Ku70-binding protein 5 (Kub5)-Hera (K-H)/RPRD1B maintains genetic integrity by 
concomitantly minimizing persistent R-loops and promoting repair of DNA double 
strand breaks (DSBs). We used tandem affinity purification-mass spectrometry, 
co-immunoprecipitation and gel-filtration chromatography to define higher-order 
protein complexes containing K-H scaffolding protein to gain insight into its 
cellular functions. We confirmed known protein partners (Ku70, RNA Pol II, 
p15RS) and discovered several novel associated proteins that function in RNA 
metabolism (Topoisomerase 1 and RNA helicases), DNA repair/replication processes 
(PARP1, MSH2, Ku, DNA-PKcs, MCM proteins, PCNA and DNA Pol δ) and in protein 
metabolic processes, including translation. Notably, this approach directed us 
to investigate an unpredicted involvement of K-H in DNA mismatch repair (MMR) 
where K-H depletion led to concomitant MMR deficiency and compromised global 
microsatellite stability. Mechanistically, MMR deficiency in K-H-depleted cells 
was a consequence of reduced stability of the core MMR proteins (MLH1 and PMS2) 
caused by elevated basal caspase-dependent proteolysis. Pan-caspase inhibitor 
treatment restored MMR protein loss. These findings represent a novel mechanism 
to acquire MMR deficiency/microsatellite alterations. A significant proportion 
of colon, endometrial and ovarian cancers exhibit k-h expression/copy number 
loss and may have severe mutator phenotypes with enhanced malignancies that are 
currently overlooked based on sporadic MSI+ screening.