RPA repair recognition of DNA containing pyrimidines bearing bulky adducts

Recognition of new DNA nucleotide excision repair (NER) substrate analogs, 48-mer ddsDNA (damaged double-stranded DNA), by human replication protein A (hRPA) has been analyzed using fluorescence spectroscopy and photoaffinity modification. The aim of the present work was to find quantitative characteristics of RPA-ddsDNA interaction and RPA subunits role in this process. The designed DNA structures bear bulky substituted pyrimidine nitrogen bases at the inner positions of duplex forming DNA chains. The photoreactive 4-azido-2,5-difluoro-3- pyridin-6-yl (FAP) and fluorescent antracenyl, pyrenyl (Antr, Pyr) groups were introduced via different linker fragments into exo-4N of deoxycytidine or 5C of deoxyuridine. J-dU-containing DNA was used as a photoactive model of undamaged DNA strands. The reporter group was a fluorescein residue, introduced into the 5'-phosphate end of one duplex-forming DNA strand. RPA-dsDNA association constants and the molar RPA/dsDNA ratio have been calculated based on fluorescence anisotropy measurements under conditions of a 1:1 RPA/dsDNA molar ratio in complexes. The evident preference for RPA binding to ddsDNA over undamaged dsDNA distinctly depends on the adduct type and varies in the following way: undamaged dsDNA < Antr-dC-ddsDNA < mmdsDNA < FAPdU-, Pyr-dU-ddsDNA < FAP-dC-ddsDNA (K(D) = 68 +/- 1; 25 +/- 6; 13 +/- 1; 8 +/- 2, and 3.5 +/- 0.5 nM correspondingly) but weakly depends on the chain integrity. Interestingly the bulkier lesions not in all cases have a greater effect on RPA affinity to ddsDNA. The experiments on photoaffinity modification demonstrated only p70 of compactly arranged RPA directly interacting with dsDNA. The formation of RPA-ddsDNA covalent adducts was drastically reduced when both strands of DNA duplex contained virtually opposite located FAP-dC and Antr-dC. Thus RPA requires undamaged DNA strand presence for the effective interaction with dsDNA bearing bulky damages and demonstrates the early NER factors characteristic features underlying strand discrimination capacity and poor activity of the NER system toward double damaged DNA.     

Stimulation of BK virus DNA replication by NFI family transcription factors

BK polyomavirus (BKV) establishes persistent, low-level, and asymptomatic infections in most humans and causes polyomavirus-associated nephropathy (PVAN) and other pathologies in some individuals. The activation of BKV replication following kidney transplantation, leading to viruria, viremia, and, ultimately, PVAN, is associated with immune suppression as well as inflammation and stress from ischemia-reperfusion injury of the allograft, but the stimuli and molecular mechanisms leading to these pathologies are not well defined. The replication of BKV DNA in cell cultures is regulated by the viral noncoding control region (NCCR) comprising the core origin and flanking sequences, to which BKV T antigen (Tag), cellular proteins, and small regulatory RNAs bind. Six nuclear factor I (NFI) binding sites occur in sequences flanking the late side of the core origin (the enhancer) of the archetype virus, and their mutation, either individually or in toto, reduces BKV DNA replication when placed in competition with templates containing intact BKV NCCRs. NFI family members interacted with the helicase domain of BKV Tag in pulldown assays, suggesting that NFI helps recruit Tag to the viral core origin and may modulate its function. However, Tag may not be the sole target of the replication-modulatory activities of NFI: the NFIC/CTF1 isotype stimulates BKV template replication in vitro at low concentrations of DNA polymerase-α primase (Pol-primase), and the p58 subunit of Pol-primase associates with NFIC/CTF1, suggesting that NFI also recruits Pol-primase to the NCCR. These results suggest that NFI proteins (and the signaling pathways that target them) activate BKV replication and contribute to the consequent pathologies caused by acute infection.     

Host-Specific Replication of BK Virus DNA in Mouse Cell Extracts Is Independently Controlled by DNA Polymerase α-Primase and Inhibitory Activities

The activation of the human polyomavirus BK causes polyomavirus-associated nephropathy in immunocompromised humans. Studies of the virus have been restricted since the virus DNA replication is species specific. Cell-based and cell-free DNA replication systems, including the BK virus (BKV) monopolymerase DNA replication system using purified proteins, reproduce the species specificity (28). Therefore, the major host proteins comprising this assay, DNA polymerase α-primase (Pol-prim) and replication protein A (RPA), were intensively studied here. We demonstrate that Pol-prim plays a major role in the species specificity of BKV DNA replication. Both large subunits p180 and p68 of the enzyme complex have central functions in modulating the host specificity. Recently, an inhibitory activity of BKV DNA replication was described (C. Mahon, B. Liang, I. Tikhanovich, J. R. Abend, M. J. Imperiale, H. P. Nasheuer, and W. R. Folk, J. Virol. 83:5708-5717, 2009), but neither mouse Pol-prim nor mouse RPA diminishes cell-free BKV DNA replication. However, the inhibition of BKV DNA replication in mouse extracts depends on sequences flanking the core origin. In the presence of human Pol-prim, the inhibitory effect of mouse cell factors is abolished with plasmid DNAs containing the murine polyomavirus early promoter region, whereas the late enhancer region and the core origin are supplied from BKV. Thus, BKV replication is regulated by both Pol-prim, as a core origin species-specific factor, and inhibitory activities, as origin-flanking sequence-dependent factor(s).BK virus (BKV) is a human polyomavirus that was first isolated in the 1970s (15). Up to 90% of adults have serologic evidence of exposure to BKV, but in most humans the virus remains latent (25, 26). Almost all disease accompanied by BKV reactivation has been found in immunocompromised patients (22). In recent years, BKV has been associated with nephropathy (polyomavirus-associated nephropathy, or PVAN) in up to 10% of renal transplant patients. Once established, the disease results in allograft loss in 45 to 70% of the patients (18). Importantly, BKV preferentially replicates in human cells and less well in cells of other primates, and the virus is highly tumorigenic in rodents (21, 41, 44). This fact and the lack of sustainable viral replication in rodents or other convenient, experimental animal models have been an enormous setback to the study of PVAN.As with other members of the Polyomaviridae family, BKