Preparation of Covalently Linked Complexes Between DNA and<i>O</i><sup>6</sup>-Alkylguanine-DNA Alkyltransferase Using Interstrand Cross-Linked DNA
Bibliographic record
Abstract
O(6)-alkylguanine-DNA alkyltransferases (AGT) are responsible for the removal of alkylation at both the O(6) atom of guanine and O(4) atom of thymine. AGT homologues show vast substrate differences with respect to the size of the adduct and which alkylated atoms they can restore. The human AGT (hAGT) has poor capabilities for removal of methylation at the O(4) atom of thymidine, which is not the case in most homologues. No structural data are available to explain this poor hAGT repair. We prepared and characterized O(6)G-butylene-O(4)T (XLGT4) and O(6)G-heptylene-O(4)T (XLGT7) interstrand cross-linked (ICL) DNA as probes for hAGT and the Escherichia coli homologues, OGT and Ada-C, for the formation of DNA-AGT covalent complexes. XLGT7 reacted only with hAGT and did so with a cross-linking efficiency of 25%, while XLGT4 was inert to all AGT tested. The hAGT mediated repair of XLGT7 occurred slowly, on the order of hours as opposed to the repair of O(6)-methyl-2'-deoxyguanosine which requires seconds. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the repair reaction revealed the formation of a covalent complex with an observed migration in accordance with a DNA-AGT complex. The identity of this covalent complex, as determined by mass spectrometry, was composed of a heptamethylene bridge between the O(4) atom of thymidine (in an 11-mer DNA strand) to residue Cys145 of hAGT. This procedure can be applied to produce well-defined covalent complexes between AGT with DNA.
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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) | 0.001 | 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 | 0.001 |
| 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".