DOI: 10.5061/dryad.15dv41p7z
title: Alphafold2 modeling of KCTD10 interactions with RNAPII machinery

description: During DNA replication, the replisome must remove barriers and roadblocks
 including the transcription machinery. Transcription-replication conflicts
 (TRCs) occur when there are collisions between the replisome and
 transcription machinery, and are increasingly recognized as an important
 source of mammalian genome instability. How cells facilitate replisome
 bypass at sites of TRCs is incompletely understood. Here, we investigated
 the role of the CUL3-KCTD10 E3 ligase as a sensor for TRCs. We
 found that the substrate adaptor KCTD10 interacts with both the replisome
 and transcription machinery and regulates both in unstressed conditions.
 These bivalent interactions allow KCTD10 to detect co-directional TRCs and
 facilitate higher-order assembly of KCTD10 complexes that can recruit CUL3
 to ubiquitinate the RNA polymerase factor TCEA2. In the absence of KCTD10
 there is increased retention of TCEA2 and RNA polymerase complex, causing
 an accumulation of TRCs and increased DNA damage. These data report
 results from a screen we performed using Alphafold2 to look for potential
 protein-protein interactions between KCTD10 and transcriptional
 regulators.
description: # Alphafold2 modeling of KCTD10 interactions with RNAPII machinery Dataset
 DOI: [10.5061/dryad.15dv41p7z](10.5061/dryad.15dv41p7z) ## Description of
 the data and file structure Screening for KCTD10 interaction partners
 FASTA sequences belonging to the gene ontology term “transcription by RNA
 polymerase II” (GO:0006366) were retrieved from the European
 Bioinformatics Institute (EMBL-EBI) Gene Ontology and GO Annotations
 database ([www.ebi.ac.uk/QuickGO/](http://www.ebi.ac.uk/QuickGO/)). Using
 linux shell commands, the FASTAs were split, concatenated with the full
 length FASTA sequence for KCTD10 (UniprotKB ID: Q9H3F6), and then pooled
 into batches of 100 files. Each batch was run through ColabFold
 (v1.5.5)9–11 using Tesla T4 GPUs as a first pass, then larger complexes
 were completed using Tesla A100 GPUs. One protein could not be completed
 due to limitations of Alphafold2 (CHD7). To prioritize top hits, an iPTM
 score &gt; 0.4 was chosen, corresponding to &gt; 1 standard deviation
 above the average iPTM scores for the complex formed between KCTD10 and
 two copies of PCNA. ### Files and variables #### Folder Structure ```
 alphafold2_kctd10-RNAPII.zip |__inputs |__dx00000 |__dx00001 |__dx00002
 |__dx00003 |__results |__dx00000 |__dx00001 |__dx00002 |__dx00003
 alphafold2_kctd10-dimer.zip |__input |__results ``` #### **Data Formats**
 .fasta file with concatenated protein sequence for the indicated protein
 pair.  ###### .bibtex citations for proteins used in multiple sequence
 alignment.  ###### config.json configuration file with command options
 used in alphafold.  ###### \_coverage.png Graph of the sequence coverage
 for the multiple sequence alignment.  ###### \_pae.png Graph of the
 predicted aligned error plots for all five models. Lower values indicate
 well-defined relative positions and orientations in the pair.  ######
 \_plddt.png Graph of the per-residue measure of local confidence (plddt).
 It is scaled from 0 to 100, with higher scores indicating higher
 confidence and usually a more accurate prediction. ######
 aligned_error_v1.json json file with the aligned error between each
 residue.  ###### \_model_x_seed_000.json json file with the per residue
 measure of local confidence and the overall predicted aligned error for
 the model.  ###### \_model_x_seed_000.pdb pdb file containing predicted
 structure of the indicated protein pair.  ###### .a3m multiple sequence
 alignment file for protein against list of references.  ## Code/software
 Protein structures can be visualized by loading pdb files into PyMOL
 ([https://www.pymol.org/](https://www.pymol.org/)) or UCSF ChimeraX
 ([https://www.rbvi.ucsf.edu/chimerax/](https://www.rbvi.ucsf.edu/chimerax/)). Error plots can be generated using the corresponding json file in ChimeraX. Bibtex files can be opened with any citation manager software (e.g. Zotero, Endnote).