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A fidelity mechanism in DNA polymerase lambda promotes error-free bypass of 8-oxo-dG.

ABSTRACT: 8-oxo-7,8-dihydroxy-2'-deoxyguanosine (8-oxo-dG) has high mutagenic potential as it is prone to mispair with deoxyadenine (dA). In order to maintain genomic integrity, post-replicative 8-oxo-dG:dA mispairs are removed through DNA polymerase lambda (Pol ?)-dependent MUTYH-initiated base excision repair (BER). Here, we describe seven novel crystal structures and kinetic data that fully characterize 8-oxo-dG bypass by Pol ?. We demonstrate that Pol ? has a flexible active site that can tolerate 8-oxo-dG in either the anti- or syn-conformation. Importantly, we show that discrimination against the pro-mutagenic syn-conformation occurs at the extension step and identify the residue responsible for this selectivity. This residue acts as a kinetic switch, shunting repair toward long-patch BER upon correct dCMP incorporation, thus enhancing repair efficiency. Moreover, this switch also provides a potential mechanism to increase repair fidelity of MUTYH-initiated BER.

SUBMITTER: Burak MJ

PROVIDER: S-EPMC5282837 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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A fidelity mechanism in DNA polymerase lambda promotes error-free bypass of 8-oxo-dG.

Burak Matthew J MJ Guja Kip E KE Hambardjieva Elena E Derkunt Burak B Garcia-Diaz Miguel M

The EMBO journal 20160801 18

8-oxo-7,8-dihydroxy-2'-deoxyguanosine (8-oxo-dG) has high mutagenic potential as it is prone to mispair with deoxyadenine (dA). In order to maintain genomic integrity, post-replicative 8-oxo-dG:dA mispairs are removed through DNA polymerase lambda (Pol λ)-dependent MUTYH-initiated base excision repair (BER). Here, we describe seven novel crystal structures and kinetic data that fully characterize 8-oxo-dG bypass by Pol λ. We demonstrate that Pol λ has a flexible active site that can tolerate 8-o ...[more]

PMID: 27481934

Similar Datasets

A switch between DNA polymerases δ and λ promotes error-free bypass of 8-oxo-G lesions.

Project description:7,8-Dihydro-8-oxoguanine (8-oxo-G) is a highly abundant and mutagenic lesion. Replicative DNA polymerases (pols) are slowed down at 8-oxo-G and insert both correct cytosine (C) and incorrect adenine (A) opposite 8-oxo-G, but they preferentially extend A:8-oxo-G mispairs. Nevertheless, 8-oxo-G bypass is fairly accurate in vivo. Thus, the question how correct bypass of 8-oxo-G lesions is accomplished despite the poor extension of C:8-oxo-G base pairs by replicative pols remains unanswered. Here we show that replicative pol δ pauses in front of 8-oxo-G and displays difficulties extending from correct C:8-oxo-G in contrast to extension from incorrect A:8-oxo-G. This leads to stalling of pol δ at 8-oxo-G after incorporation of correct C. This stalling at C:8-oxo-G can be overcome by a switch from pol δ to pols λ, β, or η, all of which are able to assist pol δ in 8-oxo-G bypass by translesion synthesis (TLS). Importantly, however, only pol λ selectively catalyzes the correct TLS past 8-oxo-G, whereas pols β and η show no selectivity and even preferentially enhance incorrect TLS. The selectivity of pol λ to promote the correct bypass depends on its N-terminal domain. Furthermore, pol λ(-/-) mouse embryonic fibroblast extracts display reduced 8-oxo-G TLS. Finally, the correct bypass of 8-oxo-G in gapped plasmids in mouse embryonic fibroblasts and HeLa cells is promoted in the presence of pol λ. Our findings suggest that even though 8-oxo-G is not a blocking lesion per se, correct replication over 8-oxo-G is promoted by a pol switch between pols δ and λ.

| S-EPMC3528542 | biostudies-literature

The Werner syndrome protein limits the error-prone 8-oxo-dG lesion bypass activity of human DNA polymerase kappa.

