Nucleic Acids Research, Vol 27, Issue 15 3057-3063, Copyright © 1999 by Oxford University Press
PG Mitsis and JG Kwagh
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.
ARTICLES
Characterization of the interaction of lambda exonuclease with the ends of DNA
Praelux Inc., 17 Princess Road, Lawrenceville, NJ 08648, USA. pmitsis@praelux.com
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