DNA Interstrand Research Articles

Structure of a Stable Interstrand DNA Cross-Link Involving a β-N-Glycosyl Linkage Between an N6-dA Amino Group and an Abasic Site.

Abasic (AP) sites are one of the most common forms of DNA damage. The deoxyribose ring of AP sites undergoes anomerization between α and β configurations, via an electrophilic aldehyde intermediate. In sequences where an adenine residue is located on the opposing strand and offset 1 nt to the 3' side of the AP site, the nucleophilic N6-dA amino group can react with the AP aldehyde residue to form an interstrand cross-link (ICL). Here, we present an experimentally determined structure of the dA-AP ICL by NMR spectroscopy. The ICL was constructed in the oligodeoxynucleotide 5'-d(T1A2T3G4T5C6T7A8A9G10T11T12C13A14T15C16T17A18)-3':5'-d(T19A20G21A22T23G24A25A26C27X28T29A30G31A32C33A34T35A36)-3' (X=AP site), with the dA-AP ICL forming between A8 and X28. The NMR spectra indicated an ordered structure for the cross-linked DNA duplex and afforded detailed spectroscopic resonance assignments. Structural refinement, using molecular dynamics calculations restrained by NOE data (rMD), revealed the structure of the ICL. In the dA-AP ICL, the 2'-deoxyribosyl ring of the AP site was ring-closed and in the β configuration. Juxtapositioning the N6-dA amino group and the aldehydic C1 of the AP site within bonding distance while simultaneously maintaining two flanking unpaired A9 and T29 bases stacked within the DNA is accomplished by the unwinding of the DNA at the ICL. The structural data is discussed in the context of recent studies describing the replication-dependent unhooking of the dA-AP ICL by the base excision repair glycosylase NEIL3.

Chromosome Instability in Fanconi Anemia: From Breaks to Phenotypic Consequences.

Fanconi anemia (FA), a chromosomal instability syndrome, is caused by inherited pathogenic variants in any of 22 FANC genes, which cooperate in the FA/BRCA pathway. This pathway regulates the repair of DNA interstrand crosslinks (ICLs) through homologous recombination. In FA proper repair of ICLs is impaired and accumulation of toxic DNA double strand breaks occurs. To repair this type of DNA damage, FA cells activate alternative error-prone DNA repair pathways, which may lead to the formation of gross structural chromosome aberrations of which radial figures are the hallmark of FA, and their segregation during cell division are the origin of subsequent aberrations such as translocations, dicentrics and acentric fragments. The deficiency in DNA repair has pleiotropic consequences in the phenotype of patients with FA, including developmental alterations, bone marrow failure and an extreme risk to develop cancer. The mechanisms leading to the physical abnormalities during embryonic development have not been clearly elucidated, however FA has features of premature aging with chronic inflammation mediated by pro-inflammatory cytokines, which results in tissue attrition, selection of malignant clones and cancer onset. Moreover, chromosomal instability and cell death are not exclusive of the somatic compartment, they also affect germinal cells, as evidenced by the infertility observed in patients with FA.

Identification of metabolic changes leading to cancer susceptibility in Fanconi anemia cells.

Fanconi anemia (FA) is a chromosomal instability disorder of bone marrow associated with aplastic anemia, congenital abnormalities and a high risk of malignancies. The identification of more than two dozen FA genes has revealed a plethora of interacting proteins that are mainly involved in repair of DNA interstrand crosslinks (ICLs). Other important findings associated with FA are inflammation, oxidative stress response, mitochondrial dysfunction and mitophagy. In this work, we performed quantitative proteomic and metabolomic analyses on defective FA cells and identified a number of metabolic abnormalities associated with cancer. In particular, an increased de novo purine biosynthesis, a high concentration of fumarate, and an accumulation of purinosomal clusters were found. This was in parallel with decreased OXPHOS and altered glycolysis. On the whole, our results indicate an association between the need for nitrogenous bases upon impaired DDR in FA cells with a subsequent increase in purine metabolism and a potential role in oncogenesis.