Project description:Human DNA polymerase kappa (hpol κ) is the only Y-family member to preferentially insert dAMP opposite 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) during translesion DNA synthesis. We have studied the mechanism of action by which hpol κ activity is modulated by the Werner syndrome protein (WRN), a RecQ helicase known to influence repair of 8-oxo-dG. Here we show that WRN stimulates the 8-oxo-dG bypass activity of hpol κ in vitro by enhancing the correct base insertion opposite the lesion, as well as extension from dC:8-oxo-dG base pairs. Steady-state kinetic analysis reveals that WRN improves hpol κ-catalyzed dCMP insertion opposite 8-oxo-dG ∼10-fold and extension from dC:8-oxo-dG by 2.4-fold. Stimulation is primarily due to an increase in the rate constant for polymerization (kpol), as assessed by pre-steady-state kinetics, and it requires the RecQ C-terminal (RQC) domain. In support of the functional data, recombinant WRN and hpol κ were found to physically interact through the exo and RQC domains of WRN, and co-localization of WRN and hpol κ was observed in human cells treated with hydrogen peroxide. Thus, WRN limits the error-prone bypass of 8-oxo-dG by hpol κ, which could influence the sensitivity to oxidative damage that has previously been observed for Werner's syndrome cells.

| S-EPMC4231769 | biostudies-literature

Structural Basis for Error-Free Bypass of the 5-N-Methylformamidopyrimidine-dG Lesion by Human DNA Polymerase ? and Sulfolobus solfataricus P2 Polymerase IV.

Project description:N(6)-(2-Deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine (MeFapy-dG) arises from N7-methylation of deoxyguanosine followed by imidazole ring opening. The lesion has been reported to persist in animal tissues. Previous in vitro replication bypass investigations of the MeFapy-dG adduct revealed predominant insertion of C opposite the lesion, dependent on the identity of the DNA polymerase (Pol) and the local sequence context. Here we report crystal structures of ternary Pol·DNA·dNTP complexes between MeFapy-dG-adducted DNA template:primer duplexes and the Y-family polymerases human Pol ? and P2 Pol IV (Dpo4) from Sulfolobus solfataricus. The structures of the hPol ? and Dpo4 complexes at the insertion and extension stages, respectively, are representative of error-free replication, with MeFapy-dG in the anti conformation and forming Watson-Crick pairs with dCTP or dC.

| S-EPMC4545269 | biostudies-literature

DNA polymerase λ promotes error-free replication through Watson-Crick impairing N1-methyl-deoxyadenosine adduct in conjunction with DNA polymerase ζ.

Project description:In a previous study, we showed that replication through the N1-methyl-deoxyadenosine (1-MeA) adduct in human cells is mediated via three different Polι/Polθ, Polη, and Polζ-dependent pathways. Based on biochemical studies with these Pols, in the Polι/Polθ pathway, we inferred a role for Polι in the insertion of a nucleotide (nt) opposite 1-MeA and of Polθ in extension of synthesis from the inserted nt; in the Polη pathway, we inferred that this Pol alone would replicate through 1-MeA; in the Polζ pathway, however, the Pol required for inserting an nt opposite 1-MeA had remained unidentified. In this study, we provide biochemical and genetic evidence for a role for Polλ in inserting the correct nt T opposite 1-MeA, from which Polζ would extend synthesis. The high proficiency of purified Polλ for inserting a T opposite 1-MeA implicates a role for Polλ-which normally uses W-C base pairing for DNA synthesis-in accommodating 1-MeA in a syn confirmation and forming a Hoogsteen base pair with T. The potential of Polλ to replicate through DNA lesions by Hoogsteen base pairing adds another novel aspect to Polλ's role in translesion synthesis in addition to its role as a scaffolding component of Polζ. We discuss how the action mechanisms of Polλ and Polζ could be restrained to inserting a T opposite 1-MeA and extending synthesis thereafter, respectively.

| S-EPMC8260881 | biostudies-literature

Unique active site promotes error-free replication opposite an 8-oxo-guanine lesion by human DNA polymerase iota.

