Human SIRT2 and SIRT3 deacetylases function in DNA homologous recombinational repair.

SIRT2 and SIRT3 protein deacetylases maintain genome integrity and stability. However, their mechanisms for maintaining the genome remain unclear. To examine the roles of SIRT2 and SIRT3 in DSB repair, I-SceI-based GFP reporter assays for HR, single-strand annealing (SSA) and nonhomologous end joining (NHEJ) repair were performed under SIRT2- or SIRT3-depleted conditions. SIRT2 or SIRT3 depletion inhibited HR repair equally to RAD52 depletion, but did not affect SSA and NHEJ repairs. SIRT2 or SIRT3 depletion disturbed the recruitment of RAD51 to DSB sites, an essential step for RAD51-dependent HR repair, but not directly through RAD52 deacetylation. SIRT2 or SIRT3 depletion decreased the colocalization of γH2AX foci with RPA1, and thus, they might be involved in initiating DSB end resection for the recruitment of RAD51 to DSB sites at an early step in HR repair. These results show the novel underlying mechanism of the SIRT2 and SIRT3 functions in HR for genome stability.

A novel mode of DnaA-DnaA interaction promotes ADP dissociation for reactivation of replication initiation activity.

ATP-DnaA is temporally increased to initiate replication during the cell cycle. Two chromosomal loci, DARS (DnaA-reactivating sequences) 1 and 2, promote ATP-DnaA production by nucleotide exchange of ADP-DnaA for timely initiation. ADP-DnaA complexes are constructed on DARS1 and DARS2, bearing a cluster of three DnaA-binding sequences (DnaA boxes I-III), promoting ADP dissociation. Although DnaA has an AAA+ domain, which ordinarily directs construction of oligomers in a head-to-tail manner, DnaA boxes I and II are oriented oppositely. In this study, we constructed a structural model of a head-to-head dimer of DnaA AAA+ domains, and analyzed residues residing on the interface of the model dimer. Gln208 was specifically required for DARS-dependent ADP dissociation in vitro, and in vivo analysis yielded consistent results. Additionally, ADP release from DnaA protomers bound to DnaA boxes I and II was dependent on Gln208 of the DnaA protomers, and DnaA box III-bound DnaA did not release ADP nor require Gln208 for ADP dissociation by DARS-DnaA complexes. Based on these and other findings, we propose a model for DARS-DnaA complex dynamics during ADP dissociation, and provide novel insight into the regulatory mechanisms of DnaA and the interaction modes of AAA+ domains.

Novel function of HATs and HDACs in homologous recombination through acetylation of human RAD52 at double-strand break sites.

The p300 and CBP histone acetyltransferases are recruited to DNA double-strand break (DSB) sites where they induce histone acetylation, thereby influencing the chromatin structure and DNA repair process. Whether p300/CBP at DSB sites also acetylate non-histone proteins, and how their acetylation affects DSB repair, remain unknown. Here we show that p300/CBP acetylate RAD52, a human homologous recombination (HR) DNA repair protein, at DSB sites. Using in vitro acetylated RAD52, we identified 13 potential acetylation sites in RAD52 by a mass spectrometry analysis. An immunofluorescence microscopy analysis revealed that RAD52 acetylation at DSBs sites is counteracted by SIRT2- and SIRT3-mediated deacetylation, and that non-acetylated RAD52 initially accumulates at DSB sites, but dissociates prematurely from them. In the absence of RAD52 acetylation, RAD51, which plays a central role in HR, also dissociates prematurely from DSB sites, and hence HR is impaired. Furthermore, inhibition of ataxia telangiectasia mutated (ATM) protein by siRNA or inhibitor treatment demonstrated that the acetylation of RAD52 at DSB sites is dependent on the ATM protein kinase activity, through the formation of RAD52, p300/CBP, SIRT2, and SIRT3 foci at DSB sites. Our findings clarify the importance of RAD52 acetylation in HR and its underlying mechanism.

Crystal structure of the human centromeric nucleosome containing CENP-A.

In eukaryotes, accurate chromosome segregation during mitosis and meiosis is coordinated by kinetochores, which are unique chromosomal sites for microtubule attachment. Centromeres specify the kinetochore formation sites on individual chromosomes, and are epigenetically marked by the assembly of nucleosomes containing the centromere-specific histone H3 variant, CENP-A. Although the underlying mechanism is unclear, centromere inheritance is probably dictated by the architecture of the centromeric nucleosome. Here we report the crystal structure of the human centromeric nucleosome containing CENP-A and its cognate α-satellite DNA derivative (147 base pairs). In the human CENP-A nucleosome, the DNA is wrapped around the histone octamer, consisting of two each of histones H2A, H2B, H4 and CENP-A, in a left-handed orientation. However, unlike the canonical H3 nucleosome, only the central 121 base pairs of the DNA are visible. The thirteen base pairs from both ends of the DNA are invisible in the crystal structure, and the αN helix of CENP-A is shorter than that of H3, which is known to be important for the orientation of the DNA ends in the canonical H3 nucleosome. A structural comparison of the CENP-A and H3 nucleosomes revealed that CENP-A contains two extra amino acid residues (Arg 80 and Gly 81) in the loop 1 region, which is completely exposed to the solvent. Mutations of the CENP-A loop 1 residues reduced CENP-A retention at the centromeres in human cells. Therefore, the CENP-A loop 1 may function in stabilizing the centromeric chromatin containing CENP-A, possibly by providing a binding site for trans-acting factors. The structure provides the first atomic-resolution picture of the centromere-specific nucleosome.

Histone chaperone activity of Fanconi anemia proteins, FANCD2 and FANCI, is required for DNA crosslink repair.

Fanconi anaemia (FA) is a rare hereditary disorder characterized by genomic instability and cancer susceptibility. A key FA protein, FANCD2, is targeted to chromatin with its partner, FANCI, and plays a critical role in DNA crosslink repair. However, the molecular function of chromatin-bound FANCD2-FANCI is still poorly understood. In the present study, we found that FANCD2 possesses nucleosome-assembly activity in vitro. The mobility of histone H3 was reduced in FANCD2-knockdown cells following treatment with an interstrand DNA crosslinker, mitomycin C. Furthermore, cells harbouring FANCD2 mutations that were defective in nucleosome assembly displayed impaired survival upon cisplatin treatment. Although FANCI by itself lacked nucleosome-assembly activity, it significantly stimulated FANCD2-mediated nucleosome assembly. These observations suggest that FANCD2-FANCI may regulate chromatin dynamics during DNA repair.

Crystal structure of the nucleosome containing ultraviolet light-induced cyclobutane pyrimidine dimer.

The cyclobutane pyrimidine dimer (CPD) is induced in genomic DNA by ultraviolet (UV) light. In mammals, this photolesion is primarily induced within nucleosomal DNA, and repaired exclusively by the nucleotide excision repair (NER) pathway. However, the mechanism by which the CPD is accommodated within the nucleosome has remained unknown. We now report the crystal structure of a nucleosome containing CPDs. In the nucleosome, the CPD induces only limited local backbone distortion, and the affected bases are accommodated within the duplex. Interestingly, one of the affected thymine bases is located within 3.0 Å from the undamaged complementary adenine base, suggesting the formation of complementary hydrogen bonds in the nucleosome. We also found that UV-DDB, which binds the CPD at the initial stage of the NER pathway, also efficiently binds to the nucleosomal CPD. These results provide important structural and biochemical information for understanding how the CPD is accommodated and recognized in chromatin.

Functional analyses of the C-terminal half of the Saccharomyces cerevisiae Rad52 protein.

The Saccharomyces cerevisiae Rad52 protein is essential for efficient homologous recombination (HR). An important role of Rad52 in HR is the loading of Rad51 onto replication protein A-coated single-stranded DNA (ssDNA), which is referred to as the recombination mediator activity. In vitro, Rad52 displays additional activities, including self-association, DNA binding and ssDNA annealing. Although Rad52 has been a subject of extensive genetic, biochemical and structural studies, the mechanisms by which these activities are coordinated in the various roles of Rad52 in HR remain largely unknown. In the present study, we found that an isolated C-terminal half of Rad52 disrupted the Rad51 oligomer and formed a heterodimeric complex with Rad51. The Rad52 fragment inhibited the binding of Rad51 to double-stranded DNA, but not to ssDNA. The phenylalanine-349 and tyrosine-409 residues present in the C-terminal half of Rad52 were critical for the interaction with Rad51, the disruption of Rad51 oligomers, the mediator activity of the full-length protein and for DNA repair in vivo in the presence of methyl methanesulfonate. Our studies suggested that phenylalanine-349 and tyrosine-409 are key residues in the C-terminal half of Rad52 and probably play an important role in the mediator activity.

