Increased Meiotic Crossovers and Reduced Genome Stability in Absence of Schizosaccharomyces pombe Rad16 (XPF)

Schizosaccharomyces pombe Rad16 is the ortholog of the XPF structure-specific endonuclease, which is required for nucleotide excision repair and implicated in the single strand annealing mechanism of recombination. We show that Rad16 is important for proper completion of meiosis. In its absence, cells suffer reduced spore viability and abnormal chromosome segregation with evidence for fragmentation. Recombination between homologous chromosomes is increased, while recombination within sister chromatids is reduced, suggesting that Rad16 is not required for typical homolog crossovers but influences the balance of recombination between the homolog and the sister. In vegetative cells, rad16 mutants show evidence for genome instability. Similar phenotypes are associated with mutants affecting Rhp14^XPA but are independent of other nucleotide excision repair proteins such as Rad13^XPG. Thus, the XPF/XPA module of the nucleotide excision repair pathway is incorporated into multiple aspects of genome maintenance even in the absence of external DNA damage.     

Identification of DNA repair gene Ercc1 as a novel target in melanoma

Increased expression of DNA repair genes contributes to the extreme resistance shown by melanoma to conventional DNA-damaging chemotherapeutics. One such chemotherapeutic effective against a range of other cancers, but not melanoma, is cisplatin. The DNA repair protein, ERCC1, is needed to remove cisplatin-induced DNA damage. We have shown that ERCC1 is essential for melanoma growth and resistance to cisplatin in a mouse xenograft model. Untreated xenografts of our transformed Ercc1-proficient melanocyte cell line grew very rapidly as malignant melanoma. Cisplatin treatment caused initial shrinkage of xenografts, but cisplatin-resistant regrowth soon followed. Cells reisolated into culture had twofold elevated levels of ERCC1 compared to both input cells and cells reisolated from untreated xenografts. An isogenic Ercc1-deficient derivative grew equally well in vitro as the Ercc1-proficient melanocyte cell line. However, in xenografts, the Ercc1-deficient melanomas were much slower to establish and were completely cured by just two cisplatin treatments.     

Structure of an XPF endonuclease with and without DNA suggests a model for substrate recognition

The XPF/Mus81 structure-specific endonucleases cleave double-stranded DNA (dsDNA) within asymmetric branched DNA substrates and play an essential role in nucleotide excision repair, recombination and genome integrity. We report the structure of an archaeal XPF homodimer alone and bound to dsDNA. Superposition of these structures reveals a large domain movement upon binding DNA, indicating how the (HhH)(2) domain and the nuclease domain are coupled to allow the recognition of double-stranded/single-stranded DNA junctions. We identify two nonequivalent DNA-binding sites and propose a model in which XPF distorts the 3' flap substrate in order to engage both binding sites and promote strand cleavage. The model rationalises published biochemical data and implies a novel role for the ERCC1 subunit of eukaryotic XPF complexes.     

Knockdown of αII spectrin in normal human cells by siRNA leads to chromosomal instability and decreased DNA interstrand cross-link repair

Nonerythroid α-spectrin (αIISp) is a structural protein involved in repair of DNA interstrand cross-links and is deficient in cells from patients with Fanconi anemia (FA), which are defective in ability to repair cross-links. In order to further demonstrate the importance of the role that αIISp plays in normal human cells and in the repair defect in FA, αIISp was knocked down in normal cells using siRNA. Depletion of αIISp in normal cells by siRNA resulted in chromosomal instability and cellular hypersensitivity to DNA interstrand cross-linking agents. An increased number of chromosomal aberrations were observed and, following treatment with a DNA interstrand cross-linking agent, mitomycin C, cells showed decreased cell growth and survival and decreased formation of damage-induced αIISp and XPF nuclear foci. Thus depletion of αIISp in normal cells leads to a number of defects observed in FA cells, such as chromosome instability and a deficiency in cross-link repair.     

