Interaction of chromosomal proteins with BrdU substituted DNA as determined by chromatin-DNA competition

Bick, Michael D.; Devine, Evelyn A.

doi:10.1093/nar/4.11.3687

Cited by 35 publications

(6 citation statements)

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“…Suggested mechanisms underlying the effects of BrdU on gene expression and cell phenotype include DNA mutations, alterations in DNA methylation, altered interactions with DNA-binding proteins and reversal of the remodelling of regulatory DNA into specific chromatin architecture that occurs during cancer. BrdU-substituted DNA has an increased capacity to bind histones [28], nonhistone proteins [29] and chromosomal proteins [30], and might become concentrated in repetitive DNA nucleotide sequences [31]; such observations have, however, not yet been linked to downstream effects. The Ayusawa group speculates that BrdU incorporation into AT-rich inactive chromatins, such as in scaffold/ nuclear matrix attachment region (S/MAR) sequences, results in altered interactions with AT-binding ligands and leads to changes in gene expression consistent with senescence [32,33].…”

Section: Introductionmentioning

confidence: 99%

5-Bromo-2-deoxyuridine activates DNA damage signalling responses and induces a senescence-like phenotype in p16-null lung cancer cells

Masterson

O’Dea

2007

Anti-Cancer Drugs

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5-Bromo-2-deoxyuridine (BrdU) is a thymidine analogue that is incorporated into replicating DNA. Although originally designed as a chemotherapeutic agent, sublethal concentrations of BrdU have long been known to alter the growth and phenotype of a wide range of cell types. Mechanisms underlying these BrdU-mediated effects remain unknown, however. We have characterized the effects of BrdU on A549 lung cancer cells by examining DNA damage responses, cell cycle effects and phenotypic changes. A549 cells express wild-type p53, but are p16-null. Sublethal concentrations of BrdU evoke a DNA damage response in these cells that involves the activation of Chk1, Chk2 and p53. Increased numbers of enlarged nuclei and multinucleated cells are evident in the treated populations. Cell cycle inhibition occurs, resulting in reduced proliferation and accumulation of cells in the S, G 2 /M and G 0 phases. BrdU induces an early inhibition of p21 expression that coincides with nuclear localization of proliferating cell nuclear antigen. Subsequently, p21 levels increase, whereas proliferating cell nuclear antigen levels decrease compared with control cells. Upregulation of p27 and p57 expression also occurs. By day 7 of exposure to BrdU, treated cells acquire a senescent-like phenotype with an increase in cell size, granularity and bgalactosidase activity. We conclude that BrdU induces a DNA damage response in A549 cells, which results in reduced proliferation mitotic exit and phenotypic changes that resemble senescence.

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Section: Introductionmentioning

confidence: 99%

5-Bromo-2-deoxyuridine activates DNA damage signalling responses and induces a senescence-like phenotype in p16-null lung cancer cells

Masterson

O’Dea

2007

Anti-Cancer Drugs

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“…Br-DNA interacts with structural and regulatory proteins in a way different from that displayed by native DNA [ 1,13,18]. Direct assessment of the interaction of Br-DNA irradiated with UVA under anoxia with cycle regulatory proteins has not been accomplished.…”

Section: Discussionmentioning

confidence: 99%

Early and late replicating DNA involved in the G₁ to S transition in Allium cepa L meristematic cells

González‐Fernández

Aller

Sans

et al. 1992

Biology of the Cell

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The involvement of portions of the genome replicated at different times of the S period in the regulation of the G1 to S transition was analyzed in Allium cepa L meristem cells. For this, DNA bromosubstitution confined to discrete portions of a previous S period followed by anoxic UVA irradiation (300-400 nm light) was performed in synchronous cells. Sequences replicated in late S appeared to be involved in the positive regulation of the initiation of replication. Hence, cells were prevented from initiating replication if irradiated at mid G1 only when the DNA sequences replicated in the last third of the previous S period were bromosubstituted. Cycloheximide-induced inhibition of protein synthesis at late G1 also prevented the G1 to S transition. Sequences replicated in mid S appeared unrelated to any control of the initiation of replication. On the other hand, sequences replicated in the first third of the S period seemed to be involved in the negative regulation of the initiation of replication, since irradiation after previous bromosubstitution of early replicating DNA sequences advanced G1 cells into the next S phase and increased the proliferative fraction of the population. Finally, the simultaneous inactivation of DNA sequences involved in both positive and negative regulation of replication allowed the cells to enter into S.