The Ubiquitin Ligase TRAIP: Double-Edged Sword at the Replisome.

In preparation for cell division, the genome must be copied with high fidelity. However, replisomes often encounter obstacles, including bulky DNA lesions caused by reactive metabolites and chemotherapeutics, as well as stable nucleoprotein complexes. Here, we discuss recent advances in our understanding of TRAIP, a replisome-associated E3 ubiquitin ligase that is mutated in microcephalic primordial dwarfism. In interphase, TRAIP helps replisomes overcome DNA interstrand crosslinks and DNA-protein crosslinks, whereas in mitosis it triggers disassembly of all replisomes that remain on chromatin. We describe a model to explain how TRAIP performs these disparate functions and how they help maintain genome integrity.

MODL-15. THE COMBINATION TREATMENT OF PARP INHIBITOR AND TMZ, OR DAG WILL BE PROMISING TREATMENT IN SF8628

Diffuse midline glioma, H3 K27M-mutant (DMG) is a newly defined entity. The prognosis of DMG is poor. Because surgical resection is often incomplete for DMG, radiotherapy and chemotherapy are important. Temozolomide (TMZ) is an alkylating agent that adds a methyl group to DNA (O6-guanine, N7-guanine, and N3-adenine). TMZ-induced cytotoxicity is mainly derived from O6-methylguanine, which is repaired by O6-methylguanine DNA methyltransferase (MGMT). It has been reported that most of DMG lacked MGMT promoter hypermethylation, which is thought to contribute to less effectiveness of TMZ to DMG. The purpose of the study is to explore the way to inhibit the proliferation of DMG. A DMG cell line, SF8628, was used for the experiments. SF8628 had the expression of MGMT and was revealed to be resistant to TMZ. Because N7-methylguanine and N3-methyladenine are repaired via base excision repair, poly (adenosine diphosphate-ribose) polymerase (PARP) inhibitor combined with TMZ was considered to be effective to suppress the proliferation of SF8628. As expected, PARP inhibitor enhanced TMZ-induced cytotoxicity in SF8628. Dianhydrogalactiol (DAG) is a bifunctional DNA-targeting agent forming N7-alkylguanine and inter-strand DNA crosslinks. DAG reduced the clonogenicity of SF8628. Moreover, inhibition of homologous recombination enhanced the DAG-induced cytotoxicity in SF8629. The combination treatment of PARP inhibitor and TMZ, or DAG were revealed to be promising treatments in SF8628.

Translesion synthesis of the major nitrogen mustard-induced DNA lesion by human DNA polymerase η.

Nitrogen mustards are among the first modern anticancer chemotherapeutics that are still widely used as non-specific anticancer alkylating agents. While the mechanism of action of mustard drugs involves the generation of DNA interstrand cross-links, the predominant lesions produced by these drugs are nitrogen half-mustard-N7-dG (NHMG) adducts. The bulky major groove lesion NHMG, if left unrepaired, can be bypassed by translesion synthesis (TLS) DNA polymerases. However, studies of the TLS past NHMG have not been reported so far. Here, we present the first synthesis of an oligonucleotide containing a site-specific NHMG. We also report kinetic and structural characterization of human DNA polymerase η (polη) bypassing NHMG. The templating NHMG slows dCTP incorporation ∼130-fold, while it increases the misincorporation frequency ∼10-30-fold, highlighting the promutagenic nature of NHMG. A crystal structure of polη incorporating dCTP opposite NHMG shows a Watson-Crick NHMG:dCTP base pair with a large propeller twist angle. The nitrogen half-mustard moiety fits snugly into an open cleft created by the Arg61-Trp64 loop of polη, suggesting a role of the Arg61-Trp64 loop in accommodating bulky major groove adducts during lesion bypass. Overall, our results presented here to provide first insights into the TLS of the major DNA adduct formed by nitrogen mustard drugs.

