Major DNA replication initiation sites in thec-myc locus in human cells

Tao, Liang; Dong, Zhifeng; Leffak, Michael; Zannis‐Hadjopoulos, Maria; Price, Gerald B.

doi:10.1002/1097-4644(20000901)78:3<442::aid-jcb9>3.0.co;2-1

Cited by 64 publications

(95 citation statements)

References 51 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To distinguish between these two possibilities, we analyzed the activities of two well-characterized replication origins, namely the lamin B2 (Giacca et al, 1994; Abdurashidova et al, 2000) and cmyc (Tao et al, 2000) (Fig. 3A), by measuring the nascent-strand abundance at these chromosomal regions, as previously described (Giacca et al, 1997;Tao et al, 2000).…”

Section: Journal Of Cell Science 592mentioning

confidence: 99%

“…Isolation of genomic and nascent-strand DNA Isolation of nascent-strand DNA was performed using the exonuclease method as previously described (Giacca et al, 1997;Tao et al, 2000). Briefly, the cells were washed twice with PBS and lysed in Hirt's lysis buffer (50 mM Tris-HCl, pH 8.0, 0.6 M NaCl, 1 mM EDTA and 0.5% SDS) (Hirt, 1967).…”

Section: Journal Of Cell Science 121 (5)mentioning

confidence: 99%

See 1 more Smart Citation

Ku is involved in cell growth, DNA replication and G1-S transition

Rampakakis

Paola

Zannis‐Hadjopoulos

2008

Journal of Cell Science

Self Cite

View full text Add to dashboard Cite

The Ku protein (Ku70-Ku80) is involved in various genomemaintenance processes such as DNA replication and repair, telomere maintenance, and chromosomal stability. We previously found that Ku80 is implicated in the loading of members of the pre-replicative complex (pre-RC) onto replication origins. Here, we report that acute reduction of Ku80 to 10% of its normal levels leads to impaired DNA replication and activation of a replication stress checkpoint. In the absence of Ku80, decreased levels of the initiator proteins Orc1 and Orc6 as well as reduced chromatin binding of Orc1, Orc4 and Cdc45 were observed, leading to decreased origin firing, whereas Orc2 and Orc3 were unaffected. Prolonged perturbation of DNA replication caused the block of cell-cycle progression in late G1 phase with low Cdk2 activity due to increased p21 expression and decreased Cdc25A and Cdk2 levels. The data suggest the interplay between the DNA-replication and cell-cycle machineries and shed light on a new role of Ku in G1-S transition.Key words: DNA replication, Ku, Cell growth, Nascent DNA, Replication stress checkpoint Journal of Cell Science 591 Ku in replication and cell cycle from 30 hours to 58.8 hours. This growth defect was independent of DNA damage and apoptosis, but reliant on impaired chromatin loading of replication factors and origin activation. Ku80-deficient cells were blocked in late G1 phase and had increased levels of cyclin E but decreased Cdk2 activity, suggesting a new role of Ku80 in G1-S progression. Results Knockdown of Ku80 leads to reduced cell proliferationInitial characterization of human Ku80-haploinsufficient cells (HCT116) revealed a defect in cell proliferation (doubling time 20.5 hours compared with 17.7 hours for the Ku80 wild-type cells) . A second round of gene targeting generated Ku80-null cells that had a more severe growth defect and underwent massive apoptosis, signifying differences between the human and the murine knockout cells, which are viable. To verify the validity of these results in different cell types, we knocked-down Ku80 levels in HeLa cells, using an RNAi approach, and analyzed the effect on cellular proliferation. At 96 hours post-transfection of an siRNA targeting Ku80 mRNA, the protein levels decreased to approximately 10% of normal levels, as measured both by western blot analysis and immunofluorescence (Fig. 1A,B). The decrease in the levels of Ku protein was gradual over time (Fig. 1A,C), thus providing us with a system to study Ku80 deficiency mimicking the generation of Ku80-knockout cells. Consistent with the growth defect observed in the human heterozygous colorectal cancer cells (HCT116 Ku80 +/-), the cervical cancer cells (HeLa) depleted of Ku80 also displayed a proliferation defect (Fig. 1D, compare panels vii, viii, ix with i, ii, iii and iv, v, vi), with their doubling time increasing twofold compared with cells transfected with a dsRNA with a scrambled sequence (hereafter referred to as scr-siRNA) or cells treated with the transfection agent (hereafter referred to as c...

