The “A” rule revisited: polymerases as determinants of mutational specificity

DNA polymerases (DNAPs) responsible for genome replication are highly faithful enzymes that nonetheless cannot deal with damaged DNA. In contrast, translesion synthesis (TLS) DNAPs are suitable for replicating modified template bases, although resulting in very lowfidelity products. Here we report the biochemical characterization of the temperate bacteriophage Bam35 DNA polymerase (B35DNAP), which belongs to the protein-primed subgroup of family B DNAPs, along with phage Φ29 and other viral and mobile element polymerases. B35DNAP is a highly faithful DNAP that can couple strand displacement to processive DNA synthesis. These properties allow it to perform multiple displacement amplification of plasmid DNA with a very low error rate. Despite its fidelity and proofreading activity, B35DNAP was able to successfully perform abasic site TLS without template realignment and inserting preferably an A opposite the abasic site (A rule). Moreover, deletion of the TPR2 subdomain, required for processivity, impaired primer extension beyond the abasic site. Taken together, these findings suggest that B35DNAP may perform faithful and processive genome replication in vivo and, when required, TLS of abasic sites.protein-primed DNA polymerase | Bam35 | abasic sites | translesion synthesis | isothermal DNA amplification R eplicative DNA polymerases (DNAPs) from A and B families, collectively termed replicases, exhibit a "tight fit" for their DNA and dNTP substrates and are wondrously adapted to form correct Watson-Crick base pairs, resulting in very pronounced fidelity (1, 2). This strict preference to produce A:T and G:C base pairs is also the Achilles heel of faithful DNA polymerases, however, because they are strongly inhibited by modified nucleotides present at sites of DNA damage, leading to the stalling of replication fork and eventually to replicative stress and cell death (3). At the stalled replication fork, the DNA polymerase may be exchanged by a translesion synthesis (TLS) polymerase, generally belonging to the Y family. These enzymes possess looser solventexposed active sites, which allows them to deal with aberrant DNA features much better, although with a very low polymerization accuracy with the risk of the accumulation of mutations and genetic instability (4, 5). Alternatively, nonbulky modified bases, such as uracil and 8-oxo-deoxyguanosine (8oxoG), can be bypassed by replicases, with faithful or mutagenic outcomes that can be modified by the sequence context and dNTP availability (6, 7).Abasic or apurinic/apyrimidinic (AP) sites are the most common DNA lesions arising in cells when the N-glycosydic bond between the sugar moiety and the nucleobase is broken, either spontaneously or by a DNA glycosylase reaction product in the base excision repair pathway (8, 9). Unrepaired abasic sites are highly blocking lesions for replicative DNA polymerases (10), although mutant polymerases with impaired proofreading activity or with mutations in the polymerization active site residues that affect the incoming nucleotide sel...

Section: Resultssupporting

confidence: 86%

DNA polymerase from temperate phage Bam35 is endowed with processive polymerization and abasic sites translesion synthesis capacity

Berjón-Otero

Villar

Vega

et al. 2015

“…In addition, the pronounced 5Ј dependency for base incorporation by Dpo4 opposite BP-dG has not been observed for Dpo4 with other types of lesions. Bypass of the minor cis-(10R)-BP-N 6 -dA (BP-dA) adduct by Dpo4 strongly follows the A rule, which shows preferential insertion of dA opposite a lesion (21,31). This is consistent with the Dpo4/BP-dA structure in which the dA* is directly paired with dA at the active site (21).…”

Section: Bp-dg Looping-out and ؊1 Frameshift Mutationssupporting

confidence: 66%

A structural gap in Dpo4 supports mutagenic bypass of a major benzo[ a ]pyrene dG adduct in DNA through template misalignment

Bauer

Xing²,

Yagi³

et al. 2007

139

Erroneous replication of lesions in DNA by DNA polymerases leads to elevated mutagenesis. To understand the molecular basis of DNA damage-induced mutagenesis, we have determined the x-ray structures of the Y-family polymerase, Dpo4, in complex with a DNA substrate containing a bulky DNA lesion and incoming nucleotides. The DNA lesion is derived from an environmentally widespread carcinogenic polycyclic aromatic hydrocarbon, benzo[ a ]pyrene (BP). The potent carcinogen BP is metabolized to diol epoxides that form covalent adducts with cellular DNA. In the present study, the major BP diol epoxide adduct in DNA, BP- N 2 -deoxyguanosine (BP–dG), was placed at a template–primer junction. Three ternary complexes reveal replication blockage, extension past a mismatched lesion, and a −1 frameshift mutation. In the productive structures, the bulky adduct is flipped/looped out of the DNA helix into a structural gap between the little finger and core domains. Sequestering of the hydrophobic BP adduct in this new substrate-binding site permits the DNA to exhibit normal geometry for primer extension. Extrusion of the lesion by template misalignment allows the base 5′ to the adduct to serve as the template, resulting in a −1 frameshift. Subsequent strand realignment produces a mismatched base opposite the lesion. These structural observations, in combination with replication and mutagenesis data, suggest a model in which the additional substrate-binding site stabilizes the extrahelical nucleotide for lesion bypass and generation of base substitutions and −1 frameshift mutations.