Comprehensive Synthetic Genetic Array Analysis of Alleles That Interact with Mutation of the Saccharomyces cerevisiae RecQ Helicases Hrq1 and Sgs1.

Most eukaryotic genomes encode multiple RecQ family helicases, including five such enzymes in humans. For many years, the yeast Saccharomyces cerevisiae was considered unusual in that it only contained a single RecQ helicase, named Sgs1. However, it has recently been discovered that a second RecQ helicase, called Hrq1, resides in yeast. Both Hrq1 and Sgs1 are involved in genome integrity, functioning in processes such as DNA inter-strand crosslink repair, double-strand break repair, and telomere maintenance. However, it is unknown if these enzymes interact at a genetic, physical, or functional level as demonstrated for their human homologs. Thus, we performed synthetic genetic array (SGA) analyses of hrq1Δ and sgs1Δ mutants. As inactive alleles of helicases can demonstrate dominant phenotypes, we also performed SGA analyses on the hrq1-K318A and sgs1-K706A ATPase/helicase-null mutants, as well as all combinations of deletion and inactive double mutants. We crossed these eight query strains (hrq1Δ, sgs1Δ, hrq1-K318A, sgs1-K706A, hrq1Δ sgs1Δ, hrq1Δ sgs1-K706A, hrq1-K318A sgs1Δ, and hrq1-K318A sgs1-K706A) to the S. cerevisiae single gene deletion and temperature-sensitive allele collections to generate double and triple mutants and scored them for synthetic positive and negative genetic effects based on colony growth. These screens identified hundreds of synthetic interactions, supporting the known roles of Hrq1 and Sgs1 in DNA repair, as well as suggesting novel connections to rRNA processing, mitochondrial DNA maintenance, transcription, and lagging strand synthesis during DNA replication.

The Genetic and Physical Interactomes of the Saccharomyces cerevisiae Hrq1 Helicase.

The human genome encodes five RecQ helicases (RECQL1, BLM, WRN, RECQL4, and RECQL5) that participate in various processes underpinning genomic stability. Of these enzymes, the disease-associated RECQL4 is comparatively understudied due to a variety of technical challenges. However, Saccharomyces cerevisiae encodes a functional homolog of RECQL4 called Hrq1, which is more amenable to experimentation and has recently been shown to be involved in DNA inter-strand crosslink (ICL) repair and telomere maintenance. To expand our understanding of Hrq1 and the RecQ4 subfamily of helicases in general, we took a multi-omics approach to define the Hrq1 interactome in yeast. Using synthetic genetic array analysis, we found that mutations of genes involved in processes such as DNA repair, chromosome segregation, and transcription synthetically interact with deletion of HRQ1 and the catalytically inactive hrq1-K318A allele. Pull-down of tagged Hrq1 and mass spectrometry identification of interacting partners similarly underscored links to these processes and others. Focusing on transcription, we found that hrq1 mutant cells are sensitive to caffeine and that mutation of HRQ1 alters the expression levels of hundreds of genes. In the case of hrq1-K318A, several of the most highly upregulated genes encode proteins of unknown function whose expression levels are also increased by DNA ICL damage. Together, our results suggest a heretofore unrecognized role for Hrq1 in transcription, as well as novel members of the Hrq1 ICL repair pathway. These data expand our understanding of RecQ4 subfamily helicase biology and help to explain why mutations in human RECQL4 cause diseases of genomic instability.

