Two sequence-specific endonucleases from Xanthomonas oryzae. Characterization and unusual properties.

Area of Science:

• Molecular Biology
• Enzymology

Background:

• Sequence-specific endonucleases are crucial tools in molecular biology for DNA manipulation.
• Isoschizomers, enzymes recognizing the same DNA sequence, offer alternative or improved properties for research.
• Xanthomas oryzae is a source of novel restriction enzymes.

Purpose of the Study:

• To partially purify and characterize two sequence-specific endonucleases, XorI and XorII, from Xanthomas oryzae.
• To investigate the properties of XorII as an isoschizomer of PvuI, focusing on its unique cleavage and stability.
• To explore the utility of XorII in molecular cloning by examining its activity on plasmid vectors.

Main Methods:

• Partial purification of endonucleases XorI and XorII from Xanthomas oryzae.
• Enzyme characterization, including identification as isoschizomers of PstI and PvuI.
• Determination of XorII's specific cleavage site within its recognition sequence (5'-CGATC G-3').
• Analysis of XorII digestion kinetics on plasmid cloning vehicles pBR313 and pBR322.
• Assessment of human DNA resistance to XorII digestion.

Main Results:

• XorI and XorII were successfully partially purified from Xanthomas oryzae.
• XorI and XorII were identified as isoschizomers of PstI and PvuI, respectively.
• XorII demonstrated high yield, stability, and a simple purification scheme, making Xanthomas oryzae a valuable source.
• XorII cleaves its recognition sequence (5'-CGATC G-3') between the C and G at the 3'-end, differing from some known isoschizomers.
• A single XorII site was found in both pBR313 and pBR322 plasmids.
• Unusual digestion kinetics of pBR313 and pBR322 by XorII were observed, along with resistance of human DNA to XorII.

Conclusions:

• Xanthomas oryzae is an excellent source for the PvuI isoschizomer, XorII, due to its favorable purification and stability.
• XorII's unique cleavage site and distinct digestion properties offer new possibilities for DNA analysis and manipulation.
• Further investigation into the unusual digestion kinetics and DNA resistance could reveal novel enzymatic mechanisms or applications.