Affinity modification of the restriction endonuclease SsoII by 2'-aldehyde-containing double stranded DNAs

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Restriction endonucleases are crucial tools in molecular biology for DNA manipulation.
• Understanding enzyme-substrate interactions is key to characterizing enzyme function and specificity.
• SsoII is a restriction endonuclease whose substrate recognition mechanism requires further elucidation.

Purpose of the Study:

• To investigate the properties of 2'-aldehyde-containing double-stranded DNAs (dsDNAs) as substrate analogs for the restriction endonuclease SsoII.
• To identify the specific amino acid residue in SsoII involved in DNA binding and recognition.

Main Methods:

• Synthesis of 2'-aldehyde-containing dsDNA analogs.
• Reductive amination for cross-linking dsDNA analogs to SsoII.
• Trypsinolysis of DNA-protein conjugates.
• Oligonucleotide-peptide conjugate purification.
• MALDI-TOF mass spectrometry for linkage analysis.

Main Results:

• Successful cross-linking of dsDNA analogs to the restriction endonuclease SsoII.
• Optimized conditions for DNA-protein conjugate processing and purification.
• Identification of a covalent linkage between the DNA's sugar moiety and Lysine 173 (Lys173) of SsoII.
• MALDI-TOF mass spectrometry confirmed the specific site of interaction.

Conclusions:

• The study provides the first characterization of 2'-aldehyde-containing dsDNAs as substrate analogs for SsoII.
• Lys173 of SsoII is identified as a key residue involved in the initial recognition and binding of dsDNA.
• This finding offers insights into the DNA readout mechanism of restriction endonucleases.