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“…This substitution replaces the 5-methyl group of thymine with a bromine, which has approximately the same van der Waals radius. Substituted DNA binds many proteins more tightly, including the lac repressor (38), the cyclic AMP receptor protein (39), histones (40), and several nonhistone chromosomal proteins (41). However, the effect on RNA polymerase binding has not been investigated carefully.…”

Section: Discussionmentioning

confidence: 99%

Contacts between Escherichia coli RNA polymerase and thymines in the lac UV5 promoter.

Simpson¹

1979

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

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I have identified those 5 positions of thymines in the lac UV5 promoter that lie close to bound Escherichia coli RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6). Although ultraviolet irradiation of DNA with 5-bromouracil substituted in place of thymine normally cleaves the DNA at the bromouracils, a protein bound to the DNA can perturb these cleavages at those locations at which the protein lies close to the bromine. In the lac promoter most of these contacts lie in three regions. Four contacts lie in the region where transcription initiates; four lie in the "Pribnow box," which is located about 10 base pairs upstream from the initiation site; and three more lie in the "-35 region," located about 35 base pairs upstream from the initiation site. The "Pribnow box" and the "-35 region" are regions whose sequences are partially conserved between promoters and in which most promoter mutations are located; thus, contacts in these two regions probably represent sites of sequence-specific recognition by RNA polymerase. Which features of a promoter direct RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) to initiate RNA synthesis? DNA sequence determination of promoters and promoter mutants provides a partial answer to this question. When the sequences of promoters are compared, two regions of prominent homology have been observed: the "Pribnow box" and the "-35 region" located about 10 and 35 base pairs, respectively, upstream from the start site of transcription (1-4). Furthermore, almost all promoter mutations are located within these regions (5-12).Another approach examines different portions of the promoter for their accessibility to various probes in the presence or absence of RNA polymerase. For example, RNA polymerase bound to any of several promoters protects about 40 base pairs of DNA from endonuclease attack (1,2,13,14). Although polymerase in the resulting complex can initiate correctly and synthesize a transcript about 20 bases long, the 40 base pair fragment does not rebind the polymerase, showing that this fragment lacks some information required for promoter recognition. And, in fact, enzymatic digestion with exonuclease (15) or restriction endonucleases (5,9,(16)(17)(18)(19) suggests that roughly 65 base pairs of DNA are required and that the approximate boundaries of a promoter are located about 45 base pairs upstream and 20 base pairs downstream from the site of initiation. A finer probe is the chemical attack on DNA by dimethyl sulfate (20), which gives a more detailed picture of the interaction because the chemical probe is much smaller than the enzymes that degrade DNA. This probe shows that contacts in one promoter extend from the point of initiation to at least 38 base pairs upstream (21). Because the dimethyl sulfate attacks only the N-7 group of guanine and the N-3 group of adenine on double-stranded DNA, other probes with different specificities are needed to get a complete picture of the crucial promoter elements.I have probed...

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Interaction of chromosomal proteins with BrdU substituted DNA as determined by chromatin-DNA competition

Cited by 35 publications

References 17 publications

5-Bromo-2-deoxyuridine activates DNA damage signalling responses and induces a senescence-like phenotype in p16-null lung cancer cells

5-Bromo-2-deoxyuridine activates DNA damage signalling responses and induces a senescence-like phenotype in p16-null lung cancer cells

Early and late replicating DNA involved in the G₁ to S transition in Allium cepa L meristematic cells

Contacts between Escherichia coli RNA polymerase and thymines in the lac UV5 promoter.

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Interaction of chromosomal proteins with BrdU substituted DNA as determined by chromatin-DNA competition

Cited by 35 publications

References 17 publications

5-Bromo-2-deoxyuridine activates DNA damage signalling responses and induces a senescence-like phenotype in p16-null lung cancer cells

5-Bromo-2-deoxyuridine activates DNA damage signalling responses and induces a senescence-like phenotype in p16-null lung cancer cells

Early and late replicating DNA involved in the G1 to S transition in Allium cepa L meristematic cells

Contacts between Escherichia coli RNA polymerase and thymines in the lac UV5 promoter.

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Early and late replicating DNA involved in the G₁ to S transition in Allium cepa L meristematic cells