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

Abstract

By using a shuttle vector system developed in our laboratory, we have carried out studies on the molecular mechanism by which 5-bromodeoxyuridine (BrdUrd) induces mutations in mammalian cells. The target for mutagenesis in these studies was the Escherichia coli gpt gene that was contained within a retroviral shuttle vector and integrated into chromosomal DNA in mouse A9 cells. Shuttle vector-transformed cells expressing the gpt gene were mutagenized with BrdUrd and cells with mutations in the gpt gene were selected.Shuttle vector sequences were recovered from the mutant cells, and the base sequence of the mutant gpt genes was determined.The great majority of the BrdUrd-induced mutations involving single-base changes were found to be GC -> A-T transitions. We have shown that mutagenesis by BrdUrd depends upon perturbation of deoxycytidine metabolism. Thus, the current results suggest that BrdUrd mutagenesis involves mispairing and misincorporation of BrdUrd opposite guanine in DNA, driven by nucleotide pool perturbation caused by BrdUrd and the resulting imbalanced supply of triphosphates available for DNA synthesis. The results also revealed a very high degree of sequence specificity for the BrdUrd mutagenesis. BrdUrdinduced G-C -> APT transitions occurred almost exclusively in sequences with two adjacent guanine residues. Furthermore, in ,9O% of the cases, the guanine residue involved in mutation was the one in the more 3' position.Our laboratory has shown that mutagenesis by the thymidine (dThd) analog 5-bromodeoxyuridine (BrdUrd) transitions (4-6).We have studied the molecular mechanisms of BrdUrd mutagenesis in mammalian cells, by using a retroviral shuttle vector system developed in our laboratory (7). As the target for mutations, the shuttle vector contains the Escherichia coli gpt gene, coding for the enzyme xanthine guanine phosphoribosyltransferase (5-phospho-a-D-ribose-l-diphosphate: xanthine phosphoribosyltransferase; EC 2.4.2.22). The shuttle vector was introduced into mouse A9 cells and a transformed line (A9I-2) was isolated that contains a single copy of the vector integrated into chromosomal DNA. Since A9 cells are deficient in the enzyme hypoxanthine guanine phosphoribosyltransferase (IMP-pyrophosphate phosphoribosyl-transferase; EC 2.4.2.8) (8), transformants with mutations in the gpt gene can be selected with 6-thioguanine (sGua). For sequencing, the gpt genes from mutant A9I-2 cells can be recovered by fusion with monkey COS cells (9).In the present study, the gpt genes were recovered and sequenced from a large number of BrdUrd-induced mutants of A91-2 cells. We have used this shuttle vector system to analyze mutations that occurred spontaneously or were induced by ethyl methanesulfonate (EtMes) (10, 11). Mutational studies with various shuttle vector systems also have been carried out by other laboratories (12-17). Our system differs from those used in the other studies in that the vector in our system is integrated into chromosomal DNA and clonal selection for cells with mutant ge...

The relationship between BCL6 bodies and nuclear sites of normal and halogenated DNA and RNA synthesis

“…Indeed, IdU has been previously shown to be less efficiently incorporated in DNA than BrdU and CldU (Franken et al, 1997). On the other hand, it has been reported that modulating the concentration of BrdU in the medium favours the occurrence of damages in the replicating DNA by an imbalancing in the ratio between the nucleotide pools leading to frequent BrdU/ guanine mispairing (Davidson et al, 1980(Davidson et al, , 1988. However, the addition of deoxycytidine, which prevents such an effect, even in excess over BrdU or CldU (Davidson et al, 1988), does not affect the association of replicating (halogenated) DNA with BCL6 bodies (unpublished data), suggesting therefore that this association is indeed correlated with the level of incorporation of the halogenated nucleotide into the DNA.…”

Sequence-Directed Base Mispairing in Human Oncogenes

“…Subsequently, dTTP, the nucleotide in excess, mispairs with guanine residues in replicating DNA molecules, resulting in GC3AT transitions (11,16,17,33). The misincorporation studies presented here are based upon previous work done in our laboratory dealing with the sequence-specific mode of dT mutagenesis in mammalian cells (17).…”

“…The misincorporation studies presented here are based upon previous work done in our laboratory dealing with the sequence-specific mode of dT mutagenesis in mammalian cells (17). Those studies had shown that when cultured mouse cells carrying a single, chromosomally integrated copy of the bacterial gpt gene were subjected to high concentrations of dTTP, GC3AT transitions occurred preferentially at the 3Ј G residue of GG doublets in the gpt gene (11,33). The mutations that were observed in these experiments thus occurred under conditions of an imbalance in two specific nucleotide pools, namely, a high-dTTP pool and a low-dCTP pool.…”

“…In cultured mammalian cells, high levels of exogenously supplied dT dramatically increase the intracellular ratio of dTTP to dCTP available for DNA synthesis (2,11,14,20). Subsequently, dTTP, the nucleotide in excess, mispairs with guanine residues in replicating DNA molecules, resulting in GC3AT transitions (11,16,17,33).…”

“…Subsequently, dTTP, now the nucleotide in excess relative to dCTP, mispairs with template guanine, leading to a GC3AT transition at the next round of replication (2,14,22). We have shown that such mutagenesis by dT (or 5-bromodeoxyuridine) exhibits strong sequence specificity in that it occurs preferentially at the 3Ј G of runs of two or more adjacent G residues, resulting in GC3AT transitions at this position in the multiple guanine run (11,16,17,33). In sequences with GG doublets, dTinduced mutations were found to occur at least 25 times more frequently at the 3Ј G residue of the GG doublet than at the 5Ј G residue of the doublet (17).…”