Edward L. Loechler

To bypass DNA damage, cells have Y-Family DNA polymerases (DNAPs). One Y-Family-class includes DNAP κ and DNAP IV, which accurately insert dCTP opposite N²-dG adducts, including from the carcinogen benzo[a]pyrene (BP). Another class includes DNAP η and DNAP V, which insert accurately opposite UV-damage, but inaccurately opposite BP-N²-dG. To investigate structural differences between Y-Family-classes, Dpo4 (a canonical η/V-class-member) is modified to make it more κ/IV-like, as evaluated via primer-extension studies with a BP-N²-dG-containing template. Three protein structural elements are identified that promote fidelity. (1) Watson-Crick-like [dCTP:BP-N²-dG] pairing requires the BP-moiety to be in the minor groove. Thus, as expected, dCTP insertion is facilitated by having large openings in the protein surface that can accommodate BP-bulk in the minor groove. The BP-moiety is also in the minor groove during dATP and dTTP misinsertion, though evidence suggests that each of these three minor groove BP-conformations differ. (2) Plugging an opening on the major groove side of the protein suppresses dGTP misinsertion, implying BP-N²-dG bulk is in the major groove during Hoogsteensyn-adduct-dG:dGTP pairing. (3) Y-Family DNAPs have non-covalent bridges (NCBs) holding their little finger-domain in contact with their catalytic core-domain; dATP/dGTP/dTTP misinsertions are suppressed by the quantity and quality of NCBs, including one near and another distal to the active site on the minor groove side. In conclusion, three protein structural elements enhance dCTP and/or suppress dATP/dGTP/dTTP insertion; four different BP-adduct conformations are responsible for the four different dNTP insertional pathways opposite BP-N²-dG; generalizations about Y-Family structure are also considered.

DNA adducts, which block replicative DNA polymerases (DNAPs), are often bypassed by lesion‐bypass DNAPs, which are mostly in the Y‐Family. Y‐Family DNAPs can do non‐mutagenic or mutagenic dNTP insertion, and understanding this difference is important, because mutations transform normal into tumorigenic cells. Y‐Family DNAP architecture that dictates mechanism, as revealed in structural and modeling studies, is considered. Steps from adduct blockage of replicative DNAPs, to bypass by a lesion‐bypass DNAP, to resumption of synthesis by a replicative DNAP are described. Catalytic steps and protein conformational changes are considered. One adduct is analyzed in greater detail: the major benzo[a]pyrene adduct (B[a]P‐N²‐dG), which is bypassed non‐mutagenically (dCTP insertion) by Y‐family DNAPs in the IV/κ‐class and mutagenically (dATP insertion) by V/η‐class Y‐Family DNAPs. Important architectural differences between IV/κ‐class versus V/η‐class DNAPs are discussed, including insights gained by analyzing ~400 sequences each for bacterial DNAPs IV and V, along with sequences from eukaryotic DNAPs kappa, eta and iota. The little finger domains of Y‐Family DNAPs do not show sequence conservation; however, their structures are remarkably similar due to the presence of a core of hydrophobic amino acids, whose exact identity is less important than the hydrophobic amino acid spacing.

Benzo[a]pyrene (B[a]P) is a polycyclic aromatic hydrocarbon (PAH) and a potent mutagen/carcinogen found ubiquitously in the environment. B[a]P is primarily metabolized to diol epoxides, which react principally at N²-dG in DNA. B[a]P–N²-dG adducts have been shown to induce a variety of mutations, notably G→T, G→A, G→C and −1 frameshifts. Four stereoisomers of B[a]P–N²-dG (designated: [+ta]-;, [+ca]-, [−ta] and [−ca]) were studied by NMR in duplex 11mers in a 5′-CGC sequence context, and each adopted a different adduct conformation (Geacintov, et al. (1997) Chem. Res. Toxicol., 10, 111). Herein these four identical B[a]P-containing 11mers are built into duplex plasmid genomes and mutagenesis studied in Escherichia coli following SOS-induction. In nucleotide excision repair (NER) proficient E.coli, no adduct-derived mutants are detected. In NER deficient E.coli, G→T mutations dominate for all four stereoisomers [+ta]-, [+ca]-, [−ta] and [−ca]-B[a]P–N²-dG, and mutation frequency is similar. Thus, the mutagenic pattern for these four B[a]P–N²-dG stereoisomers is the same, in spite of the fact that they adopt dramatically different conformations in ds-oligonucleotides as determined by NMR. These findings suggest that adduct conformation must be fluid enough in the 5′-CGC sequence that the duplex DNA conformation can interconvert to mutagenic and non-mutagenic conformations during lesion-bypass. A comparison of all published studies with these four B[a]P–N²-dG stereoisomers in E.coli reveals that B[a]P–N²-dG adduct stereochemistry tends to have a lesser impact on mutagenic pattern (e.g. G→T versus G→A mutations) than does DNA sequence context, which is discussed.

