Data with DNA damaged-induced phosphorylation of a replicative DNA helicase results in inhibition of DNA replication through attenuation of helicase function - NAR 2024

Description

SV40 DNA replication inhibition is associated with enhanced DDR kinase phosphorylation of SV40 Large T-antigen (LT), the viral DNA helicase. In this study mass spectroscopy was used to identify a novel highly conserved DDR kinase site, T518, on LT. In cell-based assays expression of a phosphomimetic form of LT at T518 (T518D) resulted in dramatically decreased levels of SV40 DNA replication, but LT-dependent transcriptional activation was unaffected. In concordance with the cell-based data, reactions using purified WT and LT-T518D, but not T518A, showed dramatic inhibition of SV40 DNA replication in vitro as well. The MS data includes all peptides identified as excel files from the UB Mass Spec Facility. The data is sorted by XCorr, which is the search engine score from Sequest HT, the higher XCorr the more confident the identification. The MS2 fragmentation of the phosphorylated and nonphosphorylated T518Q containing peptide is included in sheet 2 of the excel document. Results are also reported from MS facility as a .doc report. Raw data used to create the final figures in the paper in a pptx format is the final inclusion. Following are brief explanations of all columns in the table of the MS data (peptide list) : Sequence - the peptide sequence identified # PSMs - how many times a spectrum has been matched to this peptide sequence ( a crude quantitative value) # Proteins - how many protein(s) this peptide can be inferred to # Protein groups - similar with above but several proteins can be grouped to one protein group if no unique peptides can be identified to discriminated one from the other Protein group accessions - Uniprot (or other protein database) accession number Modifications - PTMs identified on the peptide deltaCn - the difference of XCorr (search engine score) between the best match and the second best match (which is a match to another peptide, usually only the best match is considered as the "correct" match) phosphoRS site probabilities - Predicted probabilities of PTM localization XCorr - search engine score from Sequest HT, the higher XCorr the more confident the identification Charge, MH+, RT - basic probabilities of the peptide ion deltaM - mass difference between the theoretical and detected peptide ion # Missed cleavages - how many K/Rs left in the peptide sequence, which should be cleaved by trypsin but are not.

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Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was carried out on a trapping nano-flow LC-Orbitrap MS system consisting of a Dionex Ultimate 3000 nano LC system, a Dionex Ultimate 3000 gradient micro-LC system with an WPS-3000 au-tosampler, and an Orbitrap Fusion Lumos mass spectrometer (ThermoFisher Scientific, San Jose, CA). For preparation of lyophilized LT samples, 50μL 0.5% SDS was added to each sample, and samples were sonicated for 30 sec and vortex mixed for 10 min to re-constitute protein. Protein reduction and alkylation was performed sequentially by addition of 2μL 200μM DTT and 4μL 500μM iodoacetamide (IAM), each with 45-min incubation in a covered thermomixer under 37˚C with constant shaking. Protein was precipitated. A volume of 5μL trypsin dissolved in 50mM Tris-FA (0.25μg/μL) was added to each sample, and trypsinization was performed under 37˚C overnight (~16hr) with constant shaking in a covered thermomixer. Trypsinization was terminated and derived peptide mixture was centrifuged at 18,000 g under 4˚C for 30 min, and supernatant was transferred to vials for analysis.

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