RecJ exonuclease: substrates, products and interaction with SSB

Area of Science:

• Molecular Biology
• Enzymology

Background:

• RecJ exonuclease from Escherichia coli is crucial for DNA repair pathways like homologous recombination and mismatch repair.
• It functions by degrading single-stranded DNA (ssDNA) in the 5'-3' direction.

Purpose of the Study:

• To elucidate the substrate specificity and reaction products of the RecJ exonuclease.
• To understand the binding requirements and processivity of RecJ on various ssDNA substrates.

Main Methods:

• Electrophoretic mobility shift assays were used to analyze RecJ binding to ssDNA substrates with varying tail lengths.
• Enzymatic assays were performed to determine substrate degradation and product formation in the presence of Mg2+.
• RecJ activity was assessed on both 5' phosphorylated and unphosphorylated DNA ends.

Main Results:

• RecJ requires single-stranded DNA tails of 7 nucleotides or greater for efficient binding and activity.
• RecJ is a processive exonuclease, degrading approximately 1000 nucleotides per binding event and releasing mononucleotides.
• RecJ degrades ssDNA tails up to double-stranded junctions but shows limited duplex penetration; its activity is enhanced by ssDNA-binding protein.

Conclusions:

• RecJ exhibits specific substrate length requirements for binding and degradation.
• The processive nature and product release mechanism of RecJ are characterized.
• Interaction with ssDNA-binding protein may facilitate RecJ recruitment to DNA repair sites.