UC San Diego

Transcription Coupled Nucleotide Excision repair (TC-NER) is a widely conserved mechanism that is used to repair DNA damage and maintain genome stability by targeting bulky DNA lesions during transcription. In S. Cerevisiae, TC-NER is initiated by the recognition and binding of the Rad26 protein (or CSB in H.Sapiens) directly to bulky lesion stalled Pol II and upstream of the dsDNA fork.12 The Rad26 protein can discriminate between a bulky lesion stalled Pol II from other types of stalled Pol II, determining whether downstream TC-NER factors such as UVSSA and TFIIH are needed for recruitment.1 The absence or mutation of the Rad26 homolog, the CSB protein, causes severe genetic disease such as Cockayne Syndrome (CS), highlighting its functional importance.2 The function of the Rad26 is deemed critically important to understanding bulky DNA damage repair by TC-NER. While the structure of Rad26-Pol II complexes have been determined using Cryo-EM structure studies, the Rad26 NTD and CTD flanking regions are not revealed due to flexibility. These flanking regions connect to the core domain by a flexible loop and are important for Rad26 function via interaction with different protein partners. To investigate the unknown function of the Rad26 NTD and CTD, a GST pull-down assay is performed using GST-Rad26 fusion proteins in a S. Cerevisiae system to help determine potential protein binding factors that may bring insight on the function of these flanking regions of the Rad26. The pulled-down protein bands were analyzed using LC-MS. Potential binding partners that were discovered for the Rad26 NTD and CTD regions include Elf1, TFIIH subunit SSL1, FACT complex subunit POB3, and histone associate proteins RPD3, RVB2, and BDF1. The identification of these likely protein binding factors opens the possibility for future investigation on the protein partners of the Rad26 NTD and CTD. Insight on the proteomics of the Rad26 NTD and CTD interactome will help in understanding the roles of the Rad26 flanking regions in the TC-NER pathway.