromosomes still remain enigmatic, several mammalian sequences which promote autonomous replication of plasmids in mammalian cells have been reported. A method for mapping origins that work in vivo (an origin mapping method) has been developed (32). This method has the following advantages: it avoids the use of metabolic inhibitors, does not require synchronized cells, and can detect replication origins even in single-copy sequences (31). This is thus at present the most advanced technique to identify replication initiation sites under the conditions reflecting living cells. Several replication origins were determined by this method in mammalian genes, including the region upstream from the human c-myc gene (33), the region downstream from the Chinese hamster dihydrofolate reductase gene (31), and the enhancer region of the mouse immunoglobulin heavy-chain gene (3,15).We have previously shown that a protein complex including the c-myc protein (or proteins with c-myc protein-like epitopes) specifically binds to two sites in the promoter region of the human heat shock protein 70 (hsp70) gene (Fig. 1) MATERUILS AND METHODSIn vivo mapping of DNA replication origin. All procedures were performed as previously described (31-33), except that HeLa cells were used. HeLa cells were grown in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum. For each experiment, 10 dishes (diameter, 15 cm) containing 50 to 60% confluent cells were labeled with 5-bromodeoxyuridine (BrdU) for 15 min at 37°C. Total DNA was extracted from the cells after three washes with cold phosphate-buffered saline. The DNA was denatured with NaOH (final concentration, 0.2 M) and applied on 5 to 20% (wt/vol) linear sucrose gradients containing 0.2 M NaOH and 2 mM EDTA over a 60% sucrose cushion. The gradients were centrifuged at 24,000 rpm for 18 h at 15°C in a Hitachi SRP28S rotor. The gradients were divided into 15 fractions. The top fraction with the shortest DNA fragments was discarded, because the fragments were too short to serve as a template in the following PCR amplif...

show abstract

Section: Materuils and Methodsmentioning

confidence: 99%

A novel DNA replication origin identified in the human heat shock protein 70 gene promoter.

Taira¹,

Iguchi-Ariga²,

Ariga³

1994

Mol. Cell. Biol.

View full text Add to dashboard Cite

show abstract

“…2B) confine the replication origin within a 474-bp region roughly corresponding to the nontranscribed spacer between two transcription units arranged head to tail (22). This region was shown to contain a strong promoter for the downstream transcription unit; a key feature ofthis promoter is the presence of a basic helix-loop-helix protein binding motif that was shown to bind in vitro to the USF/MLTF protein (23); interestingly, such motif also fits the binding consensus for the Myc/Max complex (24), whose function in regulating cell proliferation is suggested by several studies (25) [incidentally, the c-myc gene domain is amplified in HL-60 cells (26)]. An evolutionary conserved (A+T)-rich region (22) can be envisaged in the proximity of the origin, as is the case for well-defined replication origins of different organisms (27)(28)(29).…”

Section: ~-F )W Lmentioning

confidence: 99%

Fine mapping of a replication origin of human DNA.

Giacca

Zentilin

Norio

et al. 1994

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

179

202

View full text Add to dashboard Cite

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.

show abstract

“…In contrast, their wild-type cells continued to synthesize DNA and were able to promptly repair the DNA breaks, suggesting a role of DNA-PK in immediately repairing DNA breaks following deceleration of DNA replication. Altogether these results suggest that, in addition to its role in repairing dsDNA breaks that occur during replication fork progression (Shimura et al, 2007), Ku is also involved in the prevention of DNA breaks caused by replication fork collapse by: i) binding onto DNA replication origins at G1 phase (Novac et al, 2001;Ruiz et al, 1999), recruiting the DNA replication machinery (Rampakakis et al, 2008;Rampakakis and Zannis-Hadjopoulos, 2009;Sibani et al, 2005b) and ensuring genomic duplication and maintenance (Toth et al, 1993) (progression into S phase without the appropriate number of activated replication origins would lead to an increase of the average replicon size, resulting in stalled replication forks and chromosomal instability (Ekholm-Reed et al, 2004;Tanaka and Diffley, 2002a)); and ii) maintaining the DNA polymerase processivity factor PCNA on chromatin following ionizing radiation (Park et al, 2004).…”

Section: Ku and Mammalian Dna Replicationmentioning

confidence: 95%

“…Furthermore, K u w a s i d e n t i f i e d a s p a r t o f a h u m a n p r o t ein initiation complex, important for the replication of Kaposi's sarcoma associated HSV (KHSV) (Wang et al, 2008). Ku is an origin binding protein, binding to several replication origins, among them the adenovirus type 2 origin (de Vries et al, 1989), the Herpes Simplex Virus Type 1 (HSV1) origin (Murata et al, 2004), the B48 human origin (Toth et al, 1993), the mammalian replication origin consensus sequence, A3/4 Ruiz et al, 1999), the Chinese hamster dihydrofolate reductase (DHFR) replication origin, ori, and the monkey replication origins ors8 and ors12 (Novac et al, 2001), as well as the human origins lamin B2, -globin, c-myc (Sibani et al, 2005a, b) and dnmt1 (DNA-methyltransferase) (Araujo et al, 1998). Ku was shown to associate in vivo with replication origins in a cell cycle dependent manner (Novac et al, 2001;Ruiz et al, 1999;Sibani et al, 2005a) and its differential binding to DNA is a determining factor in its involvement in DNA replication, exhibiting distinct origin DNA binding properties from its association with DNA ends or other internal DNA sequences (Schild-Poulter et al, 2003).…”

Section: Ku and Mammalian Dna Replicationmentioning

confidence: 99%