Photoinduced DNA Interstrand Cross-Linking by Benzene Derivatives: Leaving Groups Determine the Efficiency of the Cross-Linker

We have synthesized and characterized two small libraries of 2-OMe or 2-NO2-benzene analogues 2a-i and 3a-i containing a wide variety of leaving groups. Irradiation of these compounds at 350 nm generated benzyl radicals that were spontaneously oxidized to benzyl cations directly producing DNA interstrand cross-links (ICLs). Compounds with a 2-methoxy substituent showed a faster cross-linking reaction rate and higher ICL efficiency than the corresponding 2-nitro analogues. Apart from the aromatic substituent, the benzylic leaving groups greatly affected DNA cross-linking efficiency. Higher ICL yields were observed for compounds with OCH3 (3b), OCH2Ph (3d), or Ph3P+ (3i) as leaving groups than those containing OAc (3a), NMe2 (3e), morpholine (3f), OCH2CH═CH2 (3c), SPh (3g), or SePh (3h). The heat stability study of the isolated ICL products indicated that dGs were the preferred alkylation sites in DNA for the benzyl cations produced from 2a-i, 3c, and 3e-i while 3a (L = OAc), 3b (L = OMe), and 3d (L = OCH2Ph) showed a similar photoreactivity toward dGs and dAs. Although the photogenerated benzyl cations alkylated dG, dC, and dA, ICL assay with variation of DNA sequences showed that the ICL reaction occurred with opposing dG/dC but not with staggered dA/dA.

DNA Repair Targeted Therapy

The repair of DNA harm could be a complex prepare that depends on specific pathways to cure particular sorts of harm to DNA. The run of insuperable to DNA incorporates little, humble changes in structure counting jumbled bases and straightforward methylation occasions to oxidized bases, intra- and interstrand DNA crosslinks, DNA twofold strand breaks and protein-DNA adducts. Pathways required for the repair of these injuries incorporate jumble repair, base extraction repair, nucleotide extraction repair, and the homology coordinated repair/Fanconi frailty pathway. Each of these pathways contributes to hereditary steadiness, and transformations in qualities encoding proteins included in these pathways have been illustrated to advance hereditary insecurity and cancer. In truth, it has been proposed all cancers show abandons in DNA repair. It has too been illustrated that the capacity of cancer cells to repair restoratively actuated DNA harm impacts helpful adequacy. This has driven to focusing on DNA repair pathways and proteins to create anti-cancer specialists that will increment affectability to conventional chemotherapeutics. Whereas beginning ponders mulled and were tormented by a need of specificity and a characterized instrument of activity, more later approaches to misuse engineered deadly interaction and create tall liking chemical inhibitors have demonstrated impressively more viable. In this audit we are going highlight later propels and talk about past disappointments in focusing on DNA repair to clear the way for future DNA repair focused on operators and their utilize in cancer treatment.

Clinical Perspectives of ERCC1 in Bladder Cancer.

ERCC1 is a key regulator of nucleotide excision repair (NER) pathway that repairs bulky DNA adducts, including intrastrand DNA adducts and interstrand crosslinks (ICLs). Overexpression of ERCC1 has been linked to increased DNA repair capacity and platinum resistance in solid tumors. Multiple single nucleotide polymorphisms (SNPs) have been detected in ERCC1 gene that may affect ERCC1 protein expression. Platinum-based treatment remains the cornerstone of urothelial cancer treatment. Given the expanding application of neoadjuvant and adjuvant chemotherapy in locally advanced bladder cancer, there is an emerging need for biomarkers that could distinguish potential responders to cisplatin treatment. Extensive research has been done regarding the prognostic and predictive role of ERCC1 gene expression and polymorphisms in bladder cancer. Moreover, novel compounds have been recently developed to target ERCC1 protein function in order to maximize sensitivity to cisplatin. We aim to review all the existing literature regarding the role of the ERCC1 gene in bladder cancer and address future perspectives for its clinical application.

Esophageal cancer as initial presentation of Fanconi anemia in patients with a hypomorphic FANCA variant.

