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Allosterism in the binding of AGT to short single‐ and double‐stranded DNAs
Author(s) -
Melikishvili Manana,
Fried Michael Gregory
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a943-b
Subject(s) - chemistry , circular dichroism , dna , fluorescence , biophysics , conformational change , helix (gastropod) , crystallography , sedimentation equilibrium , stereochemistry , ultracentrifuge , biochemistry , biology , ecology , physics , quantum mechanics , snail
O6‐alkylguanine‐DNA alkyltransferase (AGT) is an important cellular defense against the mutagenic effects of DNA alkylating agents. Here, we report circular dichroism (CD), fluorescence and analytical ultracentrifugation studies that characterize the interaction of AGT with 16 nt single‐stranded (ss) and 16 bp double‐stranded (ds) DNAs. Sedimentation equilibrium (SE) studies indicate that AGT binds cooperatively to these DNAs and reaches similar limiting stoichiometries (n = 4 ± 0.5) with both ss‐ and ds‐forms. Changes in circular dichroism at long wavelengths (~280 nm) suggest that at least partial unstacking and untwisting accompanies binding to both forms. Enhanced fluorescence of 2‐aminopurine‐substituted DNAs with binding is also consistent with base‐unstacking. This DNA allosterism may reflect the formation of extrahelical bases as part of the DNA‐surveillance mechanism. CD analyses suggest that the alpha‐helix content of AGT is increased slightly upon ssDNA binding. This conformational change is accompanied by a small but reproducible decrease in tryptophan fluorescence intensity but no detectable change in the wavelength of maximum emission. Since the crystal structure of a single AGT molecule bound to dsDNA is closely similar to that of the free protein, the observed changes may reflect a conformational adjustment that is necessary for cooperative binding. Supported by NIH grant GM070662.