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The article considers the theoretical and legal aspects of cyberbullying. The relevance of the research topic is connected with the lack of proper scientific study of this problem and the need to consider cyberbullying as an unlawfully socially dangerous act. Based on the study, the author's definition of cyberbullying is proposed. The results of a population survey on bullying on the Internet are presented. The main forms of cyberbullying are considered. One form of cyberbullying is doxing, which is the public disclosure of personal and confidential data via the Internet. The analysis of the motivation of cyber aggressors was carried out and two types of victims of cyberbullying were identified. The main short-term and long-term psychological and physical consequences of victims of cyberbullying are described. The differences of cyberbullying from the traditional forms of bullying are examined, the emphasis is placed on the presence of witnesses, who are divided into groups in the article, based on the roles they play in the bullying process. The current Russian legislation regarding liability for cyberbullying is analyzed. It is noted that in Russia this action is not considered an independent corpus delicti. Basic preventive measures are proposed that can eliminate or minimize the likelihood of becoming a victim of cyberbullying. Emphasis is placed on the need to complicate the registration procedure on social networks, on forums and other Internet resources by linking the profile to a mobile phone number, public services website account, and also specifying the real username or an officially registered nickname, which will eliminate online anonymity and malicious use of assumed names. The international experience of countering cyberbullying and the possibility of using it to improve domestic legislation in this area are considered.

Ongoing climate change can shift organism phenology in ways that vary depending on species, habitats and climate factors studied. To probe for large-scale patterns in associated phenological change, we use 70,709 observations from six decades of systematic monitoring across the former Union of Soviet Socialist Republics. Among 110 phenological events related to plants, birds, insects, amphibians and fungi, we find a mosaic of change, defying simple predictions of earlier springs, later autumns and stronger changes at higher latitudes and elevations. Site mean temperature emerged as a strong predictor of local phenology, but the magnitude and direction of change varied with trophic level and the relative timing of an event. Beyond temperature-associated variation, we uncover high variation among both sites and years, with some sites being characterized by disproportionately long seasons and others by short ones. Our findings emphasize concerns regarding ecosystem integrity and highlight the difficulty of predicting climate change outcomes., The work was financially supported by the Academy of Finland, grants 250243 (O.O.), 284601 (O.O.), 309581 (O.O.); the European Research Council, ERC Starting Grant 205905 (O.O.) and Synergy Grant 856506 – LIFEPLAN (to O.O. and T.R.); Nordic Environment Finance Corporation Grant (O.O.); Jane and Aatos Erkko Foundation Grant (O.O., T.R., M.H., L.A.); University of Helsinki HiLIFE Fellow Grant 2017–2020 (O.O.); and the Research Council of Norway through its Centres of Excellence Funding Scheme (223257) to O.O. via Centre for Biodiversity Dynamics; the Kone Foundation 44-6977 (M.D.) and 55-14839 (G.T.); a Spanish Ramon y Cajal grant RYC-2014-16263 (M.D.); the Federal Budget for the Forest Research Institute of Karelian Research Centre Russian Academy of Sciences 220-2017-0003, 0220-2017-0005 (L.V., S.S. and J.K.); the Russian Foundation for Basic Research Grant 16-08-00510 (L.K.), and the Ministry of Education and Science of the Russian Federation 0017-2019-0009 (Keldysh Institute of Applied Mathematics, Russian Academy of Sciences) (N.I., M. Shashkov).

