Non-catalytic Roles for XPG with BRCA1 and BRCA2 in Homologous Recombination and Genome Stability.

XPG is a structure-specific endonuclease required for nucleotide excision repair, and incision-defective XPG mutations cause the skin cancer-prone syndrome xeroderma pigmentosum. Truncating mutations instead cause the neurodevelopmental progeroid disorder Cockayne syndrome, but little is known about how XPG loss results in this devastating disease. We identify XPG as a partner of BRCA1 and BRCA2 in maintaining genomic stability through homologous recombination (HRR). XPG depletion causes DNA double-strand breaks, chromosomal abnormalities, cell-cycle delays, defective HRR, inability to overcome replication fork stalling, and replication stress. XPG directly interacts with BRCA2, RAD51, and PALB2, and XPG depletion reduces their chromatin binding and subsequent RAD51 foci formation. Upstream in HRR, XPG interacts directly with BRCA1. Its depletion causes BRCA1 hyper-phosphorylation and persistent chromatin binding. These unexpected findings establish XPG as an HRR protein with important roles in genome stability and suggest how XPG defects produce severe clinical consequences including cancer and accelerated aging.

Human XPG nuclease structure, assembly, and activities with insights for neurodegeneration and cancer from pathogenic mutations.

Xeroderma pigmentosum group G (XPG) protein is both a functional partner in multiple DNA damage responses (DDR) and a pathway coordinator and structure-specific endonuclease in nucleotide excision repair (NER). Different mutations in the XPG gene ERCC5 lead to either of two distinct human diseases: Cancer-prone xeroderma pigmentosum (XP-G) or the fatal neurodevelopmental disorder Cockayne syndrome (XP-G/CS). To address the enigmatic structural mechanism for these differing disease phenotypes and for XPG's role in multiple DDRs, here we determined the crystal structure of human XPG catalytic domain (XPGcat), revealing XPG-specific features for its activities and regulation. Furthermore, XPG DNA binding elements conserved with FEN1 superfamily members enable insights on DNA interactions. Notably, all but one of the known pathogenic point mutations map to XPGcat, and both XP-G and XP-G/CS mutations destabilize XPG and reduce its cellular protein levels. Mapping the distinct mutation classes provides structure-based predictions for disease phenotypes: Residues mutated in XP-G are positioned to reduce local stability and NER activity, whereas residues mutated in XP-G/CS have implied long-range structural defects that would likely disrupt stability of the whole protein, and thus interfere with its functional interactions. Combined data from crystallography, biochemistry, small angle X-ray scattering, and electron microscopy unveil an XPG homodimer that binds, unstacks, and sculpts duplex DNA at internal unpaired regions (bubbles) into strongly bent structures, and suggest how XPG complexes may bind both NER bubble junctions and replication forks. Collective results support XPG scaffolding and DNA sculpting functions in multiple DDR processes to maintain genome stability.

Purification and Characterization of Human DNA Ligase IIIα Complexes After Expression in Insect Cells.

With improvements in biophysical approaches, there is growing interest in characterizing large, flexible multi-protein complexes. The use of recombinant baculoviruses to express heterologous genes in cultured insect cells has advantages for the expression of human protein complexes because of the ease of co-expressing multiple proteins in insect cells and the presence of a conserved post-translational machinery that introduces many of the same modifications found in human cells. Here we describe the preparation of recombinant baculoviruses expressing DNA ligase IIIα, XRCC1, and TDP1, their subsequent co-expression in cultured insect cells, the purification of complexes containing DNA ligase IIIα from insect cell lysates, and their characterization by multi-angle light scattering linked to size exclusion chromatography and negative stain electron microscopy.

Level of visual acuity necessary to avoid false-positives on the HRR and Ishihara color vision tests.

PURPOSE: Minimizing false-positives (FPs) when evaluating color vision is important in eye care. Identification of plate 1 (demonstration plate) is often considered a way to avoid FPs. However, few studies have quantified the minimum level of visual acuity (VA) that would minimize FPs for the Ishihara and HRR color tests. METHODS: Threshold levels of optical defocus were obtained from 25 color normal subjects. Blur levels were obtained for Ishihara (38 plate) plates 1, 10, and 15 and 4th edition HRR plates 1, 7, 10, and 20 using the method of limits. Corresponding VAs were measured through these blur levels at 40 centimeters after adjusting for the dioptric distance difference. Analysis of variance testing was used to analyze the data. RESULTS: Mean optical defocus values in diopters (mean ± SD) for HRR plates 1, 7, 10, and 20 were 6.23 ± 1.61, 1.23 ± 1.16, 2.41 ± 1.31, and 7.96 ± 2.03, respectively, and for Ishihara plates 1, 10, and 15 were 5.70 ± 1.52, 3.68 ± 1.71, and 4.62 ± 1.56, respectively. There was a significant difference between the screening and demonstration plates for both tests (p<0.001). CONCLUSIONS: Based on the plate in each test that was found to be the least tolerant to blur, the average minimum VAs needed to identify the screening plates were approximately 20/180 for the Ishihara test and 20/50 for the HRR test. Identifying the demonstration plate in the Ishihara and HRR tests does not ensure FPs will be avoided.