Kinesin-5: Cross-bridging mechanism to targeted clinical therapy

Kinesin motor proteins comprise an ATPase superfamily that works hand in hand with microtubules in every eukaryote. The mitotic kinesins, by virtue of their potential therapeutic role in cancerous cells, have been a major focus of research for the past 28 years since the discovery of the canonical Kinesin-1 heavy chain. Perhaps the simplest player in mitotic spindle assembly, Kinesin-5 (also known as Kif11, Eg5, or kinesin spindle protein, KSP) is a plus-end-directed motor localized to interpolar spindle microtubules and to the spindle poles. Comprised of a homotetramer complex, its function primarily is to slide anti-parallel microtubules apart from one another. Based on multi-faceted analyses of this motor from numerous laboratories over the years, we have learned a great deal about the function of this motor at the atomic level for catalysis and as an integrated element of the cytoskeleton. These data have, in turn, informed the function of motile kinesins on the whole, as well as spearheaded integrative models of the mitotic apparatus in particular and regulation of the microtubule cytoskeleton in general. We review what is known about how this nanomotor works, its place inside the cytoskeleton of cells, and its small-molecule inhibitors that provide a toolbox for understanding motor function and for anticancer treatment in the clinic.     

Tissue specificity of senescent cell accumulation during physiologic and accelerated aging of mice

Senescent cells accumulate with age in vertebrates and promote aging largely through their senescence‐associated secretory phenotype (SASP). Many types of stress induce senescence, including genotoxic stress. ERCC1‐XPF is a DNA repair endonuclease required for multiple DNA repair mechanisms that protect the nuclear genome. Humans or mice with reduced expression of this enzyme age rapidly due to increased levels of spontaneous, genotoxic stress. Here, we asked whether this corresponds to an increased level of senescent cells. p16^Ink4a and p21^Cip1 mRNA were increased ~15‐fold in peripheral lymphocytes from 4‐ to 5‐month‐old Ercc1^?/? and 2.5‐year‐old wild‐type (WT) mice, suggesting that these animals exhibit a similar biological age. p16^Ink4a and p21^Cip1 mRNA were elevated in 10 of 13 tissues analyzed from 4‐ to 5‐month‐old Ercc1^?/? mice, indicating where endogenous DNA damage drives senescence in vivo. Aged WT mice had similar increases of p16^Ink4a and p21^Cip1 mRNA in the same 10 tissues as the mutant mice. Senescence‐associated β–galactosidase activity and p21^Cip1 protein also were increased in tissues of the progeroid and aged mice, while Lamin B1 mRNA and protein levels were diminished. In Ercc1^?/Δ mice with a p16^Ink4a luciferase reporter, bioluminescence rose steadily with age, particularly in lung, thymus, and pancreas. These data illustrate where senescence occurs with natural and accelerated aging in mice and the relative extent of senescence among tissues. Interestingly, senescence was greater in male mice until the end of life. The similarities between Ercc1^?/? and aged WT mice support the conclusion that the DNA repair‐deficient mice accurately model the age‐related accumulation of senescent cells, albeit six‐times faster.     

A nonsense mutation in the Xeroderma pigmentosum complementation group F (XPF) gene is associated with gastric carcinogenesis

XPF/ERCC1 endonuclease is required for DNA lesion repair. To assess effects of a C2169A nonsense mutation in XPF at position 2169 in gastric cancer tissues and cell lines, genomic DNA was extracted from blood samples of 488 cancer patients and 64 gastric tumors. The mutation was mapped using a TaqMan MGB probe. In addition, gastric cancer cell lines were transfected with mutated XPF to explore XPF/ERCC1 interaction, XPF degradation, and DNA repair by a comet assay. The C2169A mutation was not detected in 488 samples of blood genomic DNA, yet was found in 32 of 64 gastric cancer tissue samples (50.0%), resulting in a 194C-terminal amino acid loss in XPF protein and lower expression. Laser micro-dissection confirmed that this point mutation was not present in surrounding normal tissues from the same patients. The truncated form of XPF (tXPF) impaired interaction with ERCC1, was rapidly degraded via ubiquitination, and resulted in reduced DNA repair. In gastric cancers, the mutation was monoallelic, indicating that XPF is a haplo-insufficient DNA repair gene. As the C2169A mutation is closely associated with gastric carcinogenesis in the Chinese population, our findings shine light on it as a therapeutic target for early diagnosis and treatment of gastric cancer.