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

Abstract

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...

SV40 T antigen interacts with Nbs1 to disrupt DNA replication control

“…Expression of wild-type Nbs1 in NBS-deficient cells reduced the percentage of cells with more than four dots by more than fourfold ( Table 1A). Probes that recognize DNA regions containing known sites of DNA synthesis initiation near the HSP70 and Lamin B2 genes (Taira et al 1994;Abdurashidova et al 2000) yielded similar results (data not shown). Almost all NBS-deficient cells (GM07166) were characterized by normal chromosomal numbers.…”

Deregulated c-Myc prematurely recruits both Type I and II CD95/Fas apoptotic pathways associated with terminal myeloid differentiation

“…When the canonical cmyc-responsive element (CACGTG) of the ornithine decarboxylase (a myc target gene) promoter was replaced by the non-canonical element, the modi®ed promoter was even more responsive to c-myc. A similar or identical non-canonical myc-responsive sequence has also been identi®ed in the promoters of other genes such as HSP70 (Taira et al, 1994), c-myc itself (Ariga et al, 1989), and in the EFII enhancer of the Rous sarcoma virus LTR (Hann et al, 1994). Interestingly, the physiological importance of this myc-responsive element is further supported by the observations that the non-canonical sequence exhibits binding to in vivoderived Myc protein (Hann et al, 1994) and it e ciently binds to Myc ± Max heterodimers, but not to either Myc or Max homodimers (Hann et al, 1994;Kasibhatla et al, 2000).…”

Replication-associated strand asymmetries in mammalian genomes: Toward detection of replication origins

“…Nine replication origins were examined: namely, those situated near the genes MCM4 (13), HSPA4 (14), TOP1 (15), MYC (16), SCA-7 (17), AR (17), DNMT1 (18), LaminB2 (19), and ␤-globin (20).…”