EXCISION OF CYTOSINE AND THYMINE FROM DNA BY MUTANTS OF HUMAN URACIL-DNA GLYCOSYLASE

Uracil-DNA glycosylase (UDG) protects the genome by removing mutagenic uracil residues resulting from deamination of cytosine. Uracil binds in a rigid pocket at the base of the DNA-binding groove of human UDG a nd the specificity for uracil over the structurally related DNA bases thymine and cytosine is conferred by shape complementarity, as well as by main chain and Asn204 side chain hydrogen bonds. Here we show that replacement of Asn204 by Asp or Tyr147 by Ala, Cys or Ser results in enzymes that have cytosine-DNA glycosylase (CDG) activity or thymine-D NA glycosylase (TDG) activity, respectively. CDG and the TDG all retai n some UDG activity. CDG and TDG have k(cat) values in the same range as typical multisubstrate-DNA glycosylases, that is at least three ord ers of magnitude lower than that of the highly selective and efficient wild-type UDG. Expression of CDG or TDG in Escherichia coil causes 4- to 100-fold increases in the yield of rifampicin-resistant mutants. T hus, single amino acid substitutions in UDG result in less selective D NA glycosylases that release normal pyrimidines and confer a mutator p henotype upon the cell. Three of the four new pyrimidine-DNA glycosyla ses resulted from single nucleotide substitutions, events that may als o happen in vivo.