Kavli B, Slupphaug G, Mol C D, Arvai A S, Peterson S B, Tainer J A, Krokan H E
UNIGEN Center for Molecular Biology, The Norwegian University of Science and Technology, Trondheim, Norway.
EMBO J. 1996 Jul 1;15(13):3442-7.
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 and 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-DNA glycosylase (TDG) activity, respectively. CDG and the TDG all retain some UDG activity. CDG and TDG have kcat values in the same range as typical multisubstrate-DNA glycosylases, that is at least three orders of magnitude lower than that of the highly selective and efficient wild-type UDG. Expression of CDG or TDG in Escherichia coli causes 4- to 100-fold increases in the yield of rifampicin-resistant mutants. Thus, single amino acid substitutions in UDG result in less selective DNA glycosylases that release normal pyrimidines and confer a mutator phenotype upon the cell. Three of the four new pyrimidine-DNA glycosylases resulted from single nucleotide substitutions, events that may also happen in vivo.
尿嘧啶-DNA糖基化酶（UDG）通过去除胞嘧啶脱氨产生的诱变尿嘧啶残基来保护基因组。尿嘧啶结合在人UDG的DNA结合凹槽底部的一个刚性口袋中，与结构相关的DNA碱基胸腺嘧啶和胞嘧啶相比，对尿嘧啶的特异性是由形状互补以及主链和Asn204侧链氢键赋予的。在这里，我们表明用Asp取代Asn204或用Ala、Cys或Ser取代Tyr147会分别产生具有胞嘧啶-DNA糖基化酶（CDG）活性或胸腺嘧啶-DNA糖基化酶（TDG）活性的酶。CDG和TDG都保留了一些UDG活性。CDG和TDG的催化常数（kcat）值与典型的多底物-DNA糖基化酶在同一范围内，即至少比高度选择性和高效的野生型UDG低三个数量级。在大肠杆菌中表达CDG或TDG会使耐利福平突变体的产量增加4至100倍。因此，UDG中的单个氨基酸取代会导致选择性较低的DNA糖基化酶，这些酶会释放正常的嘧啶并赋予细胞突变表型。四种新的嘧啶-DNA糖基化酶中的三种是由单核苷酸取代产生的，这些事件在体内也可能发生。