European Research Council (ERC) Starting Grant 205905 (to O.O.) and Synergy Grant 856506—LIFEPLAN (to O.O. and T.R.); Spanish Ramón y Cajal Grant RYC-2014-16263 (to M.M.D.) (...), Delgado, M.D.M., Roslin, T., Tikhonov, G., Meyke, E., Lo, C., Gurarie, E., Abadonova, M., Abduraimov, O., Adrianova, O., Akimova, T., Akkiev, M., Ananin, A., Andreeva, E., Andriychuk, N., Antipin, M., Arzamascev, K., Babina, S., Babushkin, M., Bakin, O., Barabancova, A., Basilskaja, I., Belova, N., Belyaeva, N., Bespalova, T., Bisikalova, E., Bobretsovz, A., Bobrov, V., Bobrovskyi, V., Bochkareva, E., Bogdanov, G., Bolshakov, V., Bondarchuk, S., Bukharova, E., Butunina, A., Buyvolov, Y., Buyvolova, A., Bykov, Y., Chakhireva, E., Chashchina, O., Cherenkova, N., Chistjakov, S., Chuhontseva, S., Davydov, E.A., Demchenko, V., Diadicheva, E., Dobrolyubov, A., Dostoyevskaya, L., Drovnina, S., Drozdova, Z., Dubanaev, A., Dubrovsky, Y., Elsukov, S., Epova, L., Ermakova, O.S., Ermakova, O., Esengeldenova, A., Evstigneev, O., Fedchenko, I., Fedotova, V., Filatova, T., Gashev, S., Gavrilov, A., Gaydys, I., Golovcov, D., Goncharova, N., Gorbunova, E., Gordeeva, T., Grishchenko, V., Gromyko, L., Hohryakov, V., Hritankov, A., Ignatenko, E., Igosheva, S., Ivanova, U., Ivanova, N., Kalinkin, Y., Kaygorodova, E., Kazansky, F., Kiseleva, D., Knorrem, A., Kolpashikov, L., Korobov, E., Korolyova, H., Korotkikh, N., Kosenkov, G., Kossenko, S., Kotlugalyamova, E., Kozlovsky, E., Kozsheechkin, V., Kozurak, A., Kozyr, I., Krasnopevtseva, A., Krugliko, S., Kuberskaya, O., Kudryavtsev, A., Kulebyakina, E., Kulsha, Y., Kupriyanova, M., Kurbanbagamaev, M., Kutenkov, A., Kutenkova, N., Kuyantseva, N., Kuznetsov, A., Larin, E., Lebedev, P., Litvinov, K., Luzhkova, N., Mahmudov, A., Makovkina, L., Mamontov, V., Mayorova, S., Megalinskaja, I., Meydus, A., Minin, A., Mitrofanov, O., Motruk, M., Myslenkov, A., Nasonova, N., Nemtseva, N., Nesterova, I., Nezdoliy, T., Niroda, T., Novikova, T., Panicheva, D., Pavlov, A., Pavlova, K., Van, P., Podolski, S., Polikarpova, N., Tatiana Polyanskaya81, Pospelov, I., Pospelova, E., Prokhorov, I., Prokosheva, I., Puchnina, L., Putrashyk, I., Raiskaya, J., Rozhkov, Y., Rozhkova, O., Rudenko, M., Rybnikova, I., Rykova, S., Sahnevich, M., Samoylov, A., Sank, V., Sapelnikova, I., Sazonov, S., Selyunina, Z., Shalaeva, K., Shashkov, M., Shcherbakov, A., Shevchyk, V., Shubin, S., Shujskaja, E., Sibgatullin, R., Sikkilah, N., Sitnikova, E., Sivkov, A., Skok, N., Skorokhodova, S., Smirnova, E., Sokolova, G., Sopin, V., Spasovski, Y., Stepanov, S., Stratiy, V., Strekalovskaya, V., Sukhov, A., Suleymanova, G., Sultangareeva, L., Teleganova, V., Teplov, V., Teplova, V., Tertitsa, T., Timoshkin, V., Tirski, D., Tolmachev, A., Tomilin, A., Tselishcheva, L., Turgunov, M., Tyukh, Y., Van, V., Ershkova, E., Vasin, A., Vasina, A., Vekliuk, A., Vetchinnikova, L., Vinogradov, V., Volodchenko, N., Voloshina, I., Xoliqov, T., Yablonovska-Grishchenko, E., Yakovlev, V., Yakovleva, M., Yantser, O., Yarema, Y., Zahvatov, A., Zakharov, V., Zelenetskiy, N., Zheltukhin, A., Zubina, T., Kurhinen, J., Ovaskainen, O.

We present an extensive, large-scale, long-term and multitaxon database on phenological and climatic variation, involving 506,186 observation dates acquired in 471 localities in Russian Federation, Ukraine, Uzbekistan, Belarus and Kyrgyzstan. The data cover the period 1890–2018, with 96% of the data being from 1960 onwards. The database is rich in plants, birds and climatic events, but also includes insects, amphibians, reptiles and fungi. The database includes multiple events per species, such as the onset days of leaf unfolding and leaf fall for plants, and the days for first spring and last autumn occurrences for birds. The data were acquired using standardized methods by permanent staff of national parks and nature reserves (87% of the data) and members of a phenological observation network (13% of the data). The database is valuable for exploring how species respond in their phenology to climate change. Large-scale analyses of spatial variation in phenological response can help to better predict the consequences of species and community responses to climate change., Measurement(s)phenological event • climateTechnology Type(s)digital curationFactor Type(s)geographic location • yearSample Characteristic - LocationRussia • Ukraine • Uzbekistan • Belarus • Kyrgyzstan Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.11687169