Structural polymorphism in the L1 loop regions of human H2A.Z.1 and H2A.Z.2.

The histone H2A.Z variant is widely conserved among eukaryotes. Two isoforms, H2A.Z.1 and H2A.Z.2, have been identified in vertebrates and may have distinct functions in cell growth and gene expression. However, no structural differences between H2A.Z.1 and H2A.Z.2 have been reported. In the present study, the crystal structures of nucleosomes containing human H2A.Z.1 and H2A.Z.2 were determined. The structures of the L1 loop regions were found to clearly differ between H2A.Z.1 and H2A.Z.2, although their amino-acid sequences in this region are identical. This structural polymorphism may have been induced by a substitution that evolutionally occurred at the position of amino acid 38 and by the flexible nature of the L1 loops of H2A.Z.1 and H2A.Z.2. It was also found that in living cells nucleosomal H2A.Z.1 exchanges more rapidly than H2A.Z.2. A mutational analysis revealed that the amino-acid difference at position 38 is at least partially responsible for the distinctive dynamics of H2A.Z.1 and H2A.Z.2. These findings provide important new information for understanding the differences in the regulation and functions of H2A.Z.1 and H2A.Z.2 in cells.

Purification and characterization of the fission yeast telomere clustering factors, Bqt1 and Bqt2.

During meiosis, chromosomes adopt a bouquet arrangement, which is widely conserved among eukaryotes. This arrangement is assumed to play an important role in the normal progression of meiosis, by mediating the proper pairing of homologous chromosomes. In Schizosaccharomyces pombe, the complex of Bqt1 and Bqt2 plays a key role in telomere clustering and the subsequent bouquet arrangement of chromosomes during early meiotic prophase. Bqt1 and Bqt2 are part of a multi-protein complex that mediates the attachment of the telomere to the nuclear membrane. However, the structural details of the complex are needed to clarify the mechanism of telomere clustering. To enable biophysical studies of Bqt1 and Bqt2, we established a purification procedure for the Schizosaccharomyces japonicus Bqt1-Bqt2 complex, which is closely related to the S. pombe Bqt1-Bqt2 complex. A co-expression vector, in which one of the expressed subunits is fused to a removable SUMO tag, yielded high amounts of the proteins in the soluble fraction. The solubility of the Bqt1-Bqt2 complex after the removal of the SUMO tag was maintained by including CHAPS, a nondenaturing, zwitterionic detergent, in the purification buffers. These procedures enabled us to rapidly purify the stable Bqt1-Bqt2 complex. The co-purified Bqt1 and Bqt2 proteins formed a stable heterodimer, consistent with results from in vivo studies showing the requirement of both proteins for the bouquet arrangement. The expression and purification procedures established here will facilitate further biophysical studies of the Bqt1-Bqt2 complex.

Crystallization and preliminary X-ray diffraction analysis of the secreted protein Athe_0614 from Caldicellulosiruptor bescii.

The Athe_0614 protein is a component of the extracellular proteins secreted by the anaerobic, extremely thermophilic and cellulolytic bacterium Caldicellulosiruptor bescii. The recombinant protein was expressed in Escherichia coli, purified to near-homogeneity and crystallized using polyethylene glycol 2000 monomethyl ether as a precipitant. The crystals belonged to the monoclinic space group P2(1), with unit-cell parameters a = 48.4, b = 42.2, c = 97.8 Å, ß = 96.1°, and diffracted to 2.7 Å resolution using synchrotron radiation.

Structural basis of instability of the nucleosome containing a testis-specific histone variant, human H3T.

