Abstract
The mismatch repair (MMR) family is a highly conserved group of proteins that function in correcting base-base and insertion-deletion mismatches generated during DNA replication. Disruption of this process results in characteristic microsatellite instability (MSI), repair defects, and susceptibility to cancer. However, a significant fraction of MSI-positive cancers express MMR genes at normal levels and do not carry detectable mutation in known MMR genes, suggesting that additional factors and/or mechanisms may exist to explain these MSI phenotypes in patients. To systematically investigate the MMR pathway, we conducted a proteomic analysis and identified MMR-associated protein complexes using tandem-affinity purification coupled with mass spectrometry (TAP-MS) method. The mass spectrometry data have been deposited to the ProteomeXchange with identifier PXD003014 and DOI 10.6019/PXD003014. We identified 230 high-confidence candidate interaction proteins (HCIPs). We subsequently focused on MSH2, an essential component of the MMR pathway and uncovered a novel MSH2-binding partner, WDHD1. We further demonstrated that WDHD1 forms a stable complex with MSH2 and MSH3 or MSH6,i.e.the MutS complexes. The specific MSH2/WDHD1 interaction is mediated by the second lever domain of MSH2 and Ala(1123)site of WDHD1. Moreover, we showed that, just like MSH2-deficient cells, depletion of WDHD1 also led to 6-thioguanine (6-TG) resistance, indicating that WDHD1 likely contributes to the MMR pathway. Taken together, our study uncovers new components involved in the MMR pathway, which provides candidate genes that may be responsible for the development of MSI-positive cancers.
Highlights
Cells are equipped with a number of repair mechanisms to correct various types of DNA lesions
Proteomics Study of Mismatch Repair Protein Interactome Using TAP-MS Approach—To build the interaction network of DNA mismatch repair (MMR) pathway, we used the well-established tandem affinity purification followed by mass spectrometry (TAP-MS) strategy [33,34,35], which was described in Fig. 1A, to identify the binding proteins
Given that the MMR pathway is a critical genome maintenance pathway and MMR deficiency leads to microsatellite instability (MSI) and cancer development, we speculate that some of the MMRbinding proteins discovered in this study may be mutated or downregulated in cancer and contribute to cancer development and MSI phenotypes identified in cancer patients
Summary
Cells are equipped with a number of repair mechanisms to correct various types of DNA lesions. Proteomics Study of Mismatch Repair Protein Interactome Using TAP-MS Approach—To build the interaction network of DNA MMR pathway, we used the well-established tandem affinity purification followed by mass spectrometry (TAP-MS) strategy [33,34,35], which was described, to identify the binding proteins. To obtain the high-confidence candidate interacting proteins (HCIP) list, we submitted 20 TAP-MS results with spectra counts information for DNA MMR proteins and 233 controls with random selected unrelated control proteins for CRAPome analysis [30].
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