Mycobacterium smegmatis Ku binds DNA without free ends

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Ku protein is crucial for DNA double-strand break repair via non-homologous end-joining across all life domains.
• Eukaryotic Ku proteins exhibit diverse functions beyond the conserved core domain, unlike prokaryotic and archaeal counterparts.
• Distinct C-terminal extensions in some prokaryotic Ku proteins, like in Pseudomonas aeruginosa and Mycobacterium smegmatis, modulate DNA-binding properties.

Purpose of the Study:

• To investigate the role of the lysine-rich C-terminal extension of Mycobacterium smegmatis Ku.
• To determine how this C-terminal extension influences Ku's DNA-binding affinity, stability, and localization.
• To understand the evolutionary implications of C-terminal extensions in expanding Ku protein function.

Main Methods:

• Deletion analysis of the M. smegmatis Ku C-terminal extension.
• Assays to measure DNA-binding affinity.
• Thermal stability and intrinsic tryptophan fluorescence measurements.
• DNA binding assays to assess binding to free versus internal DNA sites.

Main Results:

• Deletion of the C-terminal extension increased DNA-binding affinity, decreased thermal stability, and reduced intrinsic tryptophan fluorescence.
• Full-length M. smegmatis Ku binds directly to DNA, including internal sites, a property absent in the C-terminal deleted mutant.
• The C-terminal deleted Ku requires free DNA ends for binding but can translocate to internal sites.

Conclusions:

• The lysine-rich C-terminal extension of M. smegmatis Ku interacts with the core domain, modulating its DNA-binding properties and enabling binding to internal DNA sites.
• This C-terminal mediated binding to internal DNA may facilitate Ku recruitment to DNA damage sites.
• The findings suggest that extensions beyond the conserved Ku core domain have evolved independently to expand protein function.