Lin J J, Phillips A M, Hearst J E, Sancar A
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599.
J Biol Chem. 1992 Sep 5;267(25):17693-700.
UvrB plays a central role in (A)BC excinuclease. To study its role in the incision reactions, conserved His and Asp residues in this subunit were mutagenized. All His and the majority of Asp mutants behaved like wild-type protein in vivo and in vitro. However, three mutants, D337A, D478A, and D510A, either completely or partially abolished UvrB activity. All three mutant proteins associate with UvrA normally but D337A and D510A were unable to bind to DNA specifically. The UvrB-D478A mutant bound to DNA specifically but failed to denature and kink the DNA. However, UvrB-D478A was efficiently loaded onto DNA preincised at the 3' site and promoted near-normal incision by UvrC at the 5' site. We propose that D478 is involved in bending DNA and catalysis of the 3' incision and that the 3' incision precedes the 5' incision. UvrB which is missing the carboxyl-terminal 43 amino acids binds to, and kinks DNA but is unable to make the 3' incision suggesting that it is missing a residue involved in catalysis. This residue was identified to be E639 by site-specific mutagenesis.
UvrB在（A）BC核酸外切酶中起核心作用。为了研究其在切口反应中的作用，对该亚基中保守的组氨酸和天冬氨酸残基进行了诱变。所有组氨酸突变体和大多数天冬氨酸突变体在体内和体外的表现都与野生型蛋白相似。然而，三个突变体D337A、D478A和D510A完全或部分消除了UvrB的活性。所有这三种突变蛋白都能正常地与UvrA结合，但D337A和D510A无法特异性结合DNA。UvrB-D478A突变体能特异性结合DNA，但无法使DNA变性和扭结。然而，UvrB-D478A能有效地加载到在3'位点预先切口的DNA上，并促进UvrC在5'位点进行接近正常的切口。我们提出，D478参与DNA弯曲和3'切口的催化作用，且3'切口先于5'切口。缺失羧基末端43个氨基酸的UvrB能结合并使DNA扭结，但无法进行3'切口，这表明它缺少一个参与催化的残基。通过位点特异性诱变确定该残基为E639。