A histone H3 variant, H3T, is highly expressed in the testis, suggesting that it may play an important role in the chromatin reorganization required for meiosis and/or spermatogenesis. In the present study, we found that the nucleosome containing human H3T is significantly unstable both in vitro and in vivo, as compared to the conventional nucleosome containing H3.1. The crystal structure of the H3T nucleosome revealed structural differences in the H3T regions on both ends of the central alpha2 helix, as compared to those of H3.1. The H3T-specific residues (Met71 and Val111) are the source of the structural differences observed between H3T and H3.1. A mutational analysis revealed that these residues are responsible for the reduced stability of the H3T-containing nucleosome. These physical and structural properties of the H3T-containing nucleosome may provide the basis of chromatin reorganization during spermatogenesis.

Biochemical characterization of the RNA-binding and RNA-DNA strand exchange activities of the human RAD52 protein.

RAD52 is a single-stranded DNA (ssDNA) binding protein that functions in the repair of DNA double-strand breaks (DSBs) by promoting the annealing of complementary DNA strands. RAD52 may also play an important role in an RNA transcript-dependent type of DSB repair, in which it reportedly binds to RNA and mediates the RNA-DNA strand exchange reaction. However, the mechanistic details of these functions are still unclear. In the present study, we utilized the domain fragments of RAD52 to biochemically characterize the single-stranded RNA (ssRNA) binding and RNA-DNA strand exchange activities of RAD52. We found that the N-terminal half of RAD52 is primarily responsible for both activities. By contrast, significant differences were observed for the roles of the C-terminal half in RNA-DNA and DNA-DNA strand exchange reactions. The C-terminal fragment stimulated the inverse RNA-DNA strand exchange activity displayed by the N-terminal fragment in trans, whereas the trans stimulatory effect by the C-terminal fragment was not observed in the inverse DNA-DNA or forward RNA-DNA strand exchange reactions. These results suggest the specific function of the C-terminal half of RAD52 in RNA-templated DSB repair.

The cryo-EM structure of full-length RAD52 protein contains an undecameric ring.

The human RAD52 protein, which forms an oligomeric ring structure, is involved in DNA double-strand break repair. The N-terminal half of RAD52 is primarily responsible for self-oligomerisation and DNA binding. Crystallographic studies have revealed the detailed structure of the N-terminal half. However, only low-resolution structures have been reported for the full-length protein, and thus the structural role of the C-terminal half in self-oligomerisation has remained elusive. In this study, we determined the solution structure of the human RAD52 protein by cryo-electron microscopy (cryo-EM), at an average resolution of 3.5 Å. The structure revealed an undecameric ring that is nearly identical to the crystal structures of the N-terminal half. The cryo-EM map for the C-terminal half was poorly defined, indicating that the region is intrinsically disordered. The present cryo-EM structure provides important insights into the mechanistic roles played by the N-terminal and C-terminal halves of RAD52 during DNA double-strand break repair.

Structural Basis of Damaged Nucleotide Recognition by Transcribing RNA Polymerase II in the Nucleosome.

In transcription-coupled repair (TCR), transcribing RNA polymerase II (RNAPII) stalls at a DNA lesion and recruits TCR proteins to the damaged site. However, the mechanism by which RNAPII recognizes a DNA lesion in the nucleosome remains enigmatic. In the present study, we inserted an apurinic/apyrimidinic DNA lesion analogue, tetrahydrofuran (THF), in the nucleosomal DNA, where RNAPII stalls at the SHL(-4), SHL(-3.5), and SHL(-3) positions, and determined the structures of these complexes by cryo-electron microscopy. In the RNAPII-nucleosome complex stalled at SHL(-3.5), the nucleosome orientation relative to RNAPII is quite different from those in the SHL(-4) and SHL(-3) complexes, which have nucleosome orientations similar to naturally paused RNAPII-nucleosome complexes. Furthermore, we found that an essential TCR protein, Rad26 (CSB), enhances the RNAPII processivity, and consequently augments the DNA damage recognition efficiency of RNAPII in the nucleosome. The cryo-EM structure of the Rad26-RNAPII-nucleosome complex revealed that Rad26 binds to the stalled RNAPII through a novel interface, which is completely different from those previously reported. These structures may provide important information to understand the mechanism by which RNAPII recognizes the nucleosomal DNA lesion and recruits TCR proteins to the stalled RNAPII on the nucleosome.