The work was funded by Academy of Finland, grants 250243, 284601, 309581 (OO); the European Research Council, ERC Starting Grant 205905 (OO); Nordic Environment Finance Corporation Grant (OO); Jane and Aatos Erkko Foundation Grant (OO and TR); University of Helsinki HiLIFE Fellow Grant 2017–2020 (OO); the Kone Foundation 44-6977 (MD); Spanish Ramon y Cajal grant RYC-2014-16263 (MD); the Federal Budget for the Forest Research Institute of Karelian Research Centre Russian Academy of Sciences 220-2017-0003,0220-2017-0005 (LV, SS and JK); the Russian Foundation for Basic Research Grant 16-08-00510 (LK), and the Ministry of Education and Science of the Russian Federation 0017-2019-0009 (Keldysh Institute of Applied Mathematics, Russian Academy of Sciences) (NI, MSh)., Ovaskainen, O., Meyke, E., Lo, C., Tikhonov, G., Delgado, M.M., Roslin, T., Gurarie, E., Abadonova, M., Abduraimov, O., Adrianova, O., Akimova, T., Akkiev, M., Ananin, A., Andreeva, E., Andriychuk, N., Antipin, M., Arzamascev, K., Babina, S., Babushkin, M., Bakin, O., Barabancova, A., Basilskaja, I., Belova, N., Belyaeva, N., Bespalova, T., Bisikalova, E., Bobretsov, A., Bobrov, V., Bobrovskyi, V., Bochkareva, E., Bogdanov, G., Bolshakov, V., Bondarchuk, S., Bukharova, E., Butunina, A., Buyvolov, Y., Buyvolova, A., Bykov, Y., Chakhireva, E., Chashchina, O., Cherenkova, N., Chistjakov, S., Chuhontseva, S., Davydov, E.A., Demchenko, V., Diadicheva, E., Dobrolyubov, A., Dostoyevskaya, L., Drovnina, S., Drozdova, Z., Dubanaev, A., Dubrovsky, Y., Elsukov, S., Epova, L., Ermakova, O.S., Ermakova, O., Esengeldenova, A., Evstigneev, O., Fedchenko, I., Fedotova, V., Filatova, T., Gashev, S., Gavrilov, A., Gaydysh, I., Golovcov, D., Goncharova, N., Gorbunova, E., Gordeeva, T., Grishchenko, V., Gromyko, L., Hohryakov, V., Hritankov, A., Ignatenko, E., Igosheva, S., Ivanova, U., Ivanova, N., Kalinkin, Y., Kaygorodova, E., Kazansky, F., Kiseleva, D., Knorre, A., Kolpashikov, L., Korobov, E., Korolyova, H., Korotkikh, N., Kosenkov, G., Kossenko, S., Kotlugalyamova, E., Kozlovsky, E., Kozsheechkin, V., Kozurak, A., Kozyr, I., Krasnopevtseva, A., Kruglikov, S., Kuberskaya, O., Kudryavtsev, A., Kulebyakina, E., Kulsha, Y., Kupriyanova, M., Kurbanbagamaev, M., Kutenkov, A., Kutenkova, N., Kuyantseva, N., Kuznetsov, A., Larin, E., Lebedev, P., Litvinov, K., Luzhkova, N., Mahmudov, A., Makovkina, L., Mamontov, V., Mayorova, S., Megalinskaja, I., Meydus, A., Minin, A., Mitrofanov, O., Motruk, M., Myslenkov, A., Nasonova, N., Nemtseva, N., Nesterova, I., Nezdoliy, T., Niroda, T., Novikova, T., Panicheva, D., Pavlov, A., Pavlova, K., Petrenko, P., Podolski, S., Polikarpova, N., Polyanskaya, T., Pospelov, I., Pospelova, E., Prokhorov, I., Prokosheva, I., Puchnina, L., Putrashyk, I., Raiskaya, J., Rozhkov, Y., Rozhkova, O., Rudenko, M., Rybnikova, I., Rykova, S., Sahnevich, M., Samoylov, A., Sanko, V., Sapelnikova, I., Sazonov, S., Selyunina, Z., Shalaeva, K., Shashkov, M., Shcherbakov, A., Shevchyk, V., Shubin, S., Shujskaja, E., Sibgatullin, R., Sikkila, N., Sitnikova, E., Sivkov, A., Skok, N., Skorokhodova, S., Smirnova, E., Sokolova, G., Sopin, V., Spasovski, Y., Stepanov, S., Stratiy, V.І., Strekalovskaya, V., Sukhov, A., Suleymanova, G., Sultangareeva, L., Teleganova, V., Teplov, V., Teplova, V., Tertitsa, T., Timoshkin, V., Tirski, D., Tolmachev, A., Tomilin, A., Tselishcheva, L., Turgunov, M., Tyukh, Y., Vladimir, V., Vargot, E., Vasin, A., Vasina, A., Vekliuk, A., Vetchinnikova, L., Vinogradov, V., Volodchenkov, N., Voloshina, I., Xoliqov, T., Yablonovska-Grishchenko, E., Yakovlev, V., Yakovleva, M., Yantser, O., Yarema, Y., Zahvatov, A., Zakharov, V., Zelenetskiy, N., Zheltukhin, A., Zubina, T., Kurhinen, J.

