3'-5' exonuclease of Klenow fragment: role of amino acid residues within the single-stranded DNA binding region in exonucleolysis and duplex DNA melting

Biochemistry
Wai-Chung LamDavid P Millar

Abstract

The mechanism of the 3'-5' exonuclease activity of the Klenow fragment of DNA polymerase I has been investigated with a combination of biochemical and spectroscopic techniques. Site-directed mutagenesis was used to make alanine substitutions of side chains that interact with the DNA substrate on the 5' side of the scissile phosphodiester bond. Kinetic parameters for 3'-5' exonuclease cleavage of single- and double-stranded DNA substrates were determined for each mutant protein in order to probe the role of the selected side chains in the exonuclease reaction. The results indicate that side chains that interact with the penultimate nucleotide (Q419, N420, and Y423) are important for anchoring the DNA substrate at the active site or ensuring proper geometry of the scissile phosphate. In contrast, side chains that interact with the third nucleotide from the DNA terminus (K422 and R455) do not participate directly in exonuclease cleavage of single-stranded DNA. Alanine substitutions of Q419, Y423, and R455 have markedly different effects on the cleavage of single- and double-stranded DNA, causing a much greater loss of activity in the case of a duplex substrate. Time-resolved fluorescence anisotropy decay measurements with a dansyl...Continue Reading

References

Jan 1, 1994·Annual Review of Biochemistry·C M Joyce, T A Steitz
Jan 1, 1995·Methods in Enzymology·H CaiM F Goodman
Feb 27, 1999·Current Opinion in Structural Biology·J Jäger, J D Pata
Jun 11, 1999·The Journal of Biological Chemistry·T A Steitz
Sep 18, 2001·Methods : a Companion to Methods in Enzymology·M F BaileyD P Millar

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Citations

Dec 4, 2009·Proceedings of the National Academy of Sciences of the United States of America·Thomas D ChristianDavid Rueda
Dec 7, 2011·Journal of Bioscience and Bioengineering·Sotaro SanoShinsuke Fujiwara
Jan 19, 2010·Biochimica Et Biophysica Acta·Kenneth A Johnson
Aug 10, 2006·Chemical Reviews·Natasa MitićGerhard Schenk

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