Structure Function Analysis of DNA Polymerase β and Discovery of Novel Interactors
Bibliographic record
Abstract
Base excision repair (BER) is the primary pathway responsible for repairing base lesions—such as uracil, deaminated, alkylated, and oxidized bases—as well as abasic sites and single-strand breaks (SSBs). Several transcription factors act as BER auxiliary factors, stimulating the enzymatic activities of DNA glycosylases that recognize oxidized DNA bases. CUT domain proteins (CUX1, CUX2, and SATB1) enhance the glycosylase and AP-lyase activities of 8-oxoguanine DNA glycosylase 1 (OGG1), while the zinc-finger proteins BCL11A and BCL11B stimulate Endonuclease III-like protein 1 (NTHL1). A recent study on the MYC interactome identified uracil-DNA glycosylase (UNG) as a MYC interactor. Our lab confirmed the MYC–UNG interaction and its stimulation in vitro. In all cases, the accessory factors were found also to interact with the DNA polymerase β (Pol β) and stimulate its polymerase and deoxyribose phosphate (dRP)-lyase activities, ensuring completion of the repair process.Based on these observations, we hypothesize that each BER accessory factor must stimulate Pol β activity in addition to enhancing a specific glycosylase. This dual stimulation ensures completion of the BER process, particularly since glycosylase activity generates single-strand breaks, which are more cytotoxic than the original base lesions. If left unrepaired, these SSBs can stall transcription and be converted into double-strand breaks during replication, contributing to genomic instability.One part of my project was to prepare reagents to perform proximity-dependent biotinylation (BioID) for Pol β. I measured the enzymatic activities of Pol β fusion proteins with the TurboID and ultraID biotin ligases positioned at the N- or C-terminal end of Pol β, and I tested several mammalian vectors to express Pol β fusion proteins in the MDA-MB-231 and HCT-116 cancer cell lines that exhibit high levels of reactive oxygen species. Subcellular localisation and the biotin ligase activity of Pol β-ultraID fusion proteins were investigated. Following these validation assay, expression of the Pol β-ultraID and ultraID-Pol β fusion proteins was induced in MDA-MB-231 cells, biotin was added to the medium and protein extracts were prepare and sent for analysis by mass spectrometry. As controls, we also prepared extracts from cells expressing eGFP-ultraID and eGFP-ultraID-NLS. Using the BioID approach, we compiled a list of proteins that come into close proximity with Pol β in cells.In parallel, structure-function analysis identified the N-terminal 8-kDa domain of Pol β as the region responsible for the interaction with, and stimulation by, MYC and CUX1. We generated a Pol β mutant that is completely unresponsive to stimulation by MYC, and we successfully pinpointed the loop 4 alpha-helix on the Pol β surface as the site of interaction with MYC
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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 | 0.001 |
| 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 | 0.001 |
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".