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The XPA protein functions together with the single-stranded DNA (ssDNA) binding protein RPA as the central scaffold to ensure proper positioning of repair factors in multi-protein nucleotide excision repair (NER) machinery. We previously determined the structure of a short motif in the disordered XPA N-terminus bound to the RPA32C domain. However, a second contact between the XPA DNA-binding domain (XPA DBD) and the RPA70AB tandem ssDNA-binding domains, which is likely to influence the orientation of XPA and RPA on the damaged DNA substrate, remains poorly characterized. NMR was used to map the binding interfaces of XPA DBD and RPA70AB. Combining NMR and X-ray scattering data with comprehensive docking and refinement revealed how XPA DBD and RPA70AB orient on model NER DNA substrates. The structural model enabled design of XPA mutations that inhibit the interaction with RPA70AB. These mutations decreased activity in cell-based NER assays, demonstrating the functional importance of XPA DBD-RPA70AB interaction. Our results inform ongoing controversy about where XPA is bound within the NER bubble, provide structural insights into the molecular basis for malfunction of disease-associated XPA missense mutations, and contribute to understanding of the structure and mechanical action of the NER machinery.
© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.
p63 is a transcriptional regulator of ectodermal development that is required for basal cell proliferation and stem cell maintenance. p73 is a closely related p53 family member that is expressed in select p63-positive basal cells and can heterodimerize with p63. p73-/- mice lack multiciliated cells and have reduced numbers of basal epithelial cells in select tissues; however, the role of p73 in basal epithelial cells is unknown. Herein, we show that p73-deficient mice exhibit delayed wound healing despite morphologically normal-appearing skin. The delay in wound healing is accompanied by decreased proliferation and increased levels of biomarkers of the DNA damage response in basal keratinocytes at the epidermal wound edge. In wild-type mice, this same cell population exhibited increased p73 expression after wounding. Analyzing single-cell transcriptomic data, we found that p73 was expressed by epidermal and hair follicle stem cells, cell types required for wound healing. Moreover, we discovered that p73 isoforms expressed in the skin (ΔNp73) enhance p63-mediated expression of keratinocyte genes during cellular reprogramming from a mesenchymal to basal keratinocyte-like cell. We identified a set of 44 genes directly or indirectly regulated by ΔNp73 that are involved in skin development, cell junctions, cornification, proliferation, and wound healing. Our results establish a role for p73 in cutaneous wound healing through regulation of basal keratinocyte function.
Cell-cycle checkpoints and DNA repair processes protect organisms from potentially lethal mutational damage. Compared to other budding yeasts in the subphylum Saccharomycotina, we noticed that a lineage in the genus Hanseniaspora exhibited very high evolutionary rates, low Guanine-Cytosine (GC) content, small genome sizes, and lower gene numbers. To better understand Hanseniaspora evolution, we analyzed 25 genomes, including 11 newly sequenced, representing 18/21 known species in the genus. Our phylogenomic analyses identify two Hanseniaspora lineages, a faster-evolving lineage (FEL), which began diversifying approximately 87 million years ago (mya), and a slower-evolving lineage (SEL), which began diversifying approximately 54 mya. Remarkably, both lineages lost genes associated with the cell cycle and genome integrity, but these losses were greater in the FEL. E.g., all species lost the cell-cycle regulator WHIskey 5 (WHI5), and the FEL lost components of the spindle checkpoint pathway (e.g., Mitotic Arrest-Deficient 1 [MAD1], Mitotic Arrest-Deficient 2 [MAD2]) and DNA-damage-checkpoint pathway (e.g., Mitosis Entry Checkpoint 3 [MEC3], RADiation sensitive 9 [RAD9]). Similarly, both lineages lost genes involved in DNA repair pathways, including the DNA glycosylase gene 3-MethylAdenine DNA Glycosylase 1 (MAG1), which is part of the base-excision repair pathway, and the DNA photolyase gene PHotoreactivation Repair deficient 1 (PHR1), which is involved in pyrimidine dimer repair. Strikingly, the FEL lost 33 additional genes, including polymerases (i.e., POLymerase 4 [POL4] and POL32) and telomere-associated genes (e.g., Repressor/activator site binding protein-Interacting Factor 1 [RIF1], Replication Factor A 3 [RFA3], Cell Division Cycle 13 [CDC13], Pbp1p Binding Protein [PBP2]). Echoing these losses, molecular evolutionary analyses reveal that, compared to the SEL, the FEL stem lineage underwent a burst of accelerated evolution, which resulted in greater mutational loads, homopolymer instabilities, and higher fractions of mutations associated with the common endogenously damaged base, 8-oxoguanine. We conclude that Hanseniaspora is an ancient lineage that has diversified and thrived, despite lacking many otherwise highly conserved cell-cycle and genome integrity genes and pathways, and may represent a novel, to our knowledge, system for studying cellular life without them.
