Bromodeoxyuridine mutagenesis in mammalian cells is stimulated by thymidine and suppressed by deoxycytidine

Davidson, Richard L.; Kaufman, Elliot R.

doi:10.1038/276722a0

Cited by 77 publications

(31 citation statements)

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“…BrdU-induced mutagenesis is attributed to low levels of dCTP (Hopkins and Goodman 1980;Meuth 1989), which is suppressed by coculture with exogenous cytidine (Meuth and Green 1974b;Davidson and Kaufman 1978). Thymidine arrest in mammalian cells (Harper 2005) is also caused by decreased dCTP levels, which are restored once excess thymidine is removed from the medium (Meuth 1989;Kunz and Kohalmi 1991).…”

Section: Discussionmentioning

confidence: 99%

A Mammalian-Like DNA Damage Response of Fission Yeast to Nucleoside Analogs

et al. 2013

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Nucleoside analogs are frequently used to label newly synthesized DNA. These analogs are toxic in many cells, with the exception of the budding yeast. We show that Schizosaccharomyces pombe behaves similarly to metazoans in response to analogs 5-bromo-29-deoxyuridine (BrdU) and 5-ethynyl-29-deoxyuridine (EdU). Incorporation causes DNA damage that activates the damage checkpoint kinase Chk1 and sensitizes cells to UV light and other DNA-damaging drugs. Replication checkpoint mutant cds1Δ shows increased DNA damage response after exposure. Finally, we demonstrate that the response to BrdU is influenced by the ribonucleotide reductase inhibitor, Spd1, suggesting that BrdU causes dNTP pool imbalance in fission yeast, as in metazoans. Consistent with this, we show that excess thymidine induces G1 arrest in wild-type fission yeast expressing thymidine kinase. Thus, fission yeast responds to nucleoside analogs similarly to mammalian cells, which has implications for their use in replication and damage research, as well as for dNTP metabolism.

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Section: Discussionmentioning

confidence: 99%

A Mammalian-Like DNA Damage Response of Fission Yeast to Nucleoside Analogs

et al. 2013

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“…Previous work from this laboratory (7,8,13,14) provided strong evidence that BrdUrd mutagenesis in mammalian cells occurred primarily by the mispairing of BrdUTP during DNA replication. Because there was no These previous results were in contrast to those found in procaryotic systems, for which, over the years, evidence has been put forth for the occurrence of both errors of incorporation (5,19) and errors of replication (11,21).…”

Section: Discussionmentioning

confidence: 99%

“…This was accomplished through the use of FBTdC medium (see above), which utilizes both dCyd and low concentrations of BrdUrd to prevent the occurrence of any pool-dependent mutagenesis during the incorporation of BrUra into DNA (7,13). The ratio of BrdUrd to dThd in the FBTdC medium was varied at a constant BrdUrd concentration to control the level of substitution of BrUra for Thy in the DNA.…”

Section: Resultsmentioning

confidence: 99%

“…This and other studies (2,7,8,14) provided evidence that BrdUrd mutagenesis in mammalian cells was driven by an increase in the intracellular ratio of BrdUTP to dCTP and occurred only during incorporation of BrdUTP into DNA and that mutations due to replication of BrUra residues in DNA did not occur. (We refer to this mutagenesis as "pool-dependent'" because it is dependent upon the BrdUTP pool levels and not the level of BrUra incorporated into DNA.…”

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confidence: 92%

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Replication of DNA containing 5-bromouracil can be mutagenic in Syrian hamster cells.

Kaufman¹

1984

Mol. Cell. Biol.

