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Volume 272, Number 43, Issue of October 24, 1997 pp. 27210-27217
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

The Role of Base Flipping in Damage Recognition and Catalysis by T4 Endonuclease V

(Received for publication, May 20, 1997, and in revised form, July 23, 1997)

Amanda K. McCullough , M. L. Dodson , Orlando D. Schärer and R. Stephen Lloyd

From the Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555-1071 and the  Department of Chemistry, Harvard University, Cambridge, Massachusetts 02138

The process of moving a DNA base extrahelical (base flipping) has been shown in the co-crystal structure of a UV-induced pyrimidine dimer-specific glycosylase, T4 endonuclease V, with its substrate DNA. Compared with other enzymes known to use base flipping, endonuclease V is unique in that it moves the base opposite the target site extrahelical, rather than moving the target base itself. Utilizing substrate analogs and catalytically inactive mutants of T4 endonuclease V, this study investigates the discrete steps involved in damage recognition by this DNA repair enzyme. Specifically, fluorescence spectroscopy analysis shows that fluorescence changes attributable to base flipping are specific for only the base directly opposite either abasic site analogs or the 5-thymine of a pyrimidine dimer, and no changes are detected if the 2-aminopurine is moved opposite the 3-thymine of the pyrimidine dimer. Interestingly, base flipping is not detectable with every specific binding event suggesting that damage recognition can be achieved without base flipping. Thus, base flipping does not add to the stability of the specific enzyme-DNA complex but rather induces a conformational change to facilitate catalysis at the appropriate target site. When used in conjunction with structural information, these types of analyses can yield detailed mechanistic models and critical amino acid residues for extrahelical base movement as a mode of damage recognition.

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