Crystal structures of a template-independent DNA polymerase: murine terminal deoxynucleotidyltransferase

Abstract

The crystal structure of the catalytic core of murine terminal deoxynucleotidyltransferase (TdT) at 2.35 A Ê resolution reveals a typical DNA polymerase b-like fold locked in a closed form. In addition, the structures of two different binary complexes, one with an oligonucleotide primer and the other with an incoming ddATP-Co 2+ complex, show that the substrates and the two divalent ions in the catalytic site are positioned in TdT in a manner similar to that described for the human DNA polymerase b ternary complex, suggesting a common two metal ions mechanism of nucleotidyl transfer in these two proteins. The inability of TdT to accommodate a template strand can be explained by steric hindrance at the catalytic site caused by a long lariat-like loop, which is absent in DNA polymerase b. However, displacement of this discriminating loop would be suf®cient to unmask a number of evolutionarily conserved residues, which could then interact with a template DNA strand. The present structure can be used to model the recently discovered human polymerase m, with which it shares 43% sequence identity.

Essential role for polymerase specialization in cellular nonhomologous end joining

“…We next addressed structural elements that could help distinguish the activities of Pol μ and Pol λ. When considering Pol μ, we and others have emphasized an extended loop insert (Loop1) in its palm subdomain (9,11,12). Deletion of this loop (Pol μ Δloop1 ), or substitution of a residue thought to help limit its mobility (Pol μ F385A ), specifically reduces activity on noncomplementary ends (its cognate substrate) in vitro (7).…”

“…All three polymerases are also active in synthesis during NHEJ in vitro, but there is an abundance of evidence for structural elements unique to each polymerase that account for striking differences in these in vitro activities (4)(5)(6)(7)(8). Most clearly, the general character of synthesis activities have a gradient of decreasing dependence on an intact template strand, Pol λ> Pol μ > TdT (9,10), that in part can be attributed to differences in a variable insert in the palm subdomain (Loop1) (9,11,12). However, evidence that these different in vitro activities effects the biological role of these polymerases is definitive only for TdT, which adds template-independent nucleotides (N additions) to broken intermediates during V(D)J recombination.…”

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

“…Loop1 is significantly larger in Pol l and TdT than in Pol b or Pol k [Delarue et al, 2002;Nick McElhinny et al, 2005;Juarez et al, 2006]. In a structure of TdT, the loop is positioned in the DNA binding site opposite the presumptive location of the primer [Delarue et al, 2002].…”

“…In a structure of TdT, the loop is positioned in the DNA binding site opposite the presumptive location of the primer [Delarue et al, 2002]. There is no comparable structure of Pol l, and loop1 is disordered in a structure of Pol l with gapped duplex [Moon et al, 2007b].…”

“…Elements that reduce dependency on continuous template. Structures from 1JMS (Delarue et al, 2002) and 2IHM (Moon et al, 2007b) were aligned in Pymol. Primer and downstream strand are in blue, template strand in green, and incoming nucleotide triphosphate in red, and are from 2IHM; the 6th and 7th nucleotides in the template strand from 2IHM were omitted.…”