Noncanonical DNA structures such as G-quadruplexes might obstruct the binding of hRPA, compromising the accuracy of replication, and be a source of genomic instability. In this study, circular dichroism (CD) and electrophoretic mobility shift assay (EMSA) experiments were used to show that hRPA can bind and melt nontelomeric, intramolecular DNA G-quadruplexes under physiologically germane conditions. EMSA results show that hRPA binds to a 58-mer that includes an embedded quadruplex with an affinity equal to or greater than to nonquadruplex forming 58-mers. Moreover, hRPA binds to a 26-mer purine-rich quadruplex-forming sequence with an affinity indistinguishable from that for binding to the complementary pyrimidine-rich sequence. Under the same conditions, hRPA does not have significant affinity for binding to the duplex formed from the two sequences. Thus, DNA secondary structures can significantly modulate the binding affinity of hRPA over and above its known preference for pyrimidine-rich single-stranded sequences, so that at least some intramolecular G-quadruplex structures may not inhibit hRPA binding during DNA replication. CD spectral changes in combination with EMSA titrations suggest that one hRPA heterotrimer is sufficient to form a stable complex with an unfolded 26-mer G-quadruplex prior to the binding of a second hRPA molecule.

The large T antigen (T-ag) protein binds to and activates DNA replication from the origin of DNA replication (ori) in simian virus 40 (SV40). Here, we determined the crystal structures of the T-ag origin-binding domain (OBD) in apo form, and bound to either a 17 bp palindrome (sites 1 and 3) or a 23 bp ori DNA palindrome comprising all four GAGGC binding sites for OBD. The T-ag OBDs were shown to interact with the DNA through a loop comprising Ser147-Thr155 (A1 loop), a combination of a DNA-binding helix and loop (His203-Asn210), and Asn227. The A1 loop traveled back-and-forth along the major groove and accounted for most of the sequence-determining contacts with the DNA. Unexpectedly, in both T-ag-DNA structures, the T-ag OBDs bound DNA independently and did not make direct protein-protein contacts. The T-ag OBD was also captured bound to a non-consensus site ATGGC even in the presence of its canonical site GAGGC. Our observations taken together with the known biochemical and structural features of the T-ag-origin interaction suggest a model for origin unwinding.

The recent structure of human replication protein A (RPA) bound to residues 38-58 of tumor suppressor p53 exemplifies several important features of protein-protein interactions involved in transcription and DNA repair. First, the N-terminal transcriptional activation domain (TAD) of p53 is multifunctional and dynamic, showing multiple interactions with partner proteins some of which are modulated by phosphorylation. Second, the binding of partner proteins is coupled with a disorder-to-order transition common to many other transcriptional activation domains. Third, the molecular features of p53 residues 47-58 imitate those of single stranded DNA in their interaction with the oligonucleotide oliogsaccharide-binding (OB) fold of the N-terminal domain of RPA70. This regulated association is implicated in transmitting the DNA damage signal to the p53 pathway of stress response. Here we review the recently reported crystal structure of the p53/RPA70N complex and the mechanism by which ssDNA can provide positive feedback to dissociate p53/RPA complexes. The binding mode and regulatory mechanisms of the p53/RPA70N interaction may represent a general paradigm for regulation of the OB folds involved in DNA repair and metabolism.

One of many protein–protein interactions modulated upon DNA damage is that of the single-stranded DNA-binding protein, replication protein A (RPA), with the p53 tumor suppressor. Here we report the crystal structure of RPA residues 1–120 (RPA70N) bound to the N-terminal transactivation domain of p53 (residues 37–57; p53N) and, by using NMR spectroscopy, characterize two mechanisms by which the RPA/p53 interaction can be modulated. RPA70N forms an oligonucleotide/oligosaccharide-binding fold, similar to that previously observed for the ssDNA-binding domains of RPA. In contrast, the N-terminal p53 transactivation domain is largely disordered in solution, but residues 37–57 fold into two amphipathic helices, H1 and H2, upon binding with RPA70N. The H2 helix of p53 structurally mimics the binding of ssDNA to the oligonucleotide/oligosaccharide-binding fold. NMR experiments confirmed that both ssDNA and an acidic peptide mimicking a phosphorylated form of RPA32N can independently compete the acidic p53N out of the binding site. Taken together, our data suggest a mechanism for DNA damage signaling that can explain a threshold response to DNA damage.

Antisense oligodeoxynucleotides (ODNs) have biological activity in treating various forms of cancer. The antisense effects of two types of 20mer ODNs, phosphorothioate-modified ODNs (S-ODNs) and S-ODNs with 12 2'-O-methyl groups (Me-S-ODNs), targeted to sites 109 and 277 of bcl-2 mRNA, were compared. Both types were at least as effective as G3139 (Genta, Inc.) in reducing the level of Bcl-2 protein in T24 cells following a 4 h transfection at a dose of 0.1 micro M. Circular dichroism spectra showed that both types formed A-form duplexes with the complementary RNA, and the melting temperatures were in the order of Me-S-ODN.RNA > normal DNA.RNA > S-ODN.RNA. In comparison with the S-ODN, the Me-S-ODN had reduced toxic growth inhibitory effects, was less prone to bind the DNA-binding domain A of human replication protein A, and was as resistant to serum nucleases. Neither type of oligomer induced apoptosis, according to a PARP-cleavage assay. Hybrids formed with Me-S-ODN sequences were less sensitive to RNase H degradation than those formed with S-ODN sequences. Despite this latter disadvantage, the addition of 2'-O-methyl groups to a phosphorothioate-modified ODN is advantageous because of increased stability of binding and reduced non-specific effects.

