Nucleotide binding interactions modulate dNTP selectivity and facilitate 8-oxo-dGTP incorporation by DNA polymerase lambda

Abstract

8-Oxo-7,8,-dihydro-2′-deoxyguanosine triphosphate (8-oxo-dGTP) is a major product of oxidative damage in the nucleotide pool. It is capable of mispairing with adenosine (dA), resulting in futile, mutagenic cycles of base excision repair. Therefore, it is critical that DNA polymerases discriminate against 8-oxo-dGTP at the insertion step. Because of its roles in oxidative DNA damage repair and non-homologous end joining, DNA polymerase lambda (Pol λ) may frequently encounter 8-oxo-dGTP. Here, we have studied the mechanisms of 8-oxo-dGMP incorporation and discrimination by Pol λ. We have solved high resolution crystal structures showing how Pol λ accommodates 8-oxo-dGTP in its active site. The structures indicate that when mispaired with dA, the oxidized nucleotide assumes the mutagenic syn-conformation, and is stabilized by multiple interactions. Steady-state kinetics reveal that two residues lining the dNTP binding pocket, Ala510 and Asn513, play differential roles in dNTP selectivity. Specifically, Ala510 and Asn513 facilitate incorporation of 8-oxo-dGMP opposite dA and dC, respectively. These residues also modulate the balance between purine and pyrimidine incorporation. Our results shed light on the mechanisms controlling 8-oxo-dGMP incorporation in Pol λ and on the importance of interactions with the incoming dNTP to determine selectivity in family X DNA polymerases.

Reading and Misreading 8-oxoguanine, a Paradigmatic Ambiguous Nucleobase

“…Pol β and Pol λ are among few polymerases for which incorporation of oxodGMP has been structurally characterized [262][263][264][265]. These structures illuminate general principles of non-mutagenic and mutagenic oxodGMP insertion and extension.…”

“…The role of the single active site residues in the stabilization of the mutagenic syn conformation of oxodGTP was also demonstrated in Pol β (Asn279) [197,198], Pol λ (Asn513) [263], Pol η (Arg61) and Pol κ (Tyr112) [266]. These residues are located in distinct positions in the active sites and modulate oxodGTP incorporation by different ways.…”

“…In contrast to the incorporation opposite oxoG, X-family Pol β and Pol λ have a clear preference both for forming pro-mutagenic A:oxoG pairs with the oxodGTP substrate [197,198,[262][263][264] and for extension from such primer termini [265]. Pol β and Pol λ incorporate oxodGMP opposite A with 11-40-fold preference over C [197,198,[262][263][264]. However, Pol β and Pol λ show reduced polymerase activity when incorporating oxodGMP.…”

“…Overall, the error-prone incorporation of oxodGMP opposite to A mostly arises from the destabilizing effect of O 8 -phosphate interactions in anti both at the insertion and the extension steps. The geometry of the A:oxodGMP mispair also causes some polymerase structure distortions in Pol λ but it still can be accommodated into the active site without major conformational rearrangements [263].…”

Structural Determinants Responsible for the Preferential Insertion of Ribonucleotides by Bacterial NHEJ PolDom

“…However, extensive structural and biochemical analyses showed that the incoming nucleotide-binding pocket is much more restrictive and specific amino acids in the active site of the DNA polymerases greatly determine the specificity for the insertion of 8oxodGMP into DNA by stabilizing either the syn or anti conformation of the glycosidic bond [reviewed in [63]]. Thus, in family X DNA polymerases β and λ an asparagine residue (Asn279 in Pol β and Asn513 in Pol λ) stabilizes the incoming 8oxodGTP (syn) through a hydrogen bonding with the C8 carbonyl [64][65][66]. The preferential insertion of 8oxodGMP in front of dC showed in the family B DNA polymerase from bacteriophage phi29 has been hypothesized to reside in the presence of a conserved lysine residue that would impair the syn conformation opposite dA [67].…”