Structural mechanism of protein recognition by the FW domain of autophagy receptor Nbr1.

Neighbor of BRCA1 (Nbr1) is a conserved autophagy receptor that provides cargo selectivity to autophagy. The four-tryptophan (FW) domain is a signature domain of Nbr1, but its exact function remains unclear. Here, we show that Nbr1 from the filamentous fungus Chaetomium thermophilum uses its FW domain to bind the α-mannosidase Ams1, a cargo of selective autophagy in both budding yeast and fission yeast, and delivers Ams1 to the vacuole by conventional autophagy in heterologous fission yeast. The structure of the Ams1-FW complex was determined at 2.2 Å resolution by cryo-electron microscopy. The FW domain adopts an immunoglobulin-like ß-sandwich structure and recognizes the quaternary structure of the Ams1 tetramer. Notably, the N-terminal di-glycine of Ams1 is specifically recognized by a conserved pocket of the FW domain. The FW domain becomes degenerated in fission yeast Nbr1, which binds Ams1 with a ZZ domain instead. Our findings illustrate the protein binding mode of the FW domain and reveal the versatility of Nbr1-mediated cargo recognition.

Genetically encoded tags for direct synthesis of EM-visible gold nanoparticles in cells.

Genetically encoded tags for single-molecule imaging in electron microscopy (EM) are long-awaited. Here, we report an approach for directly synthesizing EM-visible gold nanoparticles (AuNPs) on cysteine-rich tags for single-molecule visualization in cells. We first uncovered an auto-nucleation suppression mechanism that allows specific synthesis of AuNPs on isolated tags. Next, we exploited this mechanism to develop approaches for single-molecule detection of proteins in prokaryotic cells and achieved an unprecedented labeling efficiency. We then expanded it to more complicated eukaryotic cells and successfully detected the proteins targeted to various organelles, including the membranes of endoplasmic reticulum (ER) and nuclear envelope, ER lumen, nuclear pores, spindle pole bodies and mitochondrial matrices. We further implemented cysteine-rich tag-antibody fusion proteins as new immuno-EM probes. Thus, our approaches should allow biologists to address a wide range of biological questions at the single-molecule level in cellular ultrastructural contexts.

Tdp1 processes chromate-induced single-strand DNA breaks that collapse replication forks.

Hexavalent chromium [Cr(VI)] damages DNA and causes cancer, but it is unclear which DNA damage responses (DDRs) most critically protect cells from chromate toxicity. Here, genome-wide quantitative functional profiling, DDR measurements and genetic interaction assays in Schizosaccharomyces pombe reveal a chromate toxicogenomic profile that closely resembles the cancer chemotherapeutic drug camptothecin (CPT), which traps Topoisomerase 1 (Top1)-DNA covalent complex (Top1cc) at the 3' end of single-stand breaks (SSBs), resulting in replication fork collapse. ATR/Rad3-dependent checkpoints that detect stalled and collapsed replication forks are crucial in Cr(VI)-treated cells, as is Mus81-dependent sister chromatid recombination (SCR) that repairs single-ended double-strand breaks (seDSBs) at broken replication forks. Surprisingly, chromate resistance does not require base excision repair (BER) or interstrand crosslink (ICL) repair, nor does co-elimination of XPA-dependent nucleotide excision repair (NER) and Rad18-mediated post-replication repair (PRR) confer chromate sensitivity in fission yeast. However, co-elimination of Tdp1 tyrosyl-DNA phosphodiesterase and Rad16-Swi10 (XPF-ERCC1) NER endonuclease synergistically enhances chromate toxicity in top1Δ cells. Pnk1 polynucleotide kinase phosphatase (PNKP), which restores 3'-hydroxyl ends to SSBs processed by Tdp1, is also critical for chromate resistance. Loss of Tdp1 ameliorates pnk1Δ chromate sensitivity while enhancing the requirement for Mus81. Thus, Tdp1 and PNKP, which prevent neurodegeneration in humans, repair an important class of Cr-induced SSBs that collapse replication forks.

SUMO-Targeted DNA Translocase Rrp2 Protects the Genome from Top2-Induced DNA Damage.