Although structures of single-stranded (ss)DNA-binding proteins (SSBs) have been reported with and without ssDNA, the mechanism of ssDNA binding in eukarya remains speculative. Here we report a 2.5 Angstroms structure of the ssDNA-binding domain of human replication protein A (RPA) (eukaryotic SSB), for which we previously reported a structure in complex with ssDNA. A comparison of free and bound forms of RPA revealed that ssDNA binding is associated with a major reorientation between, and significant conformational changes within, the structural modules--OB-folds--which comprise the DNA-binding domain. Two OB-folds, whose tandem orientation was stabilized by the presence of DNA, adopted multiple orientations in its absence. Within the OB-folds, extended loops implicated in DNA binding significantly changed conformation in the absence of DNA. Analysis of intermolecular contacts suggested the possibility that other RPA molecules and/or other proteins could compete with DNA for the same binding site. Using this mechanism, protein-protein interactions can regulate, and/or be regulated by DNA binding. Combined with available biochemical data, this structure also suggested a dynamic model for the DNA-binding mechanism.

Replication protein A (RPA), the eukaryote single-stranded DNA-binding protein (SSB), is a heterotrimer. The largest subunit, RPA70, which harbours the major DNA-binding activity, has two DNA-binding domains that each adopt an OB-fold. The complex of the two smaller subunits, RPA32 and RPA14, has weak DNA-binding activity but the mechanism of DNA binding is unknown. We have determined the crystal structure of the proteolytic core of RPA32 and RPA14, which consists of the central two-thirds of RPA32 and the entire RPA14 subunit. The structure revealed that RPA14 and the central part of RPA32 are structural homologues. Each subunit contains a central OB-fold domain, which also resembles the DNA-binding domains in RPA70; an N-terminal extension that interacts with the central OB-fold domain; and a C-terminal helix that mediate heterodimerization via a helix-helix interaction. The OB-fold of RPA32, but not RPA14, possesses additional similarity to the RPA70 DNA-binding domains, supporting a DNA-binding role for RPA32. The discovery of a third and fourth OB-fold in RPA suggests that the quaternary structure of SSBs, which in Bacteria and Archaea are also tetramers of OB-folds, is conserved in evolution. The structure also suggests a mechanism for RPA trimer formation.

Epstein-Barr nuclear antigen 1 (EBNA1) binds to four recognition sites in the minimal origin of latent DNA replication of Epstein-Barr virus and activates latent-phase replication of the viral genomes. Two of these EBNA1 binding sites become sensitive to permanganate oxidation when bound by the DNA binding and dimerization domains of EBNA1. We have previously solved the co-crystal structure of this EBNA1 fragment bound to a consensus recognition site that is not sensitive to permanganate oxidation (CS). To understand the structural difference that underlies the permanganate sensitivity of EBNA1 binding sites, we have now solved the crystal structure of the EBNA1 DNA-binding and dimerization domains bound to a permanganate-sensitive site (CSA/T). Comparisons of permanganate-sensitive and insensitive EBNA1-DNA complexes have revealed only minor differences in protein and DNA structures. In the EBNA1-CSA/T structure, interstrand H-bonds for three consecutive base-pairs centered over the permanganate-sensitive thymine base are lengthened relative to the corresponding bonds in the EBNA1-CS complex, and three potential intrastrand H-bonds were observed between adjacent bases. We also observed that both the CS and CSA/T sequences are overwound by EBNA1 in the vicinity of the permanganate-sensitive thymine base. Finally, we show that the permanganate-sensitive thymine base in the CSA/T-EBNA1 complex is more accessible to solvent than the corresponding T in the EBNA-CS complex.

The Epstein–Barr virus nuclear antigen 1 (EBNA1) protein binds to and activates DNA replication from oriP , the latent origin of DNA replication in Epstein–Barr virus. The crystal structure of the DNA-binding domain of EBNA1 bound to an 18 bp binding site was solved at 2.4 A resolution. EBNA1 comprises two domains, a flanking and a core domain. The flanking domain, which includes a helix that projects into the major groove and an extended chain that travels along the minor groove, makes all of the sequence-determining contacts with the DNA. The core domain, which is structurally homologous to the complete DNA-binding domain of the bovine papilloma virus E2 protein, makes no direct contacts with the DNA bases. A model for origin unwinding is proposed that incorporates the known biochemical and structural features of the EBNA1–origin interaction.

