XPA Knockout Cell Lysate

The ubiquitin-proteasome pathway is involved in the pathogenesis of several neurogenetic diseases. We describe a Mauritanian patient harboring a homozygous deletion restricted to two contiguous genes HERC2 and OCA2 and presenting with severe developmental abnormalities. The deletion causes the complete loss of HERC2 protein function, an E3-ubiquitin ligase. HERC2 is known to target XPA and BRCA1 for degradation and a mechanism whereby it is involved in DNA repair and cell cycle regulation. We showed that loss of HERC2 function leads to the accumulation of XPA and BRCA1 in the patient's fibroblasts and generates decreased sensitivity to apoptosis and increased level of DNA repair. Our data describe for the first time the phenotypic consequences, both at the clinical and cellular levels, of a complete loss of HERC2 function in a patient. They strongly suggest that profound ubiquitin ligase - associated dysfunction is responsible for the severe phenotype in this patient, and that dysfunction of this pathway may be involved in other patients with similar neurodevelopmental diseases.

DNA replication, damage response and repair require the coordinated action of multi-domain proteins operating within dynamic multi-protein machines that act upon the DNA substrate. These modular proteins contain flexible linkers of various lengths, which enable changes in the spatial distribution of the globular domains (architecture) that harbor their essential biochemical functions. This mobile architecture is uniquely suited to follow the evolving substrate landscape present over the course of the specific process performed by the multi-protein machinery. A fundamental advance in understanding of protein machinery is the realization of the pervasive role of dynamics. Not only is the machine undergoing dynamic transformations, but the proteins themselves are flexible and constantly adapting to the progression through the steps of the overall process. Within this dynamic context the activity of the constituent proteins must be coordinated, a role typically played by hub proteins. A number of important characteristics of modular proteins and concepts about the operation of dynamic machinery have been discerned. These provide the underlying basis for the action of the machinery that reads DNA, and responds to and repairs DNA damage. Here, we introduce a number of key characteristics and concepts, including the modularity of the proteins, linkage of weak binding sites, direct competition between sites, and allostery, using the well recognized hub protein replication protein A (RPA). (C) 2014 Elsevier Ltd. All rights reserved.