Aside from abasic sites and ribonucleotides, the DNA adduct -methyl deoxyguanosine ( -CH dG) is one of the most abundant lesions in mammalian DNA. Because -CH dG is unstable, leading to deglycosylation and ring-opening, its miscoding potential is not well-understood. Here, we employed a 2'-fluoro isostere approach to synthesize an oligonucleotide containing an analog of this lesion ( -CH 2'-F dG) and examined its miscoding potential with four Y-family translesion synthesis DNA polymerases (pols): human pol (hpol) η, hpol κ, and hpol ι and Dpo4 from the archaeal thermophile We found that hpol η and Dpo4 can bypass the -CH 2'-F dG adduct, albeit with some stalling, but hpol κ is strongly blocked at this lesion site, whereas hpol ι showed no distinction with the lesion and the control templates. hpol η yielded the highest level of misincorporation opposite the adduct by inserting dATP or dTTP. Moreover, hpol η did not extend well past an -CH 2'-F dG:dT mispair. MS-based sequence analysis confirmed that hpol η catalyzes mainly error-free incorporation of dC, with misincorporation of dA and dG in 5-10% of products. We conclude that -CH 2'-F dG and, by inference, -CH dG have miscoding and mutagenic potential. The level of misincorporation arising from this abundant adduct can be considered as potentially mutagenic as a highly miscoding but rare lesion.
© 2019 Njuma et al.
Radiation therapy is often combined with androgen deprivation therapy in the treatment of aggressive localized prostate cancer. However, castration-resistant disease may not respond to testosterone deprivation approaches. Enzalutamide is a second-generation anti-androgen with high affinity and activity that is used for the treatment of metastatic disease. Although radiosensitization mechanisms are known to be mediated through androgen receptor activity, this project aims to uncover the detailed DNA damage repair factors influenced by enzalutamide using multiple models of androgen-sensitive (LNCaP) and castration-resistant human prostate cancer (22Rv1 and DU145). Enzalutamide is able to radiosensitize both androgen-dependent and androgen-independent human prostate cancer models in cell culture and xenografts in mice, as well as a treatment-resistant patient-derived xenograft. The enzalutamide-mediated mechanism of radiosensitization includes delay of DNA repair through temporal prolongation of the repair factor complexes and halting the cell cycle, which results in decreased colony survival. Altogether, these findings support the use of enzalutamide concurrently with radiotherapy to enhance the treatment efficacy for prostate cancer.
Infection by is the primary cause of gastric adenocarcinoma. The most potent virulence factor is cytotoxin-associated gene A (CagA), which is translocated by a type 4 secretion system (T4SS) into gastric epithelial cells and activates oncogenic signaling pathways. The gene encodes for a key component of the T4SS and can undergo gene rearrangements. We have shown that the cancer chemopreventive agent α-difluoromethylornithine (DFMO), known to inhibit the enzyme ornithine decarboxylase, reduces -mediated gastric cancer incidence in Mongolian gerbils. In the present study, we questioned whether DFMO might directly affect pathogenicity. We show that output strains isolated from gerbils treated with DFMO exhibit reduced ability to translocate CagA in gastric epithelial cells. Further, we frequently detected genomic modifications in the middle repeat region of the gene of output strains from DFMO-treated animals, which were associated with alterations in the CagY protein. Gerbils did not develop carcinoma when infected with a DFMO output strain containing rearranged or the parental strain in which the wild-type was replaced by with DFMO-induced rearrangements. Lastly, we demonstrate that in vitro treatment of by DFMO induces oxidative DNA damage, expression of the DNA repair enzyme MutS2, and mutations in , demonstrating that DFMO directly affects genomic stability. Deletion of abrogated the ability of DFMO to induce rearrangements directly. In conclusion, DFMO-induced oxidative stress in leads to genomic alterations and attenuates virulence.