The paper presents the results of experimental investigations of convective heat and mass transfer by sublimation of a single particle of metallorganic compounds mixture in an argon flow. The gas temperature is 180-290 ºС, the flow velocity is up to 2 m/s. The influence of the Zr(dpm)4 and Y(dpm)3 proportions on the characteristics of the transport processes was considered. An increase in the fraction of the more fusible component reduces the temperature of the particle during the sublimation process.

DNA replication is initiated upon binding of “initiators” to origins of replication. In simian virus 40 (SV40), the core origin contains four pentanucleotide binding sites organized as pairs of inverted repeats. Here we describe the crystal structures of the origin binding domain (obd) of the SV40 large T-antigen (T-ag) both with and without a subfragment of origin-containing DNA. In the co-structure, two T-ag obds are oriented in a head-to-head fashion on the same face of the DNA, and each T-ag obd engages the major groove. Although the obds are very close to each other when bound to this DNA target, they do not contact one another. These data provide a high-resolution structural model that explains site-specific binding to the origin and suggests how these interactions help direct the oligomerization events that culminate in assembly of the helicase-active dodecameric complex of T-ag., Author Summary How DNA replicates is a critical question for understanding life. DNA replication remains difficult to investigate in eukaryotes, where it involves a complex, multi-protein apparatus which initiates replication at multiple poorly-defined DNA sequences. This process is far easier to study in viral systems, where the DNA sequences at the origin of replication are well-defined and only one or two proteins are required to initiate replication. In simian virus 40 (SV40), the large T-antigen protein (T-ag) is responsible for recognizing DNA sequences required to start replication, called the origin of replication. SV40 T-ag can also cause DNA to melt or unwind. We report here the crystal structure of the DNA-binding domain of SV40 T-ag on a DNA fragment derived from the viral origin of replication. The structure shows that although T-ag and its functionally analogous protein, papilloma virus E1, share no detectable sequence homology in this region, the two domains bind the DNA in similar ways. In both cases, DNA binding is thought to initiate assembly of a complex of the full-length proteins on DNA. Interestingly, SV40 T-ag DNA-binding domains do not interact with one another when bound to DNA. In addition to describing the molecular details of the DNA–protein interactions and the alterations in protein structure induced by DNA binding, we present a model describing the subsequent assembly events., Crystal structures of the SV40 large T antigen (T-ag) origin binding domain (obd) with and without DNA show how two T-ag obds orient head-to-head and engage with the major groove.

The circular dichroism (CD) spectra of single-stranded DNAs (ssDNAs) are significantly perturbed by the binding of single-stranded DNA binding proteins such as the Ff bacteriophage gene 5 protein (g5p) and the A domain of the 70 kDa subunit of human replication protein A (RPA70-A). These two proteins have similar OB-fold secondary structures, although their CD spectra at wavelengths below 250 nm differ greatly. The spectrum of g5p is dominated by a tyrosyl L(a) band at 229 nm, while that of RPA70-A is dominated by its beta secondary structure. Despite differences in their inherent spectral properties, these two proteins similarly perturb the spectra of bound nucleic acid oligomers. CD spectra of free, non-protein-bound ssDNAs are dependent on interactions of the nearest-neighboring nucleotides in the sequence. The CD spectra (per mol of nucleotide) of simple repetitive sequences 48 nucleotides in length and containing simple combinations of A and C are related by nearest-neighbor equations. For example, 3 x Deltaepsilon[d(AAC)(16)] = 3 x Deltaepsilon[d(ACC)(16)] + Deltaepsilon[d(A)(48)] - Deltaepsilon[d(C)(48)]. Moreover, nearest-neighbor equations relate the spectra of ssDNAs when they are bound by g5p, indicating that each type of perturbed nearest neighbor has a similar average structure within the binding site of the protein.

The crystal structure of the complex between neuraminidase (NA) of influenza virus A/Memphis/31/98 (H3N2) and Fab of monoclonal antibody Mem5 has been determined at 2.1 A resolution and shows a novel pattern of interactions compared to other NA-Fab structures. The interface buries a large area of 2400 A2 and the surfaces have high complementarity. However, the interface is also highly hydrated. There are 33 water molecules in the interface ≥95% buried from bulk solvent, but only 13 of these are isolated from other water molecules. The rest are involved in an intricate network of water-mediated hydrogen bonds throughout the interface, stabilizing the complex. Glu199 on NA, the most critical side-chain to the interaction as previously determined by escape mutant analysis and site-directed mutation, is located in a non-aqueous island. Glu199 and three other residues that contribute the major part of the antigen buried surface of the complex have mutated in human influenza viruses isolated after 1998, confirming that Mem5 identifies an epidemiologically important antigenic site. We conclude that antibody selection of NA variants is a significant component of recent antigenic drift in human H3N2 influenza viruses, supporting the idea that influenza vaccines should contain NA in addition to hemagglutinin.