The potent mutagen/carcinogen benzo[a]pyrene (B[a]P) is metabolically activated to (+)-anti-B[a]PDE, which induces a full spectrum of mutations primarily at the G:C base pairs (e.g. GC→TA, GC→AT, etc.). Each of these mutations can be induced by its major adduct [+ta]-B[a]P-N²-dG, where DNA sequence context appears to influence both the quantitative and qualitative pattern of mutagenesis. We noted previously that 5′-TG sequences tend to have a higher fraction of G→T mutations for both [+ta]-B[a]P-N²-dG and (+)-anti-B[a]PDE in comparison with 5′-CG, 5′-GG or 5′-AG sequences. To investigate a possible structural element for this trend, the role (if any) of the methyl group on the 5′-T is considered. Using adduct site-specific means, the [G→T/G→A] mutational ratio for [+ta]-B[a]P-N²-dG is determined to be ∼1.08 in a 5′-TGT sequence, and ∼0.60 in a 5′-UGT sequence. (G→C mutations are minor.) Although this modest ∼1.8-fold decrease in [G→T/G→A] ratio is statistically significant (P = 0.03), it suggests that the methyl group on the 5′-T is not the main reason why a 5′-T tends to enhance G→T mutations. This study was prompted by an adduct conformational hypothesis, which predicted that the removal of the methyl group in a 5′-TG sequence would lower the fraction of G→T mutations; however, the ∼1.8-fold decrease is too small to do additional experiments to assess whether this conformational hypothesis, or other hypotheses, are the true cause of the decrease, which is discussed in this paper.

DNA-DNA interstrand cross-links are the cytotoxic lesions for many chemotherapeutic agents. A plasmid with a single nitrogen mustard (HN2) interstrand cross-link (inter-HN2-pTZSV28) was constructed and transformed into Escherichia coli , and its replication efficiency (RE = [number of transformants from inter-HN2-pTZSV28]/[number of transformants from control]) was determined to be ∼0.6. Previous work showed that RE was high because the cross-link was repaired by a pathway involving nucleotide excision repair (NER) but not recombination. (In fact, recombination was precluded because the cells do not receive lesion-free homologous DNA.) Herein, DNA polymerase II is shown to be in this new pathway, since the replication efficiency (RE) is higher in a polB ⁺ (∼0.6) than in a Δ polB (∼0.1) strain. Complementation with a polB ⁺ -containing plasmid restores RE to wild-type levels, which corroborates this conclusion. In separate experiments, E. coli was treated with HN2, and the relative sensitivity to killing was found to be as follows: wild type < polB < recA < polB recA ∼ uvrA . Because cells deficient in either recombination ( recA ) or DNA polymerase II ( polB ) are hypersensitive to nitrogen mustard killing, E. coli appears to have two pathways for cross-link repair: an NER/recombination pathway (which is possible when the cross-links are formed in cells where recombination can occur because there are multiple copies of the genome) and an NER/DNA polymerase II pathway. Furthermore, these results show that some cross-links are uniquely repaired by each pathway. This represents one of the first clearly defined pathway in which DNA polymerase II plays a role in E. coli . It remains to be determined why this new pathway prefers DNA polymerase II and why there are two pathways to repair cross-links.