Abasic sites are one of the most common DNA lesions. All known abasic site repair mechanisms operate only when the damage is in double-stranded DNA. Here, we report the discovery of 5-hydroxymethylcytosine (5hmC) binding, ESC-specific (HMCES) as a sensor of abasic sites in single-stranded DNA. HMCES acts at replication forks, binds PCNA and single-stranded DNA, and generates a DNA-protein crosslink to shield abasic sites from error-prone processing. This unusual HMCES DNA-protein crosslink intermediate is resolved by proteasome-mediated degradation. Acting as a suicide enzyme, HMCES prevents translesion DNA synthesis and the action of endonucleases that would otherwise generate mutations and double-strand breaks. HMCES is evolutionarily conserved in all domains of life, and its biochemical properties are shared with its E. coli ortholog. Thus, HMCES is an ancient DNA lesion recognition protein that preserves genome integrity by promoting error-free repair of abasic sites in single-stranded DNA.
Copyright © 2018 Elsevier Inc. All rights reserved.
DNA glycosylases remove aberrant DNA nucleobases as the first enzymatic step of the base excision repair (BER) pathway. The alkyl-DNA glycosylases AlkC and AlkD adopt a unique structure based on α-helical HEAT repeats. Both enzymes identify and excise their substrates without a base-flipping mechanism used by other glycosylases and nucleic acid processing proteins to access nucleobases that are otherwise stacked inside the double-helix. Consequently, these glycosylases act on a variety of cationic nucleobase modifications, including bulky adducts, not previously associated with BER. The related non-enzymatic HEAT-like repeat (HLR) proteins, AlkD2, and AlkF, have unique nucleic acid binding properties that expand the functions of this relatively new protein superfamily beyond DNA repair. Here, we review the phylogeny, biochemistry, and structures of the HLR proteins, which have helped broaden our understanding of the mechanisms by which DNA glycosylases locate and excise chemically modified DNA nucleobases.
© 2018 WILEY Periodicals, Inc.
DNA damage repair (DDR) pathways modulate cancer risk, progression, and therapeutic response. We systematically analyzed somatic alterations to provide a comprehensive view of DDR deficiency across 33 cancer types. Mutations with accompanying loss of heterozygosity were observed in over 1/3 of DDR genes, including TP53 and BRCA1/2. Other prevalent alterations included epigenetic silencing of the direct repair genes EXO5, MGMT, and ALKBH3 in ∼20% of samples. Homologous recombination deficiency (HRD) was present at varying frequency in many cancer types, most notably ovarian cancer. However, in contrast to ovarian cancer, HRD was associated with worse outcomes in several other cancers. Protein structure-based analyses allowed us to predict functional consequences of rare, recurrent DDR mutations. A new machine-learning-based classifier developed from gene expression data allowed us to identify alterations that phenocopy deleterious TP53 mutations. These frequent DDR gene alterations in many human cancers have functional consequences that may determine cancer progression and guide therapy.
Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.
Individuals with sporadic colorectal cancer (CRC) frequently harbor abnormalities in the composition of the gut microbiome; however, the microbiota associated with precancerous lesions in hereditary CRC remains largely unknown. We studied colonic mucosa of patients with familial adenomatous polyposis (FAP), who develop benign precursor lesions (polyps) early in life. We identified patchy bacterial biofilms composed predominately of and Genes for colibactin () and toxin (), encoding secreted oncotoxins, were highly enriched in FAP patients' colonic mucosa compared to healthy individuals. Tumor-prone mice cocolonized with (expressing colibactin), and enterotoxigenic showed increased interleukin-17 in the colon and DNA damage in colonic epithelium with faster tumor onset and greater mortality, compared to mice with either bacterial strain alone. These data suggest an unexpected link between early neoplasia of the colon and tumorigenic bacteria.
Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.