“…However, incorporation of dGTP opposite the adduct by PGM1 is rarely observed even at extremely high nucleotide concentrations (Table 1); instead, the misincorporation frequency of dATP is increased relative to dCTP. We suspect that narrowing of the groove where the BPDE is accommodated may destabilize the binding of damaged template to PGM1 and lead to increased misincorporation of dATP by the A-rule (43,44). In the modeled structure of PGM2 (Fig.…”

Section: Discussionmentioning

confidence: 99%

Variants of mouse DNA polymerase κ reveal a mechanism of efficient and accurate translesion synthesis past a benzo[ a ]pyrene dG adduct

Liu

Yang

Tang

et al. 2014

DNA polymerase κ (Polκ) is the only known Y-family DNA polymerase that bypasses the 10S (+)-trans-anti-benzo[a]pyrene diol epoxide (BPDE)-N 2 -deoxyguanine adducts efficiently and accurately. The unique features of Polκ, a large structure gap between the catalytic core and little finger domain and a 90-residue addition at the N terminus known as the N-clasp, may give rise to its special translesion capability. We designed and constructed two mouse Polκ variants, which have reduced gap size on both sides [Polκ Gap Mutant (PGM) 1] or one side flanking the template base (PGM2). These Polκ variants are nearly as efficient as WT in normal DNA synthesis, albeit with reduced accuracy. However, PGM1 is strongly blocked by the 10S (+)-trans-anti-BPDE-N 2 -dG lesion. Steady-state kinetic measurements reveal a significant reduction in efficiency of dCTP incorporation opposite the lesion by PGM1 and a moderate reduction by PGM2. Consistently, Polκ-deficient cells stably complemented with PGM1 GFP-Polκ remained hypersensitive to BPDE treatment, and complementation with WT or PGM2 GFP-Polκ restored BPDE resistance. Furthermore, deletion of the first 51 residues of the N-clasp in mouse Polκ (mPolκ 52-516 ) leads to reduced polymerization activity, and the mutant PGM2 52-516 but not PGM1 52-516 can partially compensate the N-terminal deletion and restore the catalytic activity on normal DNA. However, neither WT nor PGM2 mPolκ 52-516 retains BPDE bypass activity. We conclude that the structural gap physically accommodates the bulky aromatic adduct and the N-clasp is essential for the structural integrity and flexibility of Polκ during translesion synthesis.translesion DNA synthesis | polycyclic aromatic hydrocarbons B enzo[a]pyrene (BP) is one of the best characterized polycyclic aromatic hydrocarbons generated during incomplete fuel combustion, in tobacco smoke, and in cooked food. The most mutagenic and tumorigenic metabolite of BP in vivo is the (+)-7R,8S,9S,10R-BP dihydrodiol epoxide [(+)-anti-BPDE] (1), which reacts readily with the exocyclic amino groups of guanine residues in DNA to form the major 10S (+)-trans-anti-BPDE-N 2 -dG adduct (2) (abbreviated as BPDE-dG hereafter). This adduct typically impedes normal DNA replication. Translesion DNA synthesis (TLS), which provides one mode of DNA damage tolerance, uses specialized bypass polymerases to synthesize DNA opposite and beyond a variety of replication-blocking lesions, thus avoiding replication fork collapse (3). The Y-family DNA polymerases are specialized for TLS. To date, four of them, namely DNA polymerase (Pol) η, Polι, Polκ, and REV1, have been identified in mammals (4-6). Polκ is the only one with homologs in bacteria (DinB; also known as Pol IV in Escherichia coli), archaea [DNA polymerase IV (Dpo4) in Sulfolobus solfataricus and DinB homologue (Dbh) in Sulfolobus acidocaldarius] and all eukaryotes. The mouse and human POLK gene are expressed ubiquitously, with highest levels in testis (7).Human Polκ can accurately incorporate dCMP opposite the BPDE-dG adduct ...