Significance of Knotted Structures for Function of Proteins and Nucleic Acids
Poster Session II
31 – POS
Board 3
Cooperative DNA-binding of the Colicin E7 Nuclease Domain
Eszter Németh
1,2
, Judit Kopniczky
3
, Evelin Balázs
1
, Béla Gyurcsik
1,2
.
1
University of Szeged, Szeged, Hungary,
2
MTA-SzTE Bioinorganic Chemistry Research Group
of Hungarian Academy of Sciences, Szeged, Hungary,
3
University of Szeged, Szeged, Hungary.
Colicin E7 is a nonspecific bacterial nuclease. Although its catalytic site, i.e. the HNH motif, is
located at the C-terminus of the protein, N-terminal point mutations remarkably decreased the
nuclease activity. According to circular dichroism spectra and isothermal microcalorimetric
titrations this phenomenon could not be attributed to a large conformational change or to a
decreased metal-binding affinity. The mutant proteins bound a short double stranded DNA of 13
base pairs as strong as the wild-type enzyme in the gel mobility shift assays. However,
increasing the length of the DNA substrate (21, 188, 306, 777 base pairs) revealed differences in
the binding mode of the proteins. The gel eletrophoresis picture unexpectedly showed distinct
bands of DNA/NColE7 complexes with long substrates. This indicates the possibility of
cooperative binding process(es), resulting in favored supershifted DNA/NColE7 complexes of
outstanding stability. The influence of N-terminal mutations on the electrophoresis pattern was
also investigated. The more positive charged residues were removed from the N-terminus, the
more pronounced smear appeared. Atomic force microscopy was applied to check the DNA
conformation change upon protein binding. Our results provide better insight into the DNA-
binding mechanism of the colicin E7 nuclease domain. Furthermore, we prove the importance of
the selection of DNA molecules for protein/DNA-binding studies. It is a common way to apply
short DNA sequences in these experiments. However the natural substrates for enzymes are
mostly long DNA molecules. In this contribution we show that the K
d
value may be affected by
the length of the DNA substrates, and that the modified DNA conformation may be critical for
enzymatic activity.
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