Sequence-specific cleavage by bacteriophage T4 endonuclease II in vitro

Area of Science:

• Molecular Biology
• Enzymology
• Bacteriophage Genetics

Background:

• Bacteriophage T4 encodes various DNA repair enzymes.
• Endonuclease II (EndoII) plays a role in DNA processing and repair.
• Understanding enzyme mechanisms is crucial for molecular biology.

Purpose of the Study:

• To identify and clone the denA gene encoding bacteriophage T4 endonuclease II (EndoII).
• To characterize the DNA cleavage and nicking activity of EndoII in vitro.
• To compare in vitro and in vivo DNA processing activities of EndoII.

Main Methods:

• Gene sequencing and DNA cloning of the bacteriophage T4 denA gene.
• In vitro transcription-translation of cloned denA gene to produce EndoII.
• In vitro DNA nicking and cleavage assays using cloned DNA (pBR322).
• Comparison of in vitro and in vivo cleavage site preferences and patterns.

Main Results:

• The 136-codon denA gene encoding EndoII was successfully cloned.
• In vitro, EndoII exhibited sequence-specific nicking and cutting of DNA, primarily at CCGC motifs.
• In vitro reactions showed more frequent bottom-strand nicking than double-strand cutting compared to in vivo.
• Differences in site preference and sequence elements were observed between in vitro and in vivo cleavage.

Conclusions:

• The cloned bacteriophage T4 endonuclease II functions in a sequence-specific manner.
• Distinct differences in nicking and cleavage patterns exist between in vitro and in vivo reactions.
• A model involving sequential strand nicking and varying influencing factors explains observed reaction differences.