Fanconi anemia (FA) is a clinically heterogenous and genetically diverse disease with 22 known complementation groups (FA-A to FA-W), resulting from the inability to repair DNA interstrand cross-links. This rare disorder is characterized by congenital defects, bone marrow failure, and cancer predisposition. FANCA is the most commonly mutated gene in FA and a variety of mostly private mutations have been documented, including small and large indels and point and splicing variants. Genotype–phenotype associations in FA are complex, and a relationship between particular FANCA variants and the observed cellular phenotype or illness severity remains unclear. In this study, we describe two siblings with compound heterozygous FANCA variants (c.3788_3790delTCT and c.4199G > A) who both presented with esophageal squamous cell carcinoma at the age of 51. The proband came to medical attention when he developed pancytopenia after a single cycle of low-dose chemotherapy including platinum-based therapy. Other than a minor thumb abnormality, neither patient had prior findings to suggest FA, including normal blood counts and intact fertility. Patient fibroblasts from both siblings display increased chromosomal breakage and hypersensitivity to interstrand cross-linking agents as seen in typical FA. Based on our functional data demonstrating that the c.4199G > A/p.R1400H variant represents a hypomorphic FANCA allele, we conclude that the residual activity of the Fanconi anemia repair pathway accounts for lack of spontaneous bone marrow failure or infertility with the late presentation of malignancy as the initial disease manifestation. This and similar cases of adult-onset esophageal cancer stress the need for chromosome breakage testing in patients with early onset of aerodigestive tract squamous cell carcinomas before platinum-based therapy is initiated.

Binding of FANCD2 to SRSF1 Splicing Factor Prevents Genomic Instability through R Loop Regulation

BACKGROUND. Fanconi Anemia (FA) is a rare disease characterized by congenital abnormalities, bone marrow failure and cancer susceptibility. The pivotal role of the FA pathway is DNA interstrand crosslink (ICL) repair, where FANCD2-FANCI complex monoubiquitination is the key regulatory step. Recent studies have linked defects in the FA pathway to R-loop metabolism and implicated R-loops as an endogenous source of genomic instability that contributes to the FA phenotype, although the mechanism remains elusive. Splicing factors, such as SRSF1 (ASF/SF2), have also been linked to R loops, cancer, and myelodysplastic syndrome (MDS), so we hypothesized a link between SRSF1 and FA. METHODS. Cells: FA-D2 mutant (PD20) and corrected (D2), HeLa. siRNA transfections were used to reduce SRSF1 and FANCD2 protein levels. Survival assays were performed to determine cell sensitivity to DNA damaging agents. In vitro immunoprecipitation was performed using SRSF1 purified from bacteria and FANCD2-FANCI purified from insect cells. R-loops were studied using the Damage At RNA Transcription (DART) assay and S9.6 immunofluorescence. RESULTS. In order to study the involvement of splicing factors in the FA pathway, we knocked down SRSF1 by using siRNA. SRSF1 knockdown lead to impaired FANCD2 monoubiquitination after DNA damage as well as diminished cell survival, thus creating a FA-like phenotype (A). In addition, immunoprecipitation in cells (B) and in vitro showed direct interaction between FANCD2 and SRSF1. We found that double FANCD2+SRSF1 knockdown led to increased R-loops compared to FANCD2/SRSF1 single knockdown visualized by S9.6 immunofluorescence. In addition, DART assay revealed colocalization of SRSF1 and FANCD2 with R-loops in highly transcribed genomic sites, known to be R-loop hotspots (C). Finally, we found that SRSF1 interacts with the mRNA export factor NFX1 in a FANCD2-dependent fashion (D) through RNA, suggesting that they could cooperate in the prevention of R-loop formation by exporting accumulated RNA in the nucleus. CONCLUSION. Our findings suggest a novel non-canonical mechanism of FANCD2 via regulation of SRSF1 in R-loop prevention via facilitation of mRNA export, thus preventing genomic instability (E). CLINICAL APPLICABILITY. These results will reveal a novel mechanism for a non-canonical role of the FA pathway in prevention of genomic instability and will shed light upon the most basic cellular processes of the FA pathway, which is germane for delineating more generalized mechanisms of oncogenesis and genome maintenance. As accumulation of R-loops is associated with FA, we propose that R-loops and R-loop processing proteins (such as SRSF1) offer new avenues as relevant therapeutic targets not only for FA but also for multiple cancers. Disclosures No relevant conflicts of interest to declare.

