Structure-specific DNA binding by bacteriophage T5 5'-->3' exonuclease

Area of Science:

• Molecular Biology
• Enzymology
• Structural DNA Biology

Background:

• Phage T5 exonuclease is a DNA processing enzyme with both exo- and endonucleolytic activities.
• Its specific substrate recognition and binding mechanisms require detailed investigation.
• Understanding enzyme-DNA interactions is crucial for DNA repair and replication research.

Purpose of the Study:

• To characterize the binding preferences of T5 exonuclease to various DNA structures.
• To investigate the role of cysteine residues in T5 exonuclease activity and inhibition.
• To explore the structural basis of T5 exonuclease substrate recognition.

Main Methods:

• Synthesis of various DNA oligonucleotide structures (blunt-ended, overhangs, flaps, pseudo-Y).
• Electrophoretic mobility shift assays (EMSA) to detect enzyme-DNA binding.
• Proteolysis and chemical modification (p-hydroxymercuribenzoate) to assess enzyme stability and cysteine involvement.

Main Results:

• T5 exonuclease showed specific binding to branched DNA structures (5'-overhangs, flaps, pseudo-Y) but not blunt-ended duplexes or single-stranded DNA.
• Wild-type T5 exonuclease binding to DNA conferred partial resistance to proteolysis, yielding a 31.5 kDa active fragment.
• Mutation of cysteine residues to serine rendered the enzyme resistant to p-hydroxymercuribenzoate inhibition without significantly altering binding or catalytic activity.

Conclusions:

• Phage T5 exonuclease exhibits structural specificity, preferentially binding to branched DNA substrates.
• Cysteine residues are not essential for substrate binding but are involved in susceptibility to chemical modification.
• The study provides insights into the structure-function relationship of T5 exonuclease and its DNA interaction mechanisms.