Relating primary structure to function in the Escherichia coli XerD site-specific recombinase

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• XerC and XerD are site-specific recombinases essential for Xer recombination, involving strand exchange.
• These recombinases possess distinct functions including DNA binding, co-operative interactions, synapsis, and catalysis.

Purpose of the Study:

• To elucidate the relationship between the primary amino acid sequence of XerD and its functional domains.
• To identify specific amino acid regions responsible for DNA binding, XerC/D interactions, and catalytic activity.

Main Methods:

• Construction and analysis of internal and C-terminal deletion mutants of the XerD recombinase.
• Assessment of mutant protein functions including sequence-specific DNA binding, co-operative XerC/D interactions, and catalysis.

Main Results:

• XerD residues 1-233 retained DNA binding but lost co-operative interaction with XerC.
• Deletion of C-terminal residues abolished DNA binding, while internal deletions (32-88, 145-159) impaired DNA binding.
• A region containing residues 244-281 was necessary for catalysis, and residues 1-283 retained catalytic activity.
• A C-terminal deletion mutant (lacking five residues) retained DNA binding and catalysis but failed in vivo recombination.

Conclusions:

• The C-terminal residues of XerD are critical for in vivo recombination, independent of direct DNA binding or catalytic activity.
• Specific internal regions of XerD are essential for DNA binding and co-operative interactions with XerC.
• Understanding these functional domains provides insights into the mechanism of site-specific recombination.