Project description:The 8-oxo-guanine (8-oxo-G) lesion is the most abundant and mutagenic oxidative DNA damage existing in the genome. Due to its dual coding nature, 8-oxo-G causes most DNA polymerases to misincorporate adenine. Human Y-family DNA polymerase iota (polι) preferentially incorporates the correct cytosine nucleotide opposite 8-oxo-G. This unique specificity may contribute to polι's biological role in cellular protection against oxidative stress. However, the structural basis of this preferential cytosine incorporation is currently unknown. Here we present four crystal structures of polι in complex with DNA containing an 8-oxo-G lesion, paired with correct dCTP or incorrect dATP, dGTP, and dTTP nucleotides. An exceptionally narrow polι active site restricts the purine bases in a syn conformation, which prevents the dual coding properties of 8-oxo-G by inhibiting syn/anti conformational equilibrium. More importantly, the 8-oxo-G base in a syn conformation is not mutagenic in polι because its Hoogsteen edge does not form a stable base pair with dATP in the narrow active site. Instead, the syn 8-oxo-G template base forms the most stable replicating base pair with correct dCTP due to its small pyrimidine base size and enhanced hydrogen bonding with the Hoogsteen edge of 8-oxo-G. In combination with site directed mutagenesis, we show that Gln59 in the finger domain specifically interacts with the additional O(8) atom of the lesion base, which influences nucleotide selection, enzymatic efficiency, and replication stalling at the lesion site. Our work provides the structural mechanism of high-fidelity 8-oxo-G replication by a human DNA polymerase.

| S-EPMC3044400 | biostudies-literature

Following an environmental carcinogen N2-dG adduct through replication: elucidating blockage and bypass in a high-fidelity DNA polymerase.

Project description:We have investigated how a benzo[a]pyrene-derived N2-dG adduct, 10S(+)-trans-anti-[BP]-N2-dG ([BP]G*), is processed in a well-characterized Pol I family model replicative DNA polymerase, Bacillus fragment (BF). Experimental results are presented that reveal relatively facile nucleotide incorporation opposite the lesion, but very inefficient further extension. Computational studies follow the possible bypass of [BP]G* through the pre-insertion, insertion and post-insertion sites as BF alternates between open and closed conformations. With dG* in the normal B-DNA anti conformation, BP seriously disturbs the polymerase structure, positioning itself either deeply in the pre-insertion site or on the crowded evolving minor groove side of the modified template, consistent with a polymerase-blocking conformation. With dG* in the less prevalent syn conformation, BP causes less distortion: it is either out of the pre-insertion site or in the major groove open pocket of the polymerase. Thus, the syn conformation can account for the observed relatively easy incorporation of nucleotides, with mutagenic purines favored, opposite the [BP]G* adduct. However, with the lesion in the BF post-insertion site, more serious distortions caused by the adduct even in the syn conformation explain the very inefficient extension observed experimentally. In vivo, a switch to a potentially error-prone bypass polymerase likely dominates translesion bypass.

| S-EPMC1934992 | biostudies-literature

Mechanism of error-free and semitargeted mutagenic bypass of an aromatic amine lesion by Y-family polymerase Dpo4.

Project description:The aromatic amine carcinogen 2-aminofluorene (AF) forms covalent adducts with DNA, predominantly with guanine at the C8 position. Such lesions are bypassed by Y-family polymerases such as Dpo4 via error-free and error-prone mechanisms. We show that Dpo4 catalyzes elongation from a correct 3'-terminal cytosine opposite [AF]G in a nonrepetitive template sequence with low efficiency. This extension leads to cognate full-length product, as well as mis-elongated products containing base mutations and deletions. Crystal structures of the Dpo4 ternary complex, with the 3'-terminal primer cytosine base opposite [AF]G in the anti conformation and with the AF moiety positioned in the major groove, reveal both accurate and misalignment-mediated mutagenic extension pathways. The mutagenic template-primer-dNTP arrangement is promoted by interactions between the polymerase and the bulky lesion rather than by a base pair-stabilized misaligment. Further extension leads to semitargeted mutations via this proposed polymerase-guided mechanism.

| S-EPMC4215948 | biostudies-literature

Ring-opening of the ?-OH-PdG adduct promotes error-free bypass by the Sulfolobus solfataricus DNA polymerase Dpo4.