Recent years have witnessed tremendous progress in our structural and biophysical understanding of how replication protein A (RPA), a major nuclear ssDNA-binding protein (SSB), binds DNA. The four ssDNA-binding domains of RPA have the characteristic OB (oligonucleotide/oligosaccharide-binding) fold and contact DNA with specific polarity via a hierarchy-driven dynamic pathway. A growing mass of data suggest that many aspects of the ssDNA binding mechanism are conserved among SSBs of different origin. However, this conservation is not restricted to the SSB class. The concepts of ssDNA binding by the OB-fold, first derived from the RPA structure, have been successfully applied to the functional characterization of the BRCA2 (breast cancer susceptibility gene 2) protein. The BRCA2 structure, in its turn, has helped to better understand RPA function.

The human nuclear single-stranded (ss) DNA- binding protein, replication protein A (RPA), is a heterotrimer consisting of three subunits: p70, p32 and p14. The protein–DNA interaction is mediated by several DNA-binding domains (DBDs): two major (A and B, also known as p70A and p70B) and several minor (C and D, also known as p70C and p32D, and, presumably, by p70N). Here, using crosslinking experiments, we investigated an interaction of RPA deletion mutants containing a subset of the DBDs with partial DNA duplexes containing 5′-protruding ssDNA tails of 10, 20 and 30 nt. The crosslinks were generated using either a ‘zero-length’ photoreactive group (4-thio-2′-deoxyuridine-5′-monophosphate) embedded in the 3′ end of the DNA primer, or a group connected to the 3′ end by a lengthy linker (5-{N-[N-(4-azido-2,5-difluoro-3- chloropyridine-6-yl)-3-aminopropionyl]-trans-3-aminopropenyl-1}-2′-deoxyuridine-5′-monophosphate). In the absence of two major DBDs, p70A and p70B, the RPA trimerization core (p70C·p32D·p14) was capable of correctly recognizing the primer– template junction and adopting an orientation similar to that in native RPA. Both p70C and p32D contributed to this recognition. However, the domain contribution differed depending on the size of the ssDNA. In contrast with the trimerization core, the RPA dimerization core (p32D·p14) was incapable of detectably recognizing the DNA- junction structures, suggesting an orchestrating role for p70C in this process.

The initial high affinity binding of single-stranded DNA (ssDNA) by replication protein A (RPA) is involved in the tandem domains in the central region of the RPA70 subunit (RPA70AB). However, it was not clear whether the two domains, RPA70A and RPA70B, bind DNA simultaneously or sequentially. Here, using primarily heteronuclear NMR complemented by fluorescence spectroscopy, we have analyzed the binding characteristics of the individual RPA70A and RPA70B domains and compared them with the intact RPA70AB. NMR chemical shift comparisons confirmed that RPA70A and RPA70B tumble independently in solution in the absence of ssDNA. NMR chemical shift perturbations showed that all ssDNA oligomers bind to the same sites as observed in the x-ray crystal structure of RPA70AB complexed to d(C)8. Titrations using a variety of 5'-mer ssDNA oligomers showed that RPA70A has a 5-10-fold higher affinity for ssDNA than RPA70B. Detailed analysis of ssDNA binding to RPA70A revealed that all DNA sequences interact in a similar mode. Fluorescence binding measurements with a variety of 8-10'-mer DNA sequences showed that RPA70AB interacts with DNA with approximately 100-fold higher affinity than the isolated domains. Calculation of the theoretical "linkage effect" from the structure of RPA70AB suggests that the high overall affinity for ssDNA is a byproduct of the covalent attachment of the two domains via a short flexible tether, which increases the effective local concentration. Taken together, our data are consistent with a sequential model of DNA binding by RPA according to which RPA70A binds the majority of DNA first and subsequent loading of RPA70B domain is facilitated by the linkage effect.

The human single-stranded DNA-binding protein, replication protein A (RPA) binds DNA in at least two different modes: initial [8-10 nucleotides (nt)] and stable ( approximately 30 nt). Switching from 8 to 30 nt mode is associated with a large conformational change. Here we report the 2.8 A structure of the RPA trimerization core comprising the C-terminal DNA-binding domain of subunit RPA70 (DBD-C), the central DNA-binding domain of subunit RPA32 (DBD-D) and the entire RPA14 subunit. All three domains are built around a central oligonucleotide/oligosaccharide binding (OB)-fold and flanked by a helix at the C-terminus. Trimerization is mediated by three C-terminal helices arranged in parallel. The OB-fold of DBD-C possesses unique structural features; embedded zinc ribbon and helix-turn-helix motifs. Using time-resolved proteolysis with trypsin, we demonstrate that the trimerization core does not contribute to the binding with substrates of 10 nt, but interacts with oligonucleotides of 24 nt. Taken together, our data indicate that switching from 8-10 to 30 nt mode is mediated by DNA binding with the trimerization core.

