Characterization of the DNA- and dNTP-binding activities of the human cytomegalovirus DNA polymerase catalytic subunit UL54
Bibliographic record
Abstract
The catalytic subunit of the human cytomegalovirus DNA polymerase is critical for the replication of the virus. In the present study, we report the expression and purification of a recombinant catalytic subunit of the human cytomegalovirus DNA polymerase expressed in bacteria which retains polymerase activity. As a first step towards elucidating the nature of the interaction between the enzyme, DNA and dNTPs, we have utilized endogenous tryptophan fluorescence to evaluate the binding of ligands to the enzyme. Using this technique, we demonstrate that the minimal DNA-binding site of the enzyme is 6 nt. We also report the first detailed study of the binding kinetics and thermodynamic parameters involved in the interaction between the enzyme, DNA and dNTPs. Our thermodynamic analyses indicate that the initial formation of the enzyme-DNA binary complex is driven by a favourable entropy change, but is also clearly associated with an unfavourable enthalpic contribution. In contrast, the interaction of dNTPs to the binary complex was shown to depend on a completely different mode of binding that is dominated by a favourable enthalpy change and associated with an unfavourable entropy change. In order to provide additional insights into the structural modifications that occur during catalysis, we correlated the effect of DNA and dNTP binding on protein structure using CD. Our results indicate that the enzyme undergoes a first conformational change upon the formation of the protein-DNA binary complex, which is followed by a second structural modification upon dNTP binding. The present study provides a better understanding of the molecular basis of DNA and dNTP recognition by the catalytic subunit of the human cytomegalovirus DNA polymerase.
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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 | 0.001 |
| 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".