The action of DNA topoisomerase II (Top2) creates transient DNA breaks that are normally concealed inside Top2-DNA covalent complexes. Top2 poisons, including ubiquitously present natural compounds and clinically used anti-cancer drugs, trap Top2-DNA complexes. Here, we show that cells actively prevent Top2 degradation to avoid the exposure of concealed DNA breaks. A genome-wide screen revealed that fission yeast cells lacking Rrp2, an Snf2-family DNA translocase, are strongly sensitive to Top2 poisons. Loss of Rrp2 enhances SUMOylation-dependent ubiquitination and degradation of Top2, which in turn increases DNA damage at sites where Top2-DNA complexes are trapped. Rrp2 possesses SUMO-binding ability and prevents excessive Top2 degradation by competing against the SUMO-targeted ubiquitin ligase (STUbL) for SUMO chain binding and by displacing SUMOylated Top2 from DNA. The budding yeast homolog of Rrp2, Uls1, plays a similar role, indicating that this genome protection mechanism is widely employed, a finding with implications for cancer treatment.

Global Fitness Profiling Identifies Arsenic and Cadmium Tolerance Mechanisms in Fission Yeast.

Heavy metals and metalloids such as cadmium [Cd(II)] and arsenic [As(III)] are widespread environmental toxicants responsible for multiple adverse health effects in humans. However, the molecular mechanisms underlying metal-induced cytotoxicity and carcinogenesis, as well as the detoxification and tolerance pathways, are incompletely understood. Here, we use global fitness profiling by barcode sequencing to quantitatively survey the Schizosaccharomyces pombe haploid deletome for genes that confer tolerance of cadmium or arsenic. We identified 106 genes required for cadmium resistance and 110 genes required for arsenic resistance, with a highly significant overlap of 36 genes. A subset of these 36 genes account for almost all proteins required for incorporating sulfur into the cysteine-rich glutathione and phytochelatin peptides that chelate cadmium and arsenic. A requirement for Mms19 is explained by its role in directing iron-sulfur cluster assembly into sulfite reductase as opposed to promoting DNA repair, as DNA damage response genes were not enriched among those required for cadmium or arsenic tolerance. Ubiquinone, siroheme, and pyridoxal 5'-phosphate biosynthesis were also identified as critical for Cd/As tolerance. Arsenic-specific pathways included prefoldin-mediated assembly of unfolded proteins and protein targeting to the peroxisome, whereas cadmium-specific pathways included plasma membrane and vacuolar transporters, as well as Spt-Ada-Gcn5-acetyltransferase (SAGA) transcriptional coactivator that controls expression of key genes required for cadmium tolerance. Notable differences are apparent with corresponding screens in the budding yeast Saccharomyces cerevisiae, underscoring the utility of analyzing toxic metal defense mechanisms in both organisms.

Dimerization Mediated by a Divergent Forkhead-associated Domain Is Essential for the DNA Damage and Spindle Functions of Fission Yeast Mdb1.

MDC1 is a key factor of DNA damage response in mammalian cells. It possesses two phospho-binding domains. In its C terminus, a tandem BRCA1 C-terminal domain binds phosphorylated histone H2AX, and in its N terminus, a forkhead-associated (FHA) domain mediates a phosphorylation-enhanced homodimerization. The FHA domain of the Drosophila homolog of MDC1, MU2, also forms a homodimer but utilizes a different dimer interface. The functional importance of the dimerization of MDC1 family proteins is uncertain. In the fission yeast Schizosaccharomyces pombe, a protein sharing homology with MDC1 in the tandem BRCA1 C-terminal domain, Mdb1, regulates DNA damage response and mitotic spindle functions. Here, we report the crystal structure of the N-terminal 91 amino acids of Mdb1. Despite a lack of obvious sequence conservation to the FHA domain of MDC1, this region of Mdb1 adopts an FHA-like fold and is therefore termed Mdb1-FHA. Unlike canonical FHA domains, Mdb1-FHA lacks all the conserved phospho-binding residues. It forms a stable homodimer through an interface distinct from those of MDC1 and MU2. Mdb1-FHA is important for the localization of Mdb1 to DNA damage sites and the spindle midzone, contributes to the roles of Mdb1 in cellular responses to genotoxins and an antimicrotubule drug, and promotes in vitro binding of Mdb1 to a phospho-H2A peptide. The defects caused by the loss of Mdb1-FHA can be rescued by fusion with either of two heterologous dimerization domains, suggesting that the main function of Mdb1-FHA is mediating dimerization. Our data support that FHA-mediated dimerization is conserved for MDC1 family proteins.

Genome-wide screens for sensitivity to ionizing radiation identify the fission yeast nonhomologous end joining factor Xrc4.