Evidence for functional and regulatory cross-talk between Wnt/β-catenin signalling and Mre11-Rad50-Nbs1 complex in the repair of cisplatin-induced DNA cross-links.

The canonical Wnt/β-catenin signalling pathway plays a crucial role in a variety of functions including cell proliferation and differentiation, tumorigenic processes and radioresistance in cancer cells. The Mre11–Rad50–Nbs1 (MRN) complex has a pivotal role in sensing and repairing DNA damage. However, it remains unclear whether a connection exists between Wnt/β-catenin signalling and the MRN complex in the repair of cisplatin-induced DNA interstrand cross-links (ICLs). Here, we report that (1) cisplatin exposure results in a significant increase in the levels of MRN complex subunits in human tumour cells; (2) cisplatin treatment stimulates Wnt/β-catenin signalling through increased β-catenin expression; (3) the functional perturbation of Wnt/β-catenin signalling results in aberrant cell cycle dynamics and the activation of DNA damage response and apoptosis; (4) a treatment with CHIR99021, a potent and selective GSK3β inhibitor, augments cisplatin-induced cell death in cancer cells. On the other hand, inactivation of the Wnt/β-catenin signalling with FH535 promotes cell survival. Consistently, the staining pattern of γH2AX-foci is significantly reduced in the cells exposed simultaneously to cisplatin and FH535; and (5) inhibition of Wnt/β-catenin signalling impedes cisplatin-induced phosphorylation of Chk1, abrogates the G2/M phase arrest and impairs recombination-based DNA repair. Our data further show that Wnt signalling positively regulates the expression of β-catenin, Mre11 and FANCD2 at early time points, but declining thereafter due to negative feedback regulation. These results support a model wherein Wnt/β-catenin signalling and MRN complex crosstalk during DNA ICL repair, thereby playing an important role in the maintenance of genome stability.

Near-Infrared Light Irradiation Induced Mild Hyperthermia Enhances Glutathione Depletion and DNA Interstrand Cross-Link Formation for Efficient Chemotherapy.

DNA alkylating agents generally kill tumor cells by covalently binding with DNA to form interstrand or intrastrand cross-links. However, in the case of cisplatin, only a few DNA adducts (<1%) are highly toxic irreparable interstrand cross-links. Furthermore, cisplatin is rapidly detoxified by high levels of intracellular thiols such as glutathione (GSH). Since the discovery of its mechanism of action, people have been looking for ways to directly and efficiently remove intracellular GSH and increase interstrand cross-links to improve drug efficacy and overcome resistance, but there has been little breakthrough. Herein, we hypothesized that the anticancer efficiency of cisplatin can be enhanced through iodo-thiol click chemistry mediated GSH depletion and increased formation of DNA interstrand cross-links via mild hyperthermia triggered by near-infrared (NIR) light. This was achieved by preparing an amphiphilic polymer with platinum(IV) (Pt(IV)) prodrugs and pendant iodine atoms (iodides). The polymer was further used to encapsulate IR780 and assembled into Pt-I-IR780 nanoparticles. Induction of mild hyperthermia (43 °C) at the tumor site by NIR light irradiation had three effects: (1) it accelerated the GSH-mediated reduction of Pt(IV) in the polymer main chain to platinum(II) (Pt(II)); (2) it boosted the iodo-thiol substitution click reaction between GSH and iodide, thereby attenuating the GSH-mediated detoxification of cisplatin; (3) it increased the proportion of highly toxic and irreparable Pt-DNA interstrand cross-links. Therefore, we find that mild hyperthermia induced via NIR irradiation can enhance the killing of cancer cells and reduce the tumor burden, thus delivering efficient chemotherapy.

