In the current investigation, we demonstrated that Pol I and Pol II could replicate DNAs containing an N6-dA dodecylpeptide cross-link in an error-free manner in vitro, while Pol III* and Pol IV were strongly inhibited by this type of lesion. Although Pol V could fully and accurately extend a primer on a damaged substrate, its efficiency was low. Since Pol I is expressed constitutively, we proposed that Pol I in cells can carry out TLS past an N6-dA dodecylpeptide cross-link and that the DNA damage-inducible polymerases are not required. Consistent with this idea, in vivo studies using wild-type and Pol II− Pol IV− Pol V− E. coli strains revealed that DNA damage-inducible polymerases were dispensable.
Although these data suggested the essential role of Pol I in replication bypass of the N6
-dA dodecylpeptide cross-link, an involvement of alternative DNA polymerases in this process cannot be ruled out completely. In particular, Pol III and possibly other DNA polymerases may manifest higher bypass efficiencies in cells than those observed in vitro
due to stimulation of bypass by the β sliding clamp and additional accessory factors. It is also important to recognize that the division of labor between DNA polymerases in replication bypass is expected to be more complex in the context of chromosomal DNA, as opposed to a site-specific single lesion on plasmid DNA. Following chemical exposure, multiple structurally different DNA lesions are likely to be formed that may trigger the SOS response. The contribution of Pol II to TLS past N6
-dA polypeptide cross-links could be quite prominent under such conditions, since its intracellular levels are significantly increased (10
). Pol IV, the most abundant E. coli
DNA polymerase in stressed cells (10
), may also be involved in replication bypass of lesions, displacing more efficient but less abundant DNA polymerases from the primer termini. Such a phenomenon was recently observed during double-strand-break repair (11
). In addition, the location of the lesions on the chromosome may also influence the choice of polymerase, similar to a site-specific polymerase switch during spontaneous mutagenesis (7
The active site of Pol I is constrained, and Pol I has a stringent requirement for accommodating nucleobase pairs with the correct Watson-Crick geometry (13
). Therefore, it is intriguing that Pol I was capable of bypassing a very large N6
-dA dodecylpeptide cross-link that not only blocked replicative polymerases (E. coli
Pol III* and yeast Pol δ) but also blocked the low-fidelity polymerases specialized in lesion bypass (E. coli
Pol IV and Pol V and human Pol κ) (). One possible explanation may be that this lesion has conformational flexibility, with the bulky dodecylpeptide pointed away from the active site of Pol I, and has no effect on the Watson-Crick geometry. The DNA-peptide structure might resemble that of Lys-Trp-Lys-Lys linked at N2
-dG via the N-terminal Lys through an acrolein moiety. In the latter structure, the Trp indolyl group does not intercalate into the DNA, while the C-terminal Lys is exposed to solvent, interacting minimally with the DNA (12
). However, a detailed analysis of the mechanism of action of Pol I in the bypass of the N6
-dA dodecylpeptide cross-link awaits the co-crystal structure determination of Pol I in complex with this lesion.
Fig. 8. Replication blockage of human Pol κ by N6-dA dodecylpeptide cross-link. Primer extension reactions were catalyzed by 1 nM Pol κ under running-start conditions (−3 primer), using nondamaged (ND) and N6-dA dodecylpeptide cross-linked (more ...)
With regard to the biological significance of N6
-dA peptide cross-links, a previous report focused exclusively on the identification of E. coli
DNA polymerases that can catalyze replication bypass of N2
-dG peptide cross-links (21
). The polymerases responsible for the replication bypass of N6
-dA peptide cross-links, however, were not studied. The N6
-dA peptide lesion used in this study was derived from the ring-opened aldehydic form of the acrolein-induced γ-HO-PdA adduct. Nuclear magnetic resonance spectroscopy analyses showed that γ-HO-PdA in the ring-opened form can be detected readily (23
). This suggests that γ-HO-PdA could potentially interact with peptides, resulting in the formation of DNA-peptide cross-links. Such a mechanism would be conceptually similar to that for the aldehydic group of ring-opened γ-HO-PdG, which reacts with peptides and yields chemically identical lesions, except that the lesions are located in the minor groove of DNA (16
). Consistent with this prediction, it was demonstrated that the peptide Lys-Trp-Lys-Lys is trapped efficiently and stably with oligodeoxynucleotides containing γ-HO-PdA in the presence of a reducing agent (19
). Thus, although N6
-dA peptide cross-links have not been identified in vivo
to date, their biological relevance cannot be disregarded, and therefore the present investigation examining the identity of polymerases involved in the processing of these lesions has importance.
It is worth noting that in E. coli
, TLS past N2
-dG peptide cross-links requires the DNA damage-inducible polymerase Pol IV (21
). In contrast, such specialized polymerases were not essential for replication bypass of the N6
-dA dodecylpeptide cross-link. This is interesting given the fact that the previously investigated N2
-dG dodecylpeptide cross-link is chemically identical to the N6
-dA dodecylpeptide cross-link used here, except that the former lesion is positioned in the minor, not the major, groove of DNA. Germane to this observation, DNA polymerases of the A family interact with DNA at the minor groove (27
). Thus, Pol I may have evolved to be particularly proficient in the bypass of major groove lesions, including relatively small adducts (5
) and large degradation products of DNA-protein cross-links.
In summary, we have shown the ability of E. coli Pol I to accurately replicate past an N6-dA dodecylpeptide cross-link in vitro and have generated data suggesting its role in bypass of this lesion in vivo. Since no data about the identity of E. coli DNA polymerases responsible for processing the major groove cross-links are available to date, the findings reported here promote our understanding of how cells maintain genome integrity upon induction of DNA-peptide and DNA-protein cross-links.