Heterotrimeric human single-stranded DNA (ssDNA)-binding protein, replication protein A (RPA), is a central player in DNA replication, recombination, and repair. The C terminus of the largest subunit, RPA70, contains a putative zinc-binding motif and is implicated in complex formation with two smaller subunits, RPA14 and RPA32. The C-terminal domain of RPA70 (RPA70-CTD) was characterized using proteolysis and x-ray fluorescence emission spectroscopy. The proteolytic core of this domain comprised amino acids 432-616. X-ray fluorescence spectra revealed that RPA70-CTD possesses a coordinated Zn(II). The trimeric complex of RPA70-CTD, the ssDNA-binding domain of RPA32 (amino acids 43-171), and RPA14 had strong DNA binding activity. When properly coordinated with zinc, the trimer's affinity to ssDNA was only 3-10-fold less than that of the ssDNA-binding domain in the middle of RPA70. However, the DNA-binding activity of the trimer was dramatically reduced in the presence of chelating agents. Our data indicate that (i) Zn(II) is essential to stabilize the tertiary structure of RPA70-CTD; (ii) RPA70-CTD possesses DNA-binding activity, which is modulated by Zn(II); and (iii) ssDNA binding by the trimer is a synergistic effect generated by the RPA70-CTD and RPA32.

Replication proteinA (RPA) is a conserved nuclear single-stranded DNA (ssDNA)-binding protein. Human RPA (hRPA) comprises three subunits of approximately 70, 32, and 14 kDa (hRPA70, hRPA32 and hRPA14). RPA is known to bind ssDNA through two ssDNA-binding domains in the RPA70 subunit. Here, we demonstrate that the complex of hRPA32 and hRPA14 has an ssDNA-binding domain. Limited proteolysis of the hRPA14·32 complex defined a core dimer composed of the central region of hRPA32 (amino acids 43–171) and RPA14. The core dimer bound ssDNA with an affinity of approximately 10–50 μm, which is at least 100-fold more avid than the DNA-binding affinity of the intact dimer. Analysis of the predicted secondary structure of hRPA32 suggests that amino acids 63–150 of hRPA32 form an ssDNA-binding domain similar in structure to each of those in hRPA70. The complex of hRPA14 and hRPA32-(43–171) in turn formed a trimeric complex with the C-terminal region of hRPA70 (amino acids 436–616). The ssDNA-binding affinity of this trimeric complex was 3 to 5-fold higher than hRPA14·32-(43–171) alone, suggesting a role for the C terminus of hRPA70 in ssDNA binding.

Replication protein A (RPA), the nuclear ssDNA-binding protein in eukaryotes, is essential to DNA replication, recombination, and repair. We have shown that a globular domain at the C terminus of subunit RPA32 contains a specific surface that interacts in a similar manner with the DNA repair enzyme UNG2 and repair factors XPA and RAD52, each of which functions in a different repair pathway. NMR structures of the RPA32 domain, free and in complex with the minimal interaction domain of UNG2, were determined, defining a common structural basis for linking RPA to the nucleotide excision, base excision, and recombinational pathways of repairing damaged DNA. Our findings support a hand-off model for the assembly and coordination of different components of the DNA repair machinery.

It has been found that iodine oxidation of the complex of ribosomes with EF-G † results in the formation of a disulphide bond between the single exposed sulphydryl group of the factor and each of the ribosomal subunits. It is concluded that this EF-G region is localized on the ribosomal interface. Protein S12 is identified as the ribosomal component crosslinked with EF-G in the oxidation reaction. The possible role of the specific interaction of EF-G with protein S12 for the functioning of the factor as an activator of translocation is discussed.

The [H3]-labelled photo-activated analog of streptomycin (photo-Sm) is obtained as a result of the streptomycin reaction with 2-nitro, 4-azidobenzoylhydrazide and subsequent reduction with NaBH4 3. The analog retains the functional activity of the initial antibiotic as judged by two criteria: (1) it binds only to the 30S subparticle of ribosomes and (2) it inhibits the factor-free (“non-enzymatic”) PCMB-stimulated polyU-dependent system of translation (Gavrilova and Spirin, 1971). After irradiation of the reaction mixture containing photo-Sm and either the 30S or 50S subparticles of ribosomes under similar conditions, the analog covalently binds chiefly to the 30S subparticle. Irradiation of the photo-Sm mixture with whole 70S ribosomes leads to a uniform distribution of a covalently bound label among the subparticles. A comparison of the effects obtained allows the conclusion that the analog is located on the interface of the ribosomal subparticles. In the 30S subparticle the photo-Sm attacks mainly the protein component (more than 95% of all the covalently bound label). The proteins labelled by photo-reaction are identified as S7 (main), S14 (additional) and S16/S17 (minor).