Abstract:
: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.
journal_name
Nucleic Acids Resjournal_title
Nucleic acids researchauthors
Mitsis PG,Kwagh JGdoi
10.1093/nar/27.15.3057keywords:
subject
Has Abstractpub_date
1999-08-01 00:00:00pages
3057-63issue
15eissn
0305-1048issn
1362-4962pii
gkc490journal_volume
27pub_type
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