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A new protocol for inducing mutations in mammalian cells in culture by exposure to the thymidine analog 5-bromodeoxyuridine (BrdUrd) was established. This protocol, called "DNA-dependent" mutagenesis, involved the incorporation of BrdUrd into DNA under nonmutagenic conditions and the subsequent replication of the 5-bromouracil (BrUra)-containing DNA under mutagenic conditions but with no BrdUrd present in the culture medium. The mutagenic conditions were induced by allowing BrUra-containing DNA to replicate in the presence of high concentrations of thymidine. This generated high intracellular levels of dTTP and dGTP, causing nucleotide pool imbalance. The mutagenesis induced by this protocol was found to correlate with the level of BrUra substituted for thymine in DNA.The thymidine (dThd) analog 5-bromodeoxyuridine (BrdUrd) is an effective mutagen in procaryotic (3,9,16,20,23) and eucaryotic (1,12,13,22) systems. A model for the mutagenic action of this base analog was based on the Watson and Crick scheme of base pairing and the suggestion that rare tautomeric shifts of the bases could change their pairing properties during DNA replication and thereby lead to mutations (9). This model suggests that due to its chemical properties, 5-bromouracil (BrUra) would undergo a tautomeric shift to the rare enol state, allowing it to mispair with guanine (Gua) more frequently than would thymine (Thy). Thus, two possible mechanisms of BrUra mutagenesis were proposed, errors of incorporation and errors of replication. Errors of incorporation were thought to occur when bromodeoxyuridine triphosphate (BrdUTP) mispairs with a Gua residue in replicating DNA, resulting in GC-to-AT transitions after subsequent rounds of replication. Errors of replication were thought to occur when a BrUra residue in replicating DNA mispairs with dGTP, resulting in AT-to-GC transitions.Recent studies in mammalian systems suggested that BrdUrd mutagenesis was determined primarily by the concentration of BrdUrd to which the cells were exposed and was independent of the amount of BrUra incorporated into DNA in place of Thy residues (13). This and other studies (2,7,8,14) provided evidence that BrdUrd mutagenesis in mammalian cells was driven by an increase in the intracellular ratio of BrdUTP to dCTP and occurred only during incorporation of BrdUTP into DNA and that mutations due to replication of BrUra residues in DNA did not occur. (We refer to this mutagenesis as "pool-dependent'" because it is dependent upon the BrdUTP pool levels and not the level of BrUra incorporated into DNA.)The experiments presented in this report describe a new protocol for BrdUrd mutagenesis in mammalian cells, allowing the unambiguous detection of mutations caused by the replication of BrUra residues in DNA. (We refer to this mutagenesis as "DNA-dependent" because it was dependent upon the level of BrUra incorporated into DNA and occurred when there was no BrdUTP in the cell.) The DNAdependent protocol involved the incorporation of BrUra into DNA under nonmutagenic conditi...

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“…BUdR tolerance may depend upon a mechanism, possibly involving the specificity of thymidine kinase (Baugnet-Mahieu and Goutier, 1968), which limits the incorporation of BUdR into DNA. However, this would also lower the level of BUdR Iriphosphate, a compound recently implicated in BUdR mutagenesis and involving sites outside DNA (Davidson and Kaufman, 1979). The high level of BUdR incorporation into the DNA of the BUdR-tolerant B-16 clone (22% of total TdR) was assumed to be the cause of the observed phenotypic changes {Silagi el al., 1976), but no comparison was made with BUdR-sensitive lines or with BUdR-toierant lines which were not altered in this fashion.…”

Section: Table 3 Comparixon A/cell Ivpes For Incorporation Of H-bl'jrmentioning

confidence: 99%

Effects of Thymidine Analogues on Murine and Human Cells

Smellie

Parsons

1979

Aust J Exp Biol Med

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Summary. Three mouse tumour cell lines grew continuously in 3 |jM 5-bromoclcoxyuridinc (BUdR), One line (MC-2) produced a rcirovirus and altered in morphology in the presence of BUdR or 5-iododcoxyuridine (lUdR), These etTccls. which could be reversed by growih in normal medium, were similar to those reported for the B-16 mouse melanoma line. The B-16 line used in this study, however, as well as a variety of human cells (six melanoma lines and three fibroblast strains), were much more sensitive to BUdR. 0-03-0

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Bromodeoxyuridine mutagenesis in mammalian cells is stimulated by thymidine and suppressed by deoxycytidine

Cited by 77 publications

References 15 publications

A Mammalian-Like DNA Damage Response of Fission Yeast to Nucleoside Analogs

A Mammalian-Like DNA Damage Response of Fission Yeast to Nucleoside Analogs

Replication of DNA containing 5-bromouracil can be mutagenic in Syrian hamster cells.

Effects of Thymidine Analogues on Murine and Human Cells

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