Project description:Acrolein, a mutagenic aldehyde, reacts with deoxyguanosine (dG) to form 3-(2'-deoxy-?-d-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a] purin-10(3H)-one (?-OH-PdG). When placed opposite deoxycytosine (dC) in DNA, ?-OH-PdG undergoes ring-opening to the N(2)-(3-oxopropyl)-dG. Ring-opening of the adduct has been hypothesized to facilitate nonmutagenic bypass, particularly by DNA polymerases of the Y family. This study examined the bypass of ?-OH-PdG by Sulfolobus solfataricus Dpo4, the prototypic Y-family DNA polymerase, using templates that contained the adduct in either the 5'-CXG-3' or the 5'-TXG-3' sequence context. Although ?-OH-PdG partially blocked Dpo4-catalyzed DNA synthesis, full primer extension was observed, and the majority of bypass products were error-free. Conversion of the adduct into an irreversibly ring-opened derivative prior to reaction facilitated bypass and further improved the fidelity. Structures of ternary Dpo4·DNA·dNTP complexes were determined with primers that either were positioned immediately upstream of the lesion (preinsertion complexes) or had a 3'-terminal dC opposite the lesion (postinsertion complexes); the incoming nucleotides, either dGTP or dATP, were complementary to the template 5'-neighbor nucleotide. In both postinsertion complexes, the adduct existed as ring-opened species, and the resulting base-pair featured Watson-Crick hydrogen bonding. The incoming nucleotide paired with the 5'-neighbor template, while the primer 3'-hydroxyl was positioned to facilitate extension. In contrast, ?-OH-PdG was in the ring-closed form in both preinsertion complexes, and the overall structure did not favor catalysis. These data provide insights into ?-OH-PdG chemistry during replication bypass by the Dpo4 DNA polymerase and may explain why ?-OH-PdG-induced mutations due to primer-template misalignment are uncommon.

| S-EPMC3775444 | biostudies-literature

Stepwise translocation of Dpo4 polymerase during error-free bypass of an oxoG lesion.

Project description:7,8-dihydro-8-oxoguanine (oxoG), the predominant lesion formed following oxidative damage of DNA by reactive oxygen species, is processed differently by replicative and bypass polymerases. Our kinetic primer extension studies demonstrate that the bypass polymerase Dpo4 preferentially inserts C opposite oxoG, and also preferentially extends from the oxoG*C base pair, thus achieving error-free bypass of this lesion. We have determined the crystal structures of preinsertion binary, insertion ternary, and postinsertion binary complexes of oxoG-modified template-primer DNA and Dpo4. These structures provide insights into the translocation mechanics of the bypass polymerase during a complete cycle of nucleotide incorporation. Specifically, during noncovalent dCTP insertion opposite oxoG (or G), the little-finger domain-DNA phosphate contacts translocate by one nucleotide step, while the thumb domain-DNA phosphate contacts remain fixed. By contrast, during the nucleotidyl transfer reaction that covalently incorporates C opposite oxoG, the thumb-domain-phosphate contacts are translocated by one nucleotide step, while the little-finger contacts with phosphate groups remain fixed. These stepwise conformational transitions accompanying nucleoside triphosphate binding and covalent nucleobase incorporation during a full replication cycle of Dpo4-catalyzed bypass of the oxoG lesion are distinct from the translocation events in replicative polymerases.

| S-EPMC1325099 | biostudies-literature

Loop 1 modulates the fidelity of DNA polymerase lambda.

Project description:Differences in the substrate specificity of mammalian family X DNA polymerases are proposed to partly depend on a loop (loop 1) upstream of the polymerase active site. To examine if this is the case in DNA polymerase λ (pol λ), here we characterize a variant of the human polymerase in which nine residues of loop 1 are replaced with four residues from the equivalent position in pol β. Crystal structures of the mutant enzyme bound to gapped DNA with and without a correct dNTP reveal that the change in loop 1 does not affect the overall structure of the protein. Consistent with these structural data, the mutant enzyme has relatively normal catalytic efficiency for correct incorporation, and it efficiently participates in non-homologous end joining of double-strand DNA breaks. However, DNA junctions recovered from end-joining reactions are more diverse than normal, and the mutant enzyme is substantially less accurate than wild-type pol λ in three different biochemical assays. Comparisons of the binary and ternary complex crystal structures of mutant and wild-type pol λ suggest that loop 1 modulates pol λ's fidelity by controlling dNTP-induced movements of the template strand and the primer-terminal 3'-OH as the enzyme transitions from an inactive to an active conformation.

| S-EPMC2938210 | biostudies-literature

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