DNA distortion and specificity in a sequence-specific endonuclease.

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Biology

Background:

• The HincII enzyme is crucial for DNA manipulation.
• Previous studies focused on Ca(2+) soaking for structural analysis.

Purpose of the Study:

• To elucidate the structural basis of Q138F HincII enzyme activity.
• To investigate the role of different metal ions (Ca(2+), Mg(2+), Mn(2+)) in enzyme-DNA complex formation.
• To understand the mechanism of DNA cleavage by Q138F HincII.

Main Methods:

• X-ray crystallography to determine enzyme-DNA-metal ion complex structures.
• Cocrystallization techniques with various metal ions.
• Biochemical binding studies.

Main Results:

• Five new crystal structures of Q138F HincII with DNA and metal ions (Ca(2+), Mg(2+), Mn(2+)) were determined.
• The Mn(2+)-bound structure represents the first product complex of cleaved DNA.
• Ca(2+) was shown to trap the enzyme in a non-catalytic state with non-cognate DNA.
• Enzyme-DNA complexes exhibit asymmetry, influencing DNA cleavage rates.

Conclusions:

• The study provides a comprehensive structural model of Q138F HincII in complex with DNA and metal ions.
• DNA conformational energetics are key determinants of enzyme cleavage rates.
• Understanding these interactions advances knowledge of enzyme mechanisms and DNA processing.