New chalcone-tethered 1,3,5-triazines potentiate the anticancer effect of cisplatin against human lung adenocarcinoma A549 cells by enhancing DNA damage and cell apoptosis

In the pursuit of new compounds for co-treatment to enhance the anticancer efficacy of cisplatin against lung adenocarcinoma, a series of chalcone-tethered 1,3,5-triazines was designed and synthesized. MTT assay was used to evaluate the anticancer activity of the combinations in which two hybrids 10 and 12 were found to significantly inhibit A549 cancer cells viability and their IC50 values were 24.5 and 17 µM, respectively in reference to cisplatin (IC50 = 21.5 µM). The combined effect of cisplatin with each of 10 and 12 was analyzed according to Chou-Talalay method against both A549 and normal human fibroblast cells. Mechanistic studies employing MALDI-TOF MS and fluorescence spectroscopy using Evagreen probe inferred that 10 and 12 induced DNA double strand breaks in contrast to cisplatin which induces DNA interstrand cross-links. Also, DNA damage kinetics study demonstrated the difference in the rate of DNA damage induced by both 10 and 12 alone and in combination with cisplatin. Further Annexin V-FITC/propidium iodide dual staining assay provided evidence that 10 and 12 induced apoptosis via different pattern to cisplatin and their combination with cisplatin promoted more cells to enter late apoptosis and necrosis. Molecular docking of 10 and 12 in the active pocket of DNA dodecamer displayed their binding modes with higher number of stable hydrogen bond donor as well as π-H interactions in reference to the original ligand.

Intrinsic DNA damage repair deficiency results in progressive microglia loss and replacement.

The DNA excision repair protein Ercc1 is important for nucleotide excision, double strand DNA break, and interstrand DNA crosslink repair. In constitutive Ercc1‐knockout mice, microglia display increased phagocytosis, proliferation and an enhanced responsiveness to lipopolysaccharide (LPS)‐induced peripheral inflammation. However, the intrinsic effects of Ercc1‐deficiency on microglia are unclear. In this study, Ercc1 was specifically deleted from Cx3cr1‐expressing cells and changes in microglia morphology and immune responses at different times after deletion were determined. Microglia numbers were reduced with approximately 50% at 2–12 months after Ercc1 deletion. Larger and more ramified microglia were observed following Ercc1 deletion both in vivo and in organotypic hippocampal slice cultures. Ercc1‐deficient microglia were progressively lost, and during this period, microglia proliferation was transiently increased. Ercc1‐deficient microglia were gradually replaced by nondeficient microglia carrying a functional Ercc1 allele. In contrast to constitutive Ercc1‐deficient mice, microglia‐specific deletion of Ercc1 did not induce microglia activation or increase their responsiveness to a systemic LPS challenge. Gene expression analysis suggested that Ercc1 deletion in microglia induced a transient aging signature, which was different from a priming or disease‐associated microglia gene expression profile.

Acetylation modulates the Fanconi anemia pathway by protecting FAAP20 from ubiquitin-mediated proteasomal degradation

Fanconi anemia (FA) is a chromosome instability syndrome of children caused by inherited mutations in one of FA genes, which together constitute a DNA interstrand cross-link (ICL) repair, or the FA pathway. Monoubiquitination of Fanconi anemia group D2 protein (FANCD2) by the multisubunit ubiquitin E3 ligase, the FA core complex, is an obligate step in activation of the FA pathway, and its activity needs to be tightly regulated. FAAP20 is a key structural component of the FA core complex, and regulated proteolysis of FAAP20 mediated by prolyl cis-trans isomerization and phosphorylation at a consensus phosphodegron motif is essential for preserving the integrity of the FA core complex, and thus FANCD2 monoubiquitination. However, how ubiquitin-dependent FAAP20 degradation is modulated to fine-tune FA pathway activation remains largely un-known. Here, we present evidence that FAAP20 is acetylated by the acetyltransferase p300/CBP on lysine 152, the key residue that when polyubiquitinated results in the degradation of FAAP20. Acetylation or mutation of the lysine residue stabilizes FAAP20 by preventing its ubiquitination, thereby protecting it from proteasome-dependent FAAP20 degradation. Consequently, disruption of the FAAP20 acetylation pathway impairs FANCD2 activation. Together, our study reveals a competition mechanism between ubiquitination and acetylation of a common lysine residue that controls FAAP20 stability and highlights a complex balancing between different posttranslational modifications as a way to refine the FA pathway signaling required for DNA ICL repair and genome stability.

