Efficient and Error-Free Replication Past a Minor-Groove DNA Adduct by the Sequential Action of Human DNA Polymerases ι and κ

Abstract

DNA polymerase (Pol) is a member of the Y family of DNA polymerases, which promote replication through DNA lesions. The role of Pol in lesion bypass, however, has remained unclear. Pol is highly unusual in that it incorporates nucleotides opposite different template bases with very different efficiencies and fidelities. Since interactions of DNA polymerases with the DNA minor groove provide for the nearly equivalent efficiencies and fidelities of nucleotide incorporation opposite each of the four template bases, we considered the possibility that Pol differs from other DNA polymerases in not being as sensitive to distortions of the minor groove at the site of the incipient base pair and that this enables it to incorporate nucleotides opposite highly distorting minor-groove DNA adducts. To check the validity of this idea, we examined whether Pol could incorporate nucleotides opposite the ␥-HOPdG adduct, which is formed from an initial reaction of acrolein with the N 2 of guanine. We show here that Pol incorporates a C opposite this adduct with nearly the same efficiency as it does opposite a nonadducted template G residue. The subsequent extension step, however, is performed by Pol, which efficiently extends from the C incorporated opposite the adduct. Based upon these observations, we suggest that an important biological role of Pol and Pol is to act sequentially to carry out the efficient and accurate bypass of highly distorting minor-groove DNA adducts of the purine bases.Replicative DNA polymerases synthesize DNA with high fidelity, and because of their intolerance of geometric distortions in DNA, they are blocked by DNA lesions. The DNA polymerases belonging to the Y family, however, are low-fidelity enzymes, able to promote replication through DNA lesions. DNA polymerase (Pol) from both the yeast Saccharomyces cerevisiae and humans can efficiently and accurately replicate through a cis-syn thymine-thymine dimer (16,19,43,45), and genetic studies in yeast and humans have also indicated a role for Pol in the error-free replication through cyclobutane dimers formed at 5Ј-TC-3Ј and CC sites (39, 49). Consequently, mutational inactivation of Pol in humans causes the variant form of xeroderma pigmentosum (15, 28), characterized by a greatly enhanced predisposition for sunlight-induced skin cancers. Pol can also efficiently replicate through other DNA lesions, such as 8-oxoguanine and O 6 -methylguanine (11, 14). Pol, however, is inhibited by the N 2 -guanine adducts of 1,3-butadiene or benzo[a]pyrene diol epoxide (30, 37).In addition to Pol, humans contain two other Y-family DNA polymerases, Pol and Pol. By contrast to Pol, which promotes lesion bypass both by efficiently inserting the nucleotide opposite the lesion and by extending from the inserted nucleotide, Pol and Pol are apparently more specialized in their roles in lesion bypass (36). For example, while Pol can incorporate nucleotides opposite the 3Ј T of a (6-4) TT photoproduct or opposite an abasic site, it is unable to carry out the subsequent extensio...

Human DNA polymerase κ forms nonproductive complexes with matched primer termini but not with mismatched primer termini

“…The structures of pol in ternary complexes with DNA and dNTPs show that this enzyme uses Hoogsteen base pairing between the template residue and the incoming nucleotide (20,21). This allows pol to replicate through template lesions not capable of forming Watson-Crick base pairs or template lesions containing substantial minor groove modifications (18,19,22). The Rev1 protein is required for DNA damage-induced mutagenesis (23), and this enzyme only incorporates nucleotides opposite a narrow range of templates that include nondamaged G residues (24), abasic sites (24,25), and N 2 -adducted G residues (26).…”

“…Pol catalyzes nucleotide incorporation opposite a variety of DNA lesions, including abasic sites (15, 16), the 3Ј T of (6-4) photoproducts (15-17), and the N 2 -adducted G residues (18,19). The structures of pol in ternary complexes with DNA and dNTPs show that this enzyme uses Hoogsteen base pairing between the template residue and the incoming nucleotide (20,21).…”

“…In addition, pol can efficiently extend from aberrant primer-terminal base pairs containing certain template lesions. For example, it extends readily from nucleotides incorporated opposite the 3Ј T of a thymine dimer (31), O 6 -methylguanine and 8-oxoguanine (32), and N 2 -adducted G residues (18,19).…”

“…In addition, pol can efficiently extend from aberrant primer-terminal base pairs containing certain template lesions. For example, it extends readily from nucleotides incorporated opposite the 3Ј T of a thymine dimer (31), O 6 -methylguanine and 8-oxoguanine (32), and N 2 -adducted G residues (18,19).To better understand how pol tolerates mismatched primerterminal base pairs, we carried out pre-steady-state kinetic studies with this polymerase. Although steady-state kinetic studies are often used to quantify the efficiency of nucleotide incorporation on matched and mismatched DNA substrates, they provide no information regarding the mechanism of nucleotide incorporation.…”

Proficient and Accurate Bypass of Persistent DNA Lesions by DinB DNA Polymerases

“…34 Curiously, both DinB and pol k proficiently extend from a variety of lesions and mismatched primer ends in addition to their insertion specificities. 8,11,[35][36][37][38][39] It has been suggested, at least for pol k, that this property reflects a separate role in the extension step of TLS. 35 …”