Vital roles of the second DNA-binding site of Rad52 protein in yeast homologous recombination.

RecA/Rad51 proteins are essential in homologous DNA recombination and catalyze the ATP-dependent formation of D-loops from a single-stranded DNA and an internal homologous sequence in a double-stranded DNA. RecA and Rad51 require a "recombination mediator" to overcome the interference imposed by the prior binding of single-stranded binding protein/replication protein A to the single-stranded DNA. Rad52 is the prototype of recombination mediators, and the human Rad52 protein has two distinct DNA-binding sites: the first site binds to single-stranded DNA, and the second site binds to either double- or single-stranded DNA. We previously showed that yeast Rad52 extensively stimulates Rad51-catalyzed D-loop formation even in the absence of replication protein A, by forming a 2:1 stoichiometric complex with Rad51. However, the precise roles of Rad52 and Rad51 within the complex are unknown. In the present study, we constructed yeast Rad52 mutants in which the amino acid residues corresponding to the second DNA-binding site of the human Rad52 protein were replaced with either alanine or aspartic acid. We found that the second DNA-binding site is important for the yeast Rad52 function in vivo. Rad51-Rad52 complexes consisting of these Rad52 mutants were defective in promoting the formation of D-loops, and the ability of the complex to associate with double-stranded DNA was specifically impaired. Our studies suggest that Rad52 within the complex associates with double-stranded DNA to assist Rad51-mediated homologous pairing.

RAD51 plays a crucial role in halting cell death program induced by ionizing radiation in bovine oocytes.

Reproductive health of humans and animals exposed to daily irradiants from solar/cosmic particles remains largely understudied. We evaluated the sensitivities of bovine and mouse oocytes to bombardment by krypton-78 (1 Gy) or ultraviolet B (UV-B; 100 microjoules). Mouse oocytes responded to irradiation by undergoing massive activation of caspases, rapid loss of energy without cytochrome-c release, and subsequent necrotic death. In contrast, bovine oocytes became positive for annexin-V, exhibited cytochrome-c release, and displayed mild activation of caspases and downstream DNAses but with the absence of a complete cell death program; therefore, cytoplasmic fragmentation was never observed. However, massive cytoplasmic fragmentation and increased DNA damage were induced experimentally by both inhibiting RAD51 and increasing caspase 3 activity before irradiation. Microinjection of recombinant human RAD51 prior to irradiation markedly decreased both cytoplasmic fragmentation and DNA damage in both bovine and mouse oocytes. RAD51 response to damaged DNA occurred faster in bovine oocytes than in mouse oocytes. Therefore, we conclude that upon exposure to irradiation, bovine oocytes create a physiologically indeterminate state of partial cell death, attributed to rapid induction of DNA repair and low activation of caspases. The persistence of these damaged cells may represent an adaptive mechanism with potential implications for livestock productivity and long-term health risks associated with human activity in space.

Comprehensive structural analysis of mutant nucleosomes containing lysine to glutamine (KQ) substitutions in the H3 and H4 histone-fold domains.

Post-translational modifications (PTMs) of histones play important roles in regulating the structure and function of chromatin in eukaryotes. Although histone PTMs were considered to mainly occur at the N-terminal tails of histones, recent studies have revealed that PTMs also exist in the histone-fold domains, which are commonly shared among the core histones H2A, H2B, H3, and H4. The lysine residue is a major target for histone PTM, and the lysine to glutamine (KQ) substitution is known to mimic the acetylated states of specific histone lysine residues in vivo. Human histones H3 and H4 contain 11 lysine residues in their histone-fold domains (five for H3 and six for H4), and eight of these lysine residues are known to be targets for acetylation. In the present study, we prepared 11 mutant nucleosomes, in which each of the lysine residues of the H3 and H4 histone-fold domains was replaced by glutamine: H3 K56Q, H3 K64Q, H3 K79Q, H3 K115Q, H3 K122Q, H4 K31Q, H4 K44Q, H4 K59Q, H4 K77Q, H4 K79Q, and H4 K91Q. The crystal structures of these mutant nucleosomes were determined at 2.4-3.5 Å resolutions. Some of these amino acid substitutions altered the local protein-DNA interactions and the interactions between amino acid residues within the nucleosome. Interestingly, the C-terminal region of H2A was significantly disordered in the nucleosome containing H4 K44Q. These results provide an important structural basis for understanding how histone modifications and mutations affect chromatin structure and function.

