A comparison of BRCT domains involved in nonhomologous end-joining: Introducing the solution structure of the BRCT domain of polymerase lambda

Abstract

Three of the four family X polymerases, DNA polymerase λ, DNA polymerase μ, and TdT, have been associated with repair of double-strand DNA breaks by nonhomologous end-joining. Their involvement in this DNA repair process requires an N-terminal BRCT domain that mediates interaction with other protein factors required for recognition and binding of broken DNA ends. Here we present the NMR solution structure of the BRCT domain of DNA polymerase λ, completing the structural portrait for this family of enzymes. Analysis of the overall fold of the polymerase λ BRCT domain reveals structural similarity to the BRCT domains of polymerase μ and TdT, yet highlights some key sequence and structural differences that may account for important differences in the biological activities of these enzymes and their roles in nonhomologous end-joining. Mutagenesis studies indicate that the conserved Arg57 residue of Pol λ plays a more critical role for binding to the XRCC4-Ligase IV complex than its structural homolog in Pol μ, Arg43. In contrast, the hydrophobic Leu60 residue of Pol λ contributes less significantly to binding than the structurally homologous Phe46 residue of Pol μ. A third leucine residue involved in the binding and activity of Pol μ, is nonconservatively replaced by a glutamine in Pol λ (Gln64) and, based on binding and activity data, is apparently unimportant for Pol λ interactions with the NHEJ complex. In conclusion, both the structure of the Pol λ BRCT domain and its mode of interaction with the other components of the NHEJ complex significantly differ from the two previously studied homologs, Pol μ and TdT.