Using 2-Aminopurine Fluorescence to Measure Incorporation of Incorrect Nucleotides by Wild Type and Mutant Bacteriophage T4 DNA Polymerases
Bibliographic record
Abstract
The ability of wild type and mutant T4 DNA polymerases to discriminate in the utilization of the base analog 2-aminopurine (2AP) and the fluorescence of 2AP were used to determine how DNA polymerases distinguish between correct and incorrect nucleotides. Because T4 DNA polymerase incorporates dTMP opposite 2AP under single-turnover conditions, it was possible to compare directly the kinetic parameters for incorporation of dTMP opposite template 2AP to the parameters for incorporation of dTMP opposite template A without the complication of enzyme dissociation. The most significant difference detected was in the K(d) for dTTP, which was 10-fold higher for incorporation of dTMP opposite template 2AP (approximately 367 microm) than for incorporation of dTMP opposite template A (approximately 31 microm). In contrast, the dTMP incorporation rate was reduced only about 2-fold from about 318 s(-1) with template A to about 165 s(-1) for template 2AP. Discrimination is due to the high selectivity in the initial nucleotide-binding step. T4 DNA polymerase binding to DNA with 2AP in the template position induces formation of a nucleotide binding pocket that is preshaped to bind dTTP and to exclude other nucleotides. If nucleotide binding is hindered, initiation of the proofreading pathway acts as an error avoidance mechanism to prevent incorporation of incorrect nucleotides.
Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.
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 | 0.001 | 0.002 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.000 |
| Bibliometrics | 0.001 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.001 | 0.001 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.001 | 0.001 |
| Insufficient payload (model declined to judge) | 0.000 | 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".