Structural basis for the DNA-binding activity of the bacterial ß-propeller protein YncE.

ß-Propellers are widely utilized in nature as recognition modules. The well conserved ß-propeller fold exhibits a high degree of functional diversity, which is probably accomplished through variations in the surface properties of the proteins. Little is known about the interactions between ß-propeller proteins and nucleic acids. In the present study, it has been found that the bacterial ß-propeller protein YncE binds to DNA. Crystal structures of YncE in the free form and complexed with DNA revealed that the surface region of YncE corresponding to the `canonical' substrate-binding site forms essential contacts with DNA. A single DNA base within a single-stranded DNA region is trapped in the hydrophobic pocket located within the central channel of the ß-propeller protein. These data provide physical evidence for the DNA-binding ability of the previously uncharacterized YncE and also suggest that the `canonical' substrate-binding site may be commonly adapted to facilitate nucleic acid binding in a subset of ß-propeller proteins.

Structures of human nucleosomes containing major histone H3 variants.

The nucleosome is the fundamental repeating unit of chromatin, via which genomic DNA is packaged into the nucleus in eukaryotes. In the nucleosome, two copies of each core histone, H2A, H2B, H3 and H4, form a histone octamer which wraps 146 base pairs of DNA around itself. All of the core histones except for histone H4 have nonallelic isoforms called histone variants. In humans, eight histone H3 variants, H3.1, H3.2, H3.3, H3T, H3.5, H3.X, H3.Y and CENP-A, have been reported to date. Previous studies have suggested that histone H3 variants possess distinct functions in the formation of specific chromosome regions and/or in the regulation of transcription and replication. H3.1, H3.2 and H3.3 are the most abundant H3 variants. Here, crystal structures of human nucleosomes containing either H3.2 or H3.3 have been solved. The structures were essentially the same as that of the H3.1 nucleosome. Since the amino-acid residues specific for H3.2 and H3.3 are located on the accessible surface of the H3/H4 tetramer, they may be potential interaction sites for H3.2- and H3.3-specific chaperones.

Structural and biochemical analyses of the human PAD4 variant encoded by a functional haplotype gene.

PAD4 is a peptidylarginine deiminase that catalyzes citrullination, a type of post-translational modification. In this reaction, arginine residues in proteins are converted to citrulline. PAD4 promotes the deimination of arginine residues in histones and may regulate transcription in the context of the chromatin. Single-nucleotide polymorphisms (SNP) in the gene encoding PAD4 identified it as one of the genes associated with susceptibility to rheumatoid arthritis. The PAD4 SNP involve three amino-acid substitutions: Ser55 to Gly, Ala82 to Val and Ala112 to Gly. Autoantibodies for improperly citrullinated proteins have been found in rheumatoid arthritis patients, suggesting that the PAD4(SNP) mRNA is more stable than the conventional PAD4 mRNA and/or the PAD4(SNP) protein possesses a higher citrullination activity than the PAD4 protein. In order to study the effects of the three amino-acid substitutions found in PAD4(SNP), the crystal structure of PAD4(SNP) was determined and it was found that the amino-acid substitutions in PAD4(SNP) only induced conformational changes within the N-terminal domain, not in the active centre for citrullination located in the C-terminal domain. Biochemical analyses also suggested that the citrullination activity of PAD4(SNP) may not substantially differ from that of conventional PAD4. These structural and biochemical findings suggested that the improper protein citrullination found in rheumatoid arthritis patients is not caused by defects in the citrullination activity of PAD4(SNP) but by other reasons such as enhanced PAD4(SNP) mRNA stability.