Structural basis for human DNA polymerase kappa (polκ) to bypass cisplatin intrastrand cross-link (Pt-GG) lesion as an efficient and accurate extender
Bibliographic record
Abstract
Cisplatin (cis-diamminedichloroplatinum) is a widely used chemotherapeutic compound. It reacts with the N7 atoms of adjacent guanines in DNA to form the Pt-1,2d(GpG) intrastrand cross-link (Pt-GG) as a major product. This bulky cisplatin adduct blocks DNA replication by replicative polymerases, thereby promoting apoptosis of rapidly dividing cancer cells. However, cancer cells have developed resistance to cisplatin, partly because of the bypass of cisplatin adducts by specialized DNA polymerases (mostly belonging to the Y-family) through translesion synthesis (TLS). The human Y-family DNA polymerase eta (pol) is very efficient in replicating opposite the Pt-GG lesions, but not in extending after this adduct. Recently, it was shown that human Y-family DNA polymerase kappa (pol) is indispensable for DNA repair synthesis in dorsal root ganglion neurons exposed to cisplatin. Pol gene knockdown experiments in human cells have also indicated the involvement of pol in TLS of the Pt-GG lesion. In this study, we investigated the bypass potential and nucleotide incorporation preference of pol opposite DNA containing the Pt-GG adduct and determined two crystal structures of pol complexed with such DNA. The ternary complex structures represent two consecutive stages of lesion bypass: nucleotide insertion opposite the 5'G (Pt-GG2) and primer extension immediately after the lesion (Pt-GG3). Our in vitro DNA replication assays using pol and DNA containing the Pt-GG lesion indicate that pol inserts the correct dCTP opposite the 5'G of Pt-GG (Pt-GG2), when a nucleotide is already inserted opposite the 3'G (Pt-GG1). Pol also inserts the correct dATP opposite the T base immediately after the Pt-GG (Pt-GG3). However, pol is not able to insert a nucleotide opposite the 3'G of Pt-GG (Pt-GG1). On the other hand, pol is very efficient in replicating the Pt-GG lesion at two insertion stages (Pt-GG1 and Pt-GG2), but the enzyme is blocked at the extension stage (Pt-GG3). In terms of fidelity, we found that pol has the fewest mis-insertions compared to two other human Y-family polymerases pol and pol. Our steady-state kinetic analysis showed that pol incorporates dCTP and dATP opposite the 5'G of Pt-GG (Pt-GG2) and T template 5' to the lesion (Pt-GG3), respectively, with very similar relative efficiencies of 0.15-0.20, which are only ~5 fold lower than for normal DNA replication (GG2 and GG3). Our biochemical data indicate that pol is very proficient and accurate in inserting a nucleotide after the first G of the Pt-GG lesion. The structures revealed that the accuracy is achieved by stably accommodating the bases containing the cisplatin adducts in the enzyme's active site for proper Watson-Crick base pairing with the incoming nucleotide. Together, our biochemical and structural data provide the molecular basis for the accurate and proficient incorporation by pol of nucleotides opposite the cisplatin adduct and suggest an important role of pol as a second polymerase (extender) in the efficient bypass of the Pt-GG lesion in vivo. This work holds promise for pol as a potential target for drug design, along with pol, which together could improve the efficacy of cisplatin treatment for cancer therapy.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".