Pairing properties of bromouracil and repair of bromouracil-containing DNA. Possible utilization of bromodeoxyuridine triphosphate for site-directed mutagenesis.
5-Bromo-2'-deoxyuridine triphosphate (Br-dUTP) and dTTP are used interchangeably for DNA synthesis in vitro by the Klenow fragment of Escherichia coli DNA polymerase I. When DNA containing Br-dUMP instead of dTMP at a few preselected sites is transfected into competent bacteria, no mutation occurs, indicating that in vivo E. coli DNA polymerase always places a dAMP residue in front of any unrepaired Br-dUMP residue. On the other hand, in vitro Br-dUTP can also replace dCTP, but only with difficulty: when dCTP is absent, Br-dUMP can be forced in front of a dGMP residue, but the Klenow polymerase pauses before and after addition of Br-dUMP. Transfection into E. coli of the substituted DNA leads to the expected G----A transitions. These mutations can easily be targeted by using a suitable primer and the correctly chosen mix of deoxynucleoside triphosphates containing Br-dUTP. When Br-dUMP has been placed in front of a dGMP residue, the mutation yield is not 100%, showing a partial repair of the transfected DNA before it is replicated. Advantage can be taken of this partial repair to prepare a set of different mutations within a target region in a single experiment.
Muller
M
M
Laboratoire de Biochimie, Université de Liège, Belgium.
Martial
J
J
Verly
W G
WG
D001482:Base Composition; D001483:Base Sequence; D001976:Bromouracil / Q000378:metabolism*; D004247:DNA / Q000096:biosynthesis; D004260:DNA Repair; D003856:Deoxyuracil Nucleotides / Q000235:genetics*; D013006:Growth Hormone / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D013698:Templates, Genetic; D014162:Transfection
D003856:Deoxyuracil Nucleotides; C032549:bromodeoxyuridine triphosphate; D001976:Bromouracil; D013006:Growth Hormone; D004247:DNA