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.

Repair of Double-Strand Breaks by End Joining

“…c-NHEJ specificity of Pol X polymerases is supported by an amino-terminal BRCT domain that interacts with the core c-NHEJ enzymes (Fig. 4B) (DeRose et al 2007;Mueller et al 2008). Artemis interacts closely with DNA-PKcs (Kurosawa and Adachi 2010;Yan et al 2010) as well as Lig4 (Malu et al 2012).…”

DNA polymerases in nonhomologous end joining: Are there any benefits to standing out from the crowd?

“…As with other BRCT domains, the BRCT domain in TdT can also interact with a phosphoserine-containing motif, but whether this has functional relevance is not yet clear [Yu et al, 2003]. Rather, in all examples tested (Pol4 [Tseng and Tomkinson, 2002] as well as vertebrate Pol k [Fan and Wu, 2004;Lee et al, 2004;Ma et al, 2004;Nick McElhinny et al, 2005;Mueller et al, 2008], Pol l [Mahajan et al, 2002;Ma et al, 2004;Nick McElhinny et al, 2005;DeRose et al, 2007] and TdT), the BRCT domain is required for physical interaction with Ku and XRCC4-ligase IV at DNA ends, and there is as yet Environmental and Molecular Mutagenesis. DOI 10.1002/em no contribution of Ku, XRCC4, or ligase IV phosphorylation.…”

“…Structures are available for the BRCT domains from all three mammalian Pol X members [DeRose et al, 2007;Mueller et al, 2008]. Pol l and TdT are similar (40% identical), whereas Pol k is much less so; the BRCT domain from Pol k is only 23 and 20% identical to BRCT domains from Pol l and TdT, respectively (Fig.…”

“…An arginine (R43) and exposed hydrophobic residues (F46 and L50) in this helix are conserved through vertebrate Pol l and TdT [DeRose et al, 2007], and are important for both interaction of Pol l with Ku and XRCC4-ligase IV at DNA ends as well as Pol l activity in NHEJ. There are analogous residues in Pol k for the first two positions (R57 and L60), and these residues are also important for NHEJ interactions [Mueller et al, 2008], but R57 in Pol k is displaced relative to R43 in Pol l. Whether the surprisingly distinct interaction surfaces result in functionally significant differences in how Pol k and Pol l interact with the NHEJ complex remains unclear.…”

“…Comparison of Pol X BRCT domain structures. Molecular surfaces of Pol k (cyan) and Pol l (green) BRCT domains are represented, with amino acids critical for NHEJ labeled (Mueller et al, 2008).…”