Nonhomologous end joining (NHEJ) is the main means for repairing DNA double-strand breaks (DSBs) in human cells. Molecular understanding of NHEJ has benefited from analyses in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe. In human cells, the DNA ligation reaction of the classical NHEJ pathway is carried out by a protein complex composed of DNA ligase IV (LigIV) and XRCC4. In S. cerevisiae, this reaction is catalyzed by a homologous complex composed of Dnl4 and Lif1. Intriguingly, no homolog of XRCC4 has been found in S. pombe, raising the possibility that such a factor may not always be required for classical NHEJ. Here, through screening the ionizing radiation (IR) sensitivity phenotype of a genome-wide fission yeast deletion collection in both the vegetative growth state and the spore state, we identify Xrc4, a highly divergent homolog of human XRCC4. Like other fission yeast NHEJ factors, Xrc4 is critically important for IR resistance of spores, in which no homologous recombination templates are available. Using both extrachromosomal and chromosomal DSB repair assays, we show that Xrc4 is essential for classical NHEJ. Exogenously expressed Xrc4 colocalizes with the LigIV homolog Lig4 at the chromatin region of the nucleus in a mutually dependent manner. Furthermore, like their human counterparts, Xrc4 and Lig4 interact with each other and this interaction requires the inter-BRCT linker and the second BRCT domain of Lig4. Our discovery of Xrc4 suggests that an XRCC4 family protein is universally required for classical NHEJ in eukaryotes.

Global analysis of fission yeast mating genes reveals new autophagy factors.

Macroautophagy (autophagy) is crucial for cell survival during starvation and plays important roles in animal development and human diseases. Molecular understanding of autophagy has mainly come from the budding yeast Saccharomyces cerevisiae, and it remains unclear to what extent the mechanisms are the same in other organisms. Here, through screening the mating phenotype of a genome-wide deletion collection of the fission yeast Schizosaccharomyces pombe, we obtained a comprehensive catalog of autophagy genes in this highly tractable organism, including genes encoding three heretofore unidentified core Atg proteins, Atg10, Atg14, and Atg16, and two novel factors, Ctl1 and Fsc1. We systematically examined the subcellular localization of fission yeast autophagy factors for the first time and characterized the phenotypes of their mutants, thereby uncovering both similarities and differences between the two yeasts. Unlike budding yeast, all three Atg18/WIPI proteins in fission yeast are essential for autophagy, and we found that they play different roles, with Atg18a uniquely required for the targeting of the Atg12-Atg5·Atg16 complex. Our investigation of the two novel factors revealed unforeseen autophagy mechanisms. The choline transporter-like protein Ctl1 interacts with Atg9 and is required for autophagosome formation. The fasciclin domain protein Fsc1 localizes to the vacuole membrane and is required for autophagosome-vacuole fusion but not other vacuolar fusion events. Our study sheds new light on the evolutionary diversity of the autophagy machinery and establishes the fission yeast as a useful model for dissecting the mechanisms of autophagy.

Absence of GJB2 gene mutations, the GJB6 deletion (GJB6-D13S1830) and four common mitochondrial mutations in nonsyndromic genetic hearing loss in a South African population.

OBJECTIVE: The purpose of this study was to determine the prevalence of mutations in the GJB2 gene, the GJB6-D13S1830 deletion and the four common mitochondrial mutations (A1555G, A3243G, A7511C and A7445G) in a South African population. METHODS: Using single-strand conformation polymorphism and direct sequencing for screening GJB2 mutation; Multiplex PCR Amplification for GJB6-D13S1830 deletion and Restriction Fragment-Length Polymorphism (PCR-RFLP) analysis for the four common mtDNA mutations. We screened 182 hearing impaired students to determine the frequency of these mutations in the population. RESULTS: None of the reported disease causing mutations in GJB2 nor any novel pathogenic mutations in the coding region were detected, in contrast to the findings among Caucasians. The GJB6-D13S1830 deletion and the mitochondrial mutations were not observed in this group. CONCLUSION: These results suggest that GJB2 may not be a significant deafness gene among sub-Saharan Africans, pointing to other unidentified genes as responsible for nonsyndromic hearing loss in these populations.

The first Sino-German Symposium on DNA Repair and Human Diseases.

The first Sino-German Symposium on DNA Repair and Human Diseases was held in the Capital Normal University, Beijing, China, from October 9th to 11th, 2010. It was initiated and organized by Xingzhi Xu and Zhao-Qi Wang with strong support from top scientists in the field from China, Germany and the United States. Financially, it was fully supported by the Sino-German Center for Science Promotion jointly founded by the National Natural Science Foundation of China (NSFC) and the Deutsche Forschungsgemeinschaft (DFG). This report summarizes 35 plenary lectures presented during this three-day symposium, with topics ranging from DNA damage checkpoint signaling, DNA repair, posttranslational protein modifications in DNA damage response (DDR) to DDR in ageing and cancer. This symposium stimulated extensive discussions on science and potential collaboration among the 230 participants.

