Structure of a preternary complex involving a prokaryotic NHEJ DNA polymerase

Area of Science:

• Structural Biology
• Molecular Biology
• Biochemistry

Background:

• Nonhomologous end joining (NHEJ) is a critical DNA double-strand break (DSB) repair pathway in prokaryotes.
• A specific DNA primase/polymerase (PolDom) is essential for NHEJ in many prokaryotic organisms.

Purpose of the Study:

• To elucidate the structural and mechanistic basis of Mycobacterium tuberculosis PolDom's role in NHEJ.
• To characterize the catalytically active conformation of PolDom bound to DNA.

Main Methods:

• X-ray crystallography was used to determine the structure of Mycobacterium tuberculosis PolDom.
• Biochemical assays were employed to investigate the role of specific amino acids and metal ions in catalysis.

Main Results:

• The crystal structure revealed a preternary intermediate state of PolDom bound to a DNA end with a 3' overhang, two metal ions, and an incoming nucleotide, lacking a primer strand.
• This complex stabilizes the enzyme on DNA ends, promoting nucleotide extension of short termini.
• The invariant Arg(220) in loop 2 is crucial for catalysis, regulating the binding of a second metal ion.

Conclusions:

• The identified NHEJ intermediate facilitates extension reactions on short or noncomplementary DNA ends.
• This mechanism is vital for efficient DSB repair and minimizing sequence loss during the process.
• The findings provide insights into the molecular mechanisms of DNA repair in prokaryotes.