Structure of a preternary complex involving a prokaryotic NHEJ DNA polymerase

In many prokaryotes, a specific DNA primase/polymerase (PolDom) is required for nonhomologous end-joining repair of DNA double-strand breaks. Here, we report the crystal structure of a catalytically active conformation of Mycobacterium tuberculosis PolDom, consisting of a polymerase bound to a DNA end with a 3’ overhang, two metal ions and an incoming nucleotide but, significantly, lacking a primer strand. This structure represents a unique example of a polymerase:DNA complex in a pre-ternary intermediate state. This polymerase intermediate occurs in solution, stabilizing the enzyme on DNA ends and promoting nucleotide extension off short incoming termini. We also demonstrate that an invariant Arg220, contained in a conserved loop (Loop 2), plays an essential role in catalysis by regulating binding of a second metal ion at the active site. We propose that this NHEJ intermediate facilitates extension reactions involving critically short or non-complementary DNA ends thus promoting break repair and minimizing sequence loss during DSB repair