Inactivation of ribosomal protein S27-like impairs DNA interstrand cross-link repair by destabilization of FANCD2 and FANCI

Ribosomal protein S27-like (RPS27L), an evolutionarily conserved ribosomal protein and a direct p53 target, plays an important role in maintenance of genome integrity. We have previously reported that RPS27L regulates radiation sensitivity via the MDM2-p53 and MDM2-MRN-ATM axes. Whether and how RPS27L modulates DNA interstrand cross-link (ICL) repair is unknown. Here we identified that RPS27L binds to FANCD2 and FANCI, two Fanconi anemia (FA) proteins functioning in ICL repair pathway. Upon RPS27L knockdown, the levels of FANCD2 and FANCI are reduced due to accelerated degradation via p62-mediated autophagy-lysosome pathway, which is abrogated by chloroquine (CQ) treatment or Beclin 1 knockdown. Biologically, RPS27L knockdown suppresses FANCD2 foci formation and impairs ICL repair upon exposure to ICL-inducing agent mitomycin C (MMC) in lung cancer cells. This effect of MMC sensitization can be partially reversed by CQ treatment. Together, our study shows that RPS27L positively regulates ICL repair by binding with FANCD2 and FANCI to prevent their degradation via autophagy-lysosome system.

BRIP1 rs10744996C>A variant increases the risk of chronic obstructive pulmonary disease in the Mongolian population of northern China.

What is the central question of this study? What is the role of breast cancer type 1 interacting protein C-terminal helicase 1 (BRIP1) polymorphism in chronic obstructive pulmonary disease (COPD)? What is the main finding and its importance? Variant rs10744996C>A of BRIP1 increases the susceptibility of the Mongolian population to COPD. The expression of BRIP1 was significantly reduced in cigarette smoke extract-treated airway epithelial cells. Cigarette smoke is a major environmental pollutant that can induce DNA damage in humans. The development and progression of chronic obstructive pulmonary disease (COPD) are known to be related to the impairment of DNA repair. Breast cancer type 1 interacting protein C-terminal helicase 1 (BRIP1) plays an important role in DNA interstrand crosslink repair and double-strand break repair. However, the role of BRIP1 polymorphisms in COPD has not been previously described. In this study, whole genome sequencing was used to identify mutations, and single nucleotide polymorphism (SNP) genotyping was used to verify the selected SNPs. In addition the BRIP1 expression levels in 16HBE and A549 airway epithelial cells treated with or without cigarette smoke extract (CSE) were measured using western blotting and RT-qPCR. Rs10744996C>A in the 3'-untranslated region (3'UTR) of BRIP1 was then genotyped in 1296 COPD cases and 988 healthy control subjects from a Mongolian population in northern China. Significant differences in the distribution of rs10744996C>A variants between COPD and control groups (P=0.001) were identified. Rs10744996C>A was found to be associated with significantly increased COPD risk (adjusted odds ratio=1.60, 95% CI=1.30-1.98, P<0.0001). Additionally, rs10744996A genotype was found to interact with a family history of cancer and a history of x-ray exposure (P=0.028 and 0.009, respectively). BRIP1 expression levels in 16HBE and A549 cells treated with CSE were significantly lower compared to the control treated cells. The rs10744996C>A variant of BRIP1 increased the COPD susceptibility of the Mongolian population cohort. BRIP1 mRNA and protein expression levels were significantly reduced in conjunction with CSE-induced DNA damage in 16HBE and A549 cells.