show abstract

Section: Journal Of Cell Science 592mentioning

confidence: 99%

Section: Journal Of Cell Science 121 (5)mentioning

confidence: 99%

Ku is involved in cell growth, DNA replication and G1-S transition

Rampakakis

Paola

Zannis‐Hadjopoulos

2008

Journal of Cell Science

Self Cite

View full text Add to dashboard Cite

show abstract

“…PCR Analysis for Nascent Strand Abundance DNA replication initiation frequencies at the human c-Myc and ß-globin locus were determined based on short nascent DNA abundance (Aladjem et al, 1998;Kamath and Leffak, 2001;Malott and Leffak, 1999;Tao et al, 2000). The 1-2 kb fraction of nascent DNA was Isolated by alkaline gel lysis of cells In logarithmic growth and quantitated by slot blot hybridization.…”

Section: Methodsmentioning

confidence: 99%

“…To examine the initiation activity at chromosomal sites, the abundance of short nascent DNA strands was quantitated by PCR at two loci containing known origins of DNA replication, c-myc and ß-globin ( Figure 4). In both of these regions, the abundance and size distribution of DNA nascent strands suggests that there are multiple, nonrandom sites of replication initiation (Kamath and Leffak, 2001;Malott and Leffak, 1999;Tao et al, 2000). The pattern of nascent strand abundance at these specific initiation sites was unchanged between +/+ and A/-cells, suggesting that at least for these two origins, replication initiated normally in the A/-cells that do enter S phase.…”

Section: Replication From Orip Of Epstein-barr Virusmentioning

confidence: 99%

Mapping Critical DNA Sequence Elements Required for Amplification of erbB2 in Breast Cancer

Machida¹,

Dutta²

2002

View full text Add to dashboard Cite

Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Washington Headquarters Services, Directorate for Information Operations and Reports, 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA 22202-4302, and FUNDING NUMBERSDAMD17-00-1-0166 PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES)Brigham and Women's Hospital Boston, MA 02115 E-Mail:ymachida@rics. bwh.Harvard.edu Recent studies show that 80-90% of breast carcinomas are associated with gene amplification. Each amplicon most likely has an origin of replication. In Drosophila, DmORC (Origin Recognition Complex) binds to specific DNA sequences and this binding is essential for chorion gene amplification. To study the implications of replication initiator proteins to gene amplification, we characterized two proteins required for DNA replication (ORC2 and McmlO). We created hypomorphic mutation in the ORC2 gene of a cancer cell line through homologous recombination. We found that these cells failed to support the replication of an extrachromosomal plasmid bearing the oriP replicator of Epstein Barr virus (EBV). Geminin, an inhibitor of replication initiation complex, inhibited replication of this episome. The result identifies a novel means by which to cure cancer with gene amplification where the amplicons are carried as episomes. We also report that Xenopus McmlO (XMcmlO) is not required for origin binding of XMcm2-7. Instead, the chromatin binding of XMcmlO at the onset of DNA replication requires chromatin-bound XMcm2-7, and it is independent of Cdk2 and Cdc7. In the absence of XMcmlO, XCdc45 binding, XRPA binding, and initiation-dependent plasmid supercoiling are blocked. Therefore, XMcmlO performs its function after pre-RC assembly and before origin unwinding. PERFORMING ORGANIZATION REPORT NUMBER SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES)U IntroductionRecent studies show that 80-90% of breast carcinomas are associated with gene amplification (1,2). Unfortunately, no effort has been put so far to study the mechanism of gene amplification from a view of DNA replication. Origins of replication are first recognized and bound by a sixsubunit origin recognition complex (ORC), which was first identified in yeast. In Drosophila, DmORC (Origin Recognition Complex) binds to specific DNA sequences and this binding is essential for chorion gene amplification. One certain way to diminish the amount of amplification is to inhibit the initiation of replication in the double minute chromosome. To study the implications of replication initiator proteins to gene amplification, we characterized two proteins required for DNA replication (ORC2 and Mem 10). We also report that inh...

show abstract

“…Extensive mapping of replication start sites in mammalian chromosomes has revealed that replication at most, but not all, loci begins at a few high frequency start sites contained within a broad zone of initiation (83,63,82,37,4,59, and other studies reviewed in 40,24,12). One of the most thoroughly mapped high frequency initiation Altaian, A.L.…”

Section: Introductionmentioning

confidence: 99%

Genetic Analysis of a Mammalian Chromosomal Origin of Replication

Altman¹

2002

View full text Add to dashboard Cite

Major DNA replication initiation sites in thec-myc locus in human cells

Cited by 64 publications

References 51 publications

Ku is involved in cell growth, DNA replication and G1-S transition

Ku is involved in cell growth, DNA replication and G1-S transition

Mapping Critical DNA Sequence Elements Required for Amplification of erbB2 in Breast Cancer

Genetic Analysis of a Mammalian Chromosomal Origin of Replication

Contact Info

Product

Resources

About