Search tips
Search criteria 


Logo of narLink to Publisher's site
Nucleic Acids Res. 1999 August 1; 27(15): 3057–3063.
PMCID: PMC148530

Characterization of the interaction of lambda exonuclease with the ends of DNA.


Lambda exonuclease processively degrades one strand of double-stranded DNA (dsDNA) in the 5"-3" direction. To understand the mechanism through which this enzyme generates high processivity we are analyzing the first step in the reaction, namely the interaction of lambda exonuclease with the ends of substrate DNA. Endonuclease mapping of lambda exonuclease bound to DNA has shown that the enzyme protects approximately 13-14 bp on dsDNA, and no nucleo-tides on the single-stranded tail of the DNA product. We have developed a rapid fluorescence-based assay using 2-aminopurine and measured the steady-state rate constants for different end-structures of DNA. The relative k(cat)for 5" ends decreases in the order 5" recessed > blunt >> 5" overhang. However, k(cat)/K(m)remains relatively constant for these different structures suggesting they are all used equally efficiently as substrates. From these data we propose that a single-stranded 5" overhang end can bind non-productively to the enzyme and the non-hydrolyzed strand is required to aid in the proper alignment of the 5" end. We have also measured the length-dependence of the steady-state rate para-meters and find that they are consistent with a high degree of processivity.

Full Text

The Full Text of this article is available as a PDF (350K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Radding CM. Regulation of lambda exonuclease. I. Properties of lambda exonuclease purified from lysogens of lambda T11 and wild type. J Mol Biol. 1966 Jul;18(2):235–250. [PubMed]
  • Carter DM, Radding CM. The role of exonuclease and beta protein of phage lambda in genetic recombination. II. Substrate specificity and the mode of action of lambda exonuclease. J Biol Chem. 1971 Apr 25;246(8):2502–2512. [PubMed]
  • Little JW. Lambda exonuclease. Gene Amplif Anal. 1981;2:135–145. [PubMed]
  • Little JW, Lehman IR, Kaiser AD. An exonuclease induced by bacteriophage lambda. I. Preparation of the crystalline enzyme. J Biol Chem. 1967 Feb 25;242(4):672–678. [PubMed]
  • Little JW. An exonuclease induced by bacteriophage lambda. II. Nature of the enzymatic reaction. J Biol Chem. 1967 Feb 25;242(4):679–686. [PubMed]
  • Cassuto E, Radding CM. Mechanism for the action of lambda exonuclease in genetic recombination. Nat New Biol. 1971 Jan 6;229(1):13–16. [PubMed]
  • Kovall R, Matthews BW. Toroidal structure of lambda-exonuclease. Science. 1997 Sep 19;277(5333):1824–1827. [PubMed]
  • Kelman Z, O'Donnell M. Structural and functional similarities of prokaryotic and eukaryotic DNA polymerase sliding clamps. Nucleic Acids Res. 1995 Sep 25;23(18):3613–3620. [PMC free article] [PubMed]
  • Sriprakash KS, Lundh N, Huh MM-O, Radding CM. The specificity of lambda exonuclease. Interactions with single-stranded DNA. J Biol Chem. 1975 Jul 25;250(14):5438–5445. [PubMed]
  • Radding CM, Carter DM. The role of exonuclease and beta protein of phage lambda in genetic recombination. 3. Binding to deoxyribonucleic acid. J Biol Chem. 1971 Apr 25;246(8):2513–2518. [PubMed]
  • Mach H, Middaugh CR, Lewis RV. Statistical determination of the average values of the extinction coefficients of tryptophan and tyrosine in native proteins. Anal Biochem. 1992 Jan;200(1):74–80. [PubMed]
  • Gill SC, von Hippel PH. Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem. 1989 Nov 1;182(2):319–326. [PubMed]
  • Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. [PubMed]
  • Read SM, Northcote DH. Minimization of variation in the response to different proteins of the Coomassie blue G dye-binding assay for protein. Anal Biochem. 1981 Sep 1;116(1):53–64. [PubMed]
  • Ward DC, Reich E, Stryer L. Fluorescence studies of nucleotides and polynucleotides. I. Formycin, 2-aminopurine riboside, 2,6-diaminopurine riboside, and their derivatives. J Biol Chem. 1969 Mar 10;244(5):1228–1237. [PubMed]
  • Suck D. DNA recognition by DNase I. J Mol Recognit. 1994 Jun;7(2):65–70. [PubMed]
  • Frey MW, Sowers LC, Millar DP, Benkovic SJ. The nucleotide analog 2-aminopurine as a spectroscopic probe of nucleotide incorporation by the Klenow fragment of Escherichia coli polymerase I and bacteriophage T4 DNA polymerase. Biochemistry. 1995 Jul 18;34(28):9185–9192. [PubMed]
  • Bloom LB, Otto MR, Eritja R, Reha-Krantz LJ, Goodman MF, Beechem JM. Pre-steady-state kinetic analysis of sequence-dependent nucleotide excision by the 3'-exonuclease activity of bacteriophage T4 DNA polymerase. Biochemistry. 1994 Jun 21;33(24):7576–7586. [PubMed]
  • Sastry SS, Ross BM. A direct real-time spectroscopic investigation of the mechanism of open complex formation by T7 RNA polymerase. Biochemistry. 1996 Dec 10;35(49):15715–15725. [PubMed]
  • Raney KD, Sowers LC, Millar DP, Benkovic SJ. A fluorescence-based assay for monitoring helicase activity. Proc Natl Acad Sci U S A. 1994 Jul 5;91(14):6644–6648. [PubMed]
  • Sowers LC, Fazakerley GV, Eritja R, Kaplan BE, Goodman MF. Base pairing and mutagenesis: observation of a protonated base pair between 2-aminopurine and cytosine in an oligonucleotide by proton NMR. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5434–5438. [PubMed]
  • Nordlund TM, Andersson S, Nilsson L, Rigler R, Gräslund A, McLaughlin LW. Structure and dynamics of a fluorescent DNA oligomer containing the EcoRI recognition sequence: fluorescence, molecular dynamics, and NMR studies. Biochemistry. 1989 Nov 14;28(23):9095–9103. [PubMed]
  • Brody RS, Doherty KG, Zimmerman PD. Processivity and kinetics of the reaction of exonuclease I from Escherichia coli with polydeoxyribonucleotides. J Biol Chem. 1986 Jun 5;261(16):7136–7143. [PubMed]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press