Mechanism Of Genome Instability Mediated By Human DNA Polymerase Mu Misincorporation

In order to cope with different types of DNA damages,cells have evolved distinct mechanisms for DNA repair.In addition to the homologous recombination,non-homologous end joining(NHEJ)is involved in the repair of double-strand breaks,which is one of the most severe types of DNA damage.Because of the lack of paired DNA ends,NHEJ is generally thought as an error-prone pathway.Pol is capable of performing gap-filling repair synthesis in the NHEJ pathway.However,Pol lacks proofreading activity,with an intrinsically high error rate of 10-3 to 10-5 for dNTP misincorporation.Together with DNA ligase,the misincorporation of dGTP opposite the templating T by Polμ results in a promutagenic T:G mispair,leading to genomic instability.Here,crystal structures and kinetics of Pol substituting dGTP for dATP on gapped DNA substrates containing templating T were determined and compared.The main conclusions are:1.With different conditions,including different DNA substrates,catalyzed metal ions and correct/incorrect nucleotide incorporation,the active site configuration of Pol is almost identical,indicating the conserved two-metal-ion catalysis during dGTP misincorporation.2.Polμ is more effective for gap-filling synthesis on a 1-nt gapped DNA substrate.while for a 2-nt gapped DNA substrate,Pol prefers to insert opposite the 5’ unpaired template base of the gap(the so called“skipping ahead" mechanism).However,the mis-inserted nucleotide could be stabilized in the active site and form hydrogen bonds with the templating base T,which increases the misincorporation rate of Polμ.3.For Pol misincorporating dGTP opposite the templating T on a 1-nt gapped DNA substrate,the guanine base is not stable in the active site and causes the flipping conformation of 3’-end of the primer strand,which prevents the misincorporation.4.Two unique residues,Lys438 and Gln441,are involved in the dGTP misincorporation of Polμ.During the misincorporation,these two residues directly interact with the T:dGTP mispair.Alanine substitutions of either of these two residues drastically increase the dGTP misincorporation rate of Polμ.Moreover,alanine substitutions of both residues completely abolished the discrimination of the correct(dATP)and incorrect(dGTP)insertions of Polμ.5.Structural and biochemical analysis indicate that Pol could mis-insert two dGTPs on a 1-nt gapped DNA substrate,suggesting its potential mutagenic role among human X family polymerases in NHEJ.Taken together,based on the measurements of kinetic parameters and structural determination of Polμ in complexed with gapped DNA substrates containing templating T and incoming dGTP,our results revealed the mechanism of Polμ dGTP misincorporation,which further causes the genomic instability.