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

Abstract

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...

Reversion analysis of mutations induced by 5-bromodeoxyuridine mutagenesis in mammalian cells.

“…Biol. 4:2449-2454, 1984. The first protocol, termed incorporational (INC) mutagenesis, utilizes high concentrations of BrdUrd in the culture medium to generate a high intracellular ratio of BrdUTP/dCTP.…”

DNA base sequence changes and sequence specificity of bromodeoxyuridine-induced mutations in mammalian cells.

“…Previous results from this laboratory showed (4) that the conditions of incorporation mutagenesis result in an increase in the dGTP/dATP molar ratio. However, this ratio apparently increases to a much smaller extent during incorporation mutagenesis than replication mutagenesis (6). Thus, the occurrence of a small number of A-T -> G-C transition mutations in the present study could be due to a low level of replication mutagenesis, and these mutations could be explained by the following mechanism.…”

“…These mutations also can be explained by a mechanism involving base mispairing driven by pool perturbation. There are two modes for BrdUrd mutagenesis in mammalian cells and these two modes can produce reciprocal genetic events, as judged by reversion analysis (6,28). The protocol for mutagenesis used in the current study favors the induction of mutations during the incorporation of BrdUrd into DNA, with mutagenesis dependent upon the ratio of BrdUTP to dCTP in the intracellular pools.…”

“…These studies showed that BrdUrd mutagenesis in mammalian cells is (i) determined by the concentration of exogenous BrdUrd to which cells are exposed and not by the amount of BrdUrd incorporated into DNA, (ii) suppressed by exogenous deoxycytidine, and (iii) quantitatively related to the ratio of BrdUrd to deoxycytidine in the intracellular pools. Thus, we have suggested that BrdUrd mutagenesis in mammalian cells is dependent upon the perturbation of endogenous deoxycytidine metabolism, that mutations could arise from misincorporation of BrdUrd into DNA, driven by the unbalanced deoxyribonucleoside triphosphate (dNTP) pools available for DNA synthesis, and that mutagenesis under such conditions would result primarily in G'C --A-T transitions (4)(5)(6).…”

Stacking of the mutagenic base analogue 5-bromouracil: energy landscapes of pyrimidine dimers in gas phase and water

“…It is a well-known mutagen, causing transition mutations by mispairing with guanine (G) rather than pairing with adenine (A) during replication. [1][2][3][4][5][6][7] This behaviour is most commonly attributed to enolisation of BrU -either in the template strand or the nucleotide pool -converting the major diketo tautomer into the ''rare'' enol (O4-hydroxy) tautomer, which is apparently stabilised by the bromine substituent. 4,[8][9][10][11][12][13][14][15][16] The rare tautomer mimics cytosine (C) in its hydrogen-bonding affinity, and mispairs with G in Watson-Crick geometry (Fig.…”