Mismatch repair proteins collaborate with methyltransferases in the repair of O(6)-methylguanine

DNA repair is essential for combatting the adverse effects of damage to the genome. One example of base damage is O(6)-methylguanine (O(6)mG), which stably pairs with thymine during replication and thereby creates a promutagenic O(6)mG:T mismatch. This mismatch has also been linked with cellular toxicity. Therefore, in the absence of repair, O(6)mG:T mismatches can lead to cell death or result in G:C-->A:T transition mutations upon the next round of replication. Cysteine thiolate residues on the Ada and Ogt methyltransferase (MTase) proteins directly reverse the O(6)mG base damage to yield guanine. When a cytosine is opposite the lesion, MTase repair restores a normal G:C pairing. However, if replication past the lesion has produced an O(6)mG:T mismatch, MTase conversion to a G:T mispair must still undergo correction to avoid mutation. Two mismatch repair pathways in E. coli that convert G:T mispairs to native G:C pairings are methyl-directed mismatch repair (MMR) and very short patch repair (VSPR). This work examined the possible roles that proteins in these pathways play in coordination with the canonical MTase repair of O(6)mG:T mismatches. The possibility of this repair network was analyzed by probing the efficiency of MTase repair of a single O(6)mG residue in cells deficient in individual mismatch repair proteins (Dam, MutH, MutS, MutL, or Vsr). We found that MTase repair in cells deficient in Dam or MutH showed wild-type levels of MTase repair. In contrast, cells lacking any of the VSPR proteins MutS, MutL, or Vsr showed a decrease in repair of O(6)mG by the Ada and Ogt MTases. Evidence is presented that the VSPR pathway positively influences MTase repair of O(6)mG:T mismatches, and assists the efficiency of restoring these mismatches to native G:C base pairs.     

Quantitative collision‐induced unfolding differentiates model antibody–drug conjugates

Antibody–drug conjugates (ADCs) are antibody‐based therapeutics that have proven to be highly effective cancer treatment platforms. They are composed of monoclonal antibodies conjugated with highly potent drugs via chemical linkers. Compared to cysteine‐targeted chemistries, conjugation at native lysine residues can lead to a higher degree of structural heterogeneity, and thus it is important to evaluate the impact of conjugation on antibody conformation. Here, we present a workflow involving native ion mobility (IM)‐MS and gas‐phase unfolding for the structural characterization of lysine‐linked monoclonal antibody (mAb)–biotin conjugates. Following the determination of conjugation states via denaturing Liquid Chromatography‐Mass Spectrometry (LC–MS) measurements, we performed both size exclusion chromatography (SEC) and native IM‐MS measurements in order to compare the structures of biotinylated and unmodified IgG1 molecules. Hydrodynamic radii (Rh) and collision cross‐sectional (CCS) values were insufficient to distinguish the conformational changes in these antibody–biotin conjugates owing to their flexible structures and limited instrument resolution. In contrast, collision induced unfolding (CIU) analyses were able to detect subtle structural and stability differences in the mAb upon biotin conjugation, exhibiting a sensitivity to mAb conjugation that exceeds native MS analysis alone. Destabilization of mAb–biotin conjugates was detected by both CIU and differential scanning calorimetry (DSC) data, suggesting a previously unknown correlation between the two measurement tools. We conclude by discussing the impact of IM‐MS and CIU technologies on the future of ADC development pipelines.