Mechanistic Basis for the Bypass of a Bulky DNA Adduct Catalyzed by a Y-Family DNA Polymerase.

1-Nitropyrene (1-NP), an environmental pollutant, induces DNA damage in vivo and is considered to be carcinogenic. The DNA adducts formed by the 1-NP metabolites stall replicative DNA polymerases but are presumably bypassed by error-prone Y-family DNA polymerases at the expense of replication fidelity and efficiency in vivo. Our running start assays confirmed that a site-specifically placed 8-(deoxyguanosin-N(2)-yl)-1-aminopyrene (dG(1,8)), one of the DNA adducts derived from 1-NP, can be bypassed by Sulfolobus solfataricus DNA polymerase IV (Dpo4), although this representative Y-family enzyme was paused strongly by the lesion. Pre-steady-state kinetic assays were employed to determine the low nucleotide incorporation fidelity and establish a minimal kinetic mechanism for the dG(1,8) bypass by Dpo4. To reveal a structural basis for dCTP incorporation opposite dG(1,8), we solved the crystal structures of the complexes of Dpo4 and DNA containing a templating dG(1,8) lesion in the absence or presence of dCTP. The Dpo4·DNA-dG(1,8) binary structure shows that the aminopyrene moiety of the lesion stacks against the primer/template junction pair, while its dG moiety projected into the cleft between the Finger and Little Finger domains of Dpo4. In the Dpo4·DNA-dG(1,8)·dCTP ternary structure, the aminopyrene moiety of the dG(1,8) lesion, is sandwiched between the nascent and junction base pairs, while its base is present in the major groove. Moreover, dCTP forms a Watson-Crick base pair with dG, two nucleotides upstream from the dG(1,8) site, creating a complex for "-2" frameshift mutation. Mechanistically, these crystal structures provide additional insight into the aforementioned minimal kinetic mechanism.

Structural and kinetic insights into binding and incorporation of L-nucleotide analogs by a Y-family DNA polymerase.

Considering that all natural nucleotides (D-dNTPs) and the building blocks (D-dNMPs) of DNA chains possess D-stereochemistry, DNA polymerases and reverse transcriptases (RTs) likely possess strongD-stereoselectivity by preferably binding and incorporating D-dNTPs over unnatural L-dNTPs during DNA synthesis. Surprisingly, a structural basis for the discrimination against L-dNTPs by DNA polymerases or RTs has not been established although L-deoxycytidine analogs (lamivudine and emtricitabine) and L-thymidine (telbivudine) have been widely used as antiviral drugs for years. Here we report seven high-resolution ternary crystal structures of a prototype Y-family DNA polymerase, DNA, and D-dCTP, D-dCDP, L-dCDP, or the diphosphates and triphosphates of lamivudine and emtricitabine. These structures reveal that relative to D-dCTP, each of these L-nucleotides has its sugar ring rotated by 180° with an unusual O4'-endo sugar puckering and exhibits multiple triphosphate-binding conformations within the active site of the polymerase. Such rare binding modes significantly decrease the incorporation rates and efficiencies of these L-nucleotides catalyzed by the polymerase.

Kinetic analysis of the bypass of a bulky DNA lesion catalyzed by human Y-family DNA polymerases.

1-Nitropyrene (1-NP), a mutagen and potential carcinogen, is the most abundant nitro polyaromatic hydrocarbon in diesel exhaust, which reacts with DNA to form predominantly N-(deoxyguanosin-8-yl)-1-aminopyrene (dG(AP)). If not repaired, this DNA lesion is presumably bypassed in vivo by any of human Y-family DNA polymerases kappa (hPolκ), iota (hPolι), eta (hPolη), and Rev1 (hRev1). Our running start assays demonstrated that each of these enzymes was indeed capable of traversing a site-specifically placed dG(AP) on a synthetic DNA template but that hRev1 was stopped after lesion bypass. The time required to bypass 50% of the dG(AP) sites (t(50)(bypass)) encountered by hPolη, hPolκ, and hPolι was determined to be 2.5 s, 4.1 s, and 106.5 s, respectively. The efficiency order of catalyzing translesion synthesis of dG(AP) (hPolη > hPolκ > hPolι ≫ hRev1) is the same as the order for these human Y-family enzymes to elongate undamaged DNA. Although hPolη bypassed dG(AP) efficiently, replication by both hPolκ and hPolι was strongly stalled at the lesion site and at a site immediately downstream from dG(AP). By employing presteady state kinetic methods, a kinetic basis was established for polymerase pausing at these DNA template sites. Besides efficiency of bypass, the fidelity of those low-fidelity polymerases at these pause sites was also significantly decreased. Thus, if the translesion DNA synthesis of dG(AP)in vivo is catalyzed by a human Y-family DNA polymerase, e.g., hPolη, the process is certainly mutagenic.

3-Oxocyclo-butane-carboxylic acid: hydrogen bonding in a small-ring γ-keto acid.

The title ketocarboxylic acid, C(5)H(6)O(3), is the smallest carboxy-cyclanone to have its crystal structure determined. It adopts a chiral conformation, by rotation of its carboxyl O atoms away from the plane of skeletal symmetry that passes through the carboxyl carbon and both atoms of the ketone carbonyl. The four-membered ring is non-planar, with a shallow fold of 14.3 (1)° along a line connecting the two α-carbons of the ketone group. In the crystal, the molecules are linked by centrosymmetric hydrogen-bond pairing of ordered carboxylic acid groups [O⋯O = 2.6392 (12) Šand O-H⋯O = 175.74 (15)°], yielding two different sets of dimers, related by by a 2(1) screw axis in c, in the cell. A C-H⋯O interaction is also present.

(±)-2-Oxocyclo-penta-neacetic acid: catemeric hydrogen bonding in a γ-keto acid.

The title racemate, C(7)H(10)O(3), aggregates in the solid as acid-to-ketone hydrogen-bonding catemers [O⋯O = 2.7050 (13) Šand O-H⋯O = 166.1 (17)°] having glide-related components. Four such heterochiral chains, paired centrosymmetrically about (, , ) in the cell, proceed through the cell in the 010 direction, with alignment with respect to the c axis of ++--.

(2RS,8aRS)-6-Oxo-1,2,3,4,6,7,8,8a-octa-hydro-naphthalene-2-carboxylic acid.

The title racemate, C(11)H(14)O(3), aggregates in the crystal structure as acid-to-ketone O-H⋯O hydrogen-bonding catemers whose components are glide-related. The relative stereochemistry at the carboxyl group arises spontaneously during the synthesis. Two inter-molecular C-H⋯O=C close contacts were found, both involving the acid group.

(2SR,4aSR,8aSR)-6-Oxoperhydro-naphthalene-2-carboxylic acid.

In the title racemic compound, C(11)H(16)O(3), the mol-ecule adopts a conformation that places its carboxyl group in an equatorial position. Mol-ecules aggregate by hydrogen-bond pairing of carboxyl groups, yielding centrosymmetric dimers that are arranged into layers in the (020) planes.