Antioxidant enzymes, presbycusis, and ethnic variability.

OBJECTIVE: A proposed mechanism for presbycusis is a significant increase in oxidative stress in the cochlea. The enzymes glutathione S-transferase (GST) and N-acetyltransferase (NAT) are two classes of antioxidant enzymes active in the cochlea. In this work, we sought to investigate the association of different polymorphisms of GSTM1, GSTT1, and NAT2 and presbycusis and analyze whether ethnicity has an effect in the genotype-phenotype associations. STUDY DESIGN: Case-control study of 134 DNA samples. SETTING: University-based tertiary care center. SUBJECTS AND METHODS: Clinical, audiometric, and DNA testing of 55 adults with presbycusis and 79 control patients with normal hearing. RESULTS: The GSTM1 null genotype was present in 77 percent of white Hispanics and 51 percent of white non-Hispanics (Fisher's exact test, 2-tail, P = 0.0262). The GSTT1 null genotype was present in 34 percent of control patients and in 60 percent of white presbycusis subjects (P = 0.0067, odds ratio [OR] = 2.843, 95% confidence interval [95% CI] = 1.379-5.860). The GSTM1 null genotype was more frequent in presbycusis subjects, i.e., 48 percent of control patients and 69 percent of white subjects carried this deletion (P = 0.0198, OR = 2.43, 95% CI = 1.163-5.067). The NAT2*6A mutant genotype was more frequent among subjects with presbycusis (60%) than in control patients (34%; P = 0.0086, OR = 2.88, 95% CI = 1.355-6.141). CONCLUSION: We showed an increased risk of presbycusis among white subjects carrying the GSTM1 and the GSTT1 null genotype and the NAT*6A mutant allele. Subjects with the GSTT1 null genotypes are almost three times more likely to develop presbycusis than those with the wild type. The GSTM1 null genotype was more prevalent in white Hispanics than in white non-Hispanics, but the GSTT1 and NAT2 polymorphisms were equally represented in the two groups.

Mutation analysis of SLC26A4 in mainland Chinese patients with enlarged vestibular aqueduct.

OBJECTIVE: We have characterized the spectrum of SLC26A4 mutations and clinical features in a population of mainland Chinese patients with nonsyndromic sensorineural hearing loss (SNHL) and enlarged vestibular aqueduct (EVA). STUDY DESIGN: Cross-sectional clinical genetic study. SETTING: Tertiary care outpatient otolaryngology clinic. METHODS: A total of 32 subjects identified with bilateral EVA using high-resolution CT were screened for mutations in SLC26A4 by denaturing high-performance liquid chromatography and direct sequencing methods. RESULTS: A total of 13 different mutations were identified in the SLC26A4 gene, five of which are novel. A total of 88 percent of the patients harbored biallelic mutations, 11 patients were homozygotes, and 17 were compound heterozygotes. Four patients were found to carry a single SLC26A4 mutation. The IVS7-2A>G mutation was the most frequent, accounting for 60 percent of the mutant alleles. We have not found any correlations between the type of SLC26A4 mutations and the type, degree, and progression of hearing loss. There are significant proportions of patients with asymmetric (26%), progressive (32%), or fluctuating hearing loss (21%). CONCLUSION: Our data confirm the high prevalence of SLC26A4 mutations in Chinese patients with SNHL and EVA. We could not establish any relationship between genotype and phenotype. However, the high incidence of asymmetric, progressive, and fluctuating hearing loss found in the current study indicates that patients with those features should be routinely screened for SLC26A4 mutation in addition to diagnosis of EVA using CT or MRI.

Cooperative control of Crb2 by ATM family and Cdc2 kinases is essential for the DNA damage checkpoint in fission yeast.

The cellular responses to double-stranded breaks (DSBs) typically involve the extensive accumulation of checkpoint proteins in chromatin surrounding the damaged DNA. One well-characterized example involves the checkpoint protein Crb2 in the fission yeast Schizosaccharomyces pombe. The accumulation of Crb2 at DSBs requires the C-terminal phosphorylation of histone H2A (known as gamma-H2A) by ATM family kinases in chromatin surrounding the break. It also requires the constitutive methylation of histone H4 on lysine-20 (K20). Interestingly, neither type of histone modification is essential for the Crb2-dependent checkpoint response. However, H4-K20 methylation is essential in a crb2-T215A strain that lacks a cyclin-dependent kinase phosphorylation site in Crb2. Here we explain this genetic interaction by describing a previously overlooked effect of the crb2-T215A mutation. We show that crb2-T215A cells are able to initiate but not sustain a checkpoint response. We also report that gamma-H2A is essential for the DNA damage checkpoint in crb2-T215A cells. Importantly, we show that inactivation of Cdc2 in gamma-H2A-defective cells impairs Crb2-dependent signaling to the checkpoint kinase Chk1. These findings demonstrate that full Crb2 activity requires phosphorylation of threonine-215 by Cdc2. This regulation of Crb2 is independent of the histone modifications that are required for the hyperaccumulation of Crb2 at DSBs.

Histone H2A phosphorylation controls Crb2 recruitment at DNA breaks, maintains checkpoint arrest, and influences DNA repair in fission yeast.

Mammalian ATR and ATM checkpoint kinases modulate chromatin structures near DNA breaks by phosphorylating a serine residue in the carboxy-terminal tail SQE motif of histone H2AX. Histone H2A is similarly regulated in Saccharomyces cerevisiae. The phosphorylated forms of H2AX and H2A, known as gamma-H2AX and gamma-H2A, are thought to be important for DNA repair, although their evolutionarily conserved roles are unknown. Here, we investigate gamma-H2A in the fission yeast Schizosaccharomyces pombe. We show that formation of gamma-H2A redundantly requires the ATR/ATM-related kinases Rad3 and Tel1. Mutation of the SQE motif to AQE (H2A-AQE) in the two histone H2A genes caused sensitivity to a wide range of genotoxic agents, increased spontaneous DNA damage, and impaired checkpoint maintenance. The H2A-AQE mutations displayed a striking synergistic interaction with rad22Delta (Rad52 homolog) in ionizing radiation (IR) survival. These phenotypes correlated with defective phosphorylation of the checkpoint proteins Crb2 and Chk1 and a failure to recruit large amounts of Crb2 to damaged DNA. Surprisingly, the H2A-AQE mutations substantially suppressed the IR hypersensitivity of crb2Delta cells by a mechanism that required the RecQ-like DNA helicase Rqh1. We propose that gamma-H2A modulates checkpoint and DNA repair through large-scale recruitment of Crb2 to damaged DNA. This function correlates with evidence that gamma-H2AX regulates recruitment of several BRCA1 carboxyl terminus domain-containing proteins (NBS1, 53BP1, MDC1/NFBD1, and BRCA1) in mammals.

Homo-oligomerization is the essential function of the tandem BRCT domains in the checkpoint protein Crb2.

BRCT (BRCA1 C terminus) domains are frequently found as a tandem repeat in proteins involved in DNA damage responses, such as Saccharomyces cerevisiae Rad9, human 53BP1 and BRCA1. Tandem BRCT domains mediate protein-protein and protein-DNA interactions. However, the functional significance of these interactions is largely unknown. Here we report the oligomerization of Schizosaccharomyces pombe checkpoint protein Crb2 through its tandem BRCT domains. Truncated Crb2 without BRCT domains is defective in DNA damage checkpoint signaling. However, addition of either of two heterologous dimerization motifs largely restores the functions of truncated Crb2 without BRCT domains. Replacement of Crb2 BRCT domains with a dimerization motif also renders cells resistant to the dominant negative effect of overexpressing Crb2 BRCT domains. These results demonstrate that the crucial function of the tandem BRCT domains is to oligomerize Crb2.

Retention but not recruitment of Crb2 at double-strand breaks requires Rad1 and Rad3 complexes.

The fission yeast checkpoint protein Crb2, related to budding yeast Rad9 and human 53BP1 and BRCA1, has been suggested to act as an adapter protein facilitating the phosphorylation of specific substrates by Rad3-Rad26 kinase. To further understand its role in checkpoint signaling, we examined its localization in live cells by using fluorescence microscopy. In response to DNA damage, Crb2 localizes to distinct nuclear foci, which represent sites of DNA double-strand breaks (DSBs). Crb2 colocalizes with Rad22 at persistent foci, suggesting that Crb2 is retained at sites of DNA damage during repair. Damage-induced Crb2 foci still form in cells defective in Rad1, Rad3, and Rad17 complexes, but these foci do not persist as long as in wild-type cells. Our results suggest that Crb2 functions at the sites of DNA damage, and its regulated persistent localization at damage sites may be involved in facilitating DNA repair and/or maintaining the checkpoint arrest while DNA repair is under way.