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Photo-activatable Ub-PCNA probes reveal new structural features of the S. cerevisiae Polη/PCNA complex

Citation

Shen, Siqi; Davidson, Gregory A.; Yang, Kun; Zhuang, Zhihao (2021), Photo-activatable Ub-PCNA probes reveal new structural features of the S. cerevisiae Polη/PCNA complex, Dryad, Dataset, https://doi.org/10.5061/dryad.nk98sf7s3

Abstract

The Y-family DNA polymerase η (Polη) is critical for the synthesis past damaged DNA nucleotides in yeast through translesion DNA synthesis (TLS). TLS is initiated by monoubiquitination of proliferating cell nuclear antigen (PCNA) and the subsequent recruitment of TLS polymerases. Although individual structures of the Polη catalytic core and PCNA have been solved, a high-resolution structure of the complex of Polη/PCNA or Polη/monoubiquitinated PCNA (Ub-PCNA) still remains elusive, partly due to the disordered Polη C-terminal region and the flexibility of ubiquitin on PCNA. To circumvent these obstacles and obtain structural insights into this important TLS polymerase complex, we developed photo-activatable PCNA and Ub-PCNA probes containing a p-benzoyl-L-phenylalanine (pBpa) crosslinker at selected positions on PCNA. By photo-crosslinking the probes with full-length Polη, specific crosslinking sites were identified following tryptic digestion and tandem mass spectrometry analysis. We discovered direct interactions of the Polη catalytic core and its C-terminal region with both sides of the PCNA ring. Model building using the crosslinking site information as a restraint revealed multiple conformations of Polη in the polymerase complex. Availability of the photo-activatable PCNA and Ub-PCNA probes will also facilitate investigations into other PCNA-containing complexes important for DNA replication, repair and damage tolerance. 

Methods

Dataset was collected by Q-exative Orbitrap. In-gel digestion was carried out for crosslinking products. Samples were reduced by DTT, alkylated by iodoacetamide, trypsin digested and desalted by C18 ZipTip before Q-exative Orbitrap analysis.

Mgf files are spectrum files used for MeroX analysis. Zhrm files are result from MeroX analysis. Protein docking models are saved as cif files.

Usage Notes

The following data contains the mass spectrometry file in the .raw, .mgf and . zhrm file. The .raw file can be opened by the Thermo Fisher Xcalibur software. The .mgf fils is generated by the Thermo Fisher Proteome Disocoverer 1.4 for the analysis of the crosslinking peptides by the MeroX Software. The .zhrm file is the result file of the identified crosslinking peptides and can be opened by the MeroX software. The .cif file is the docking result from HADDOCK which can be opened by the PyMOL software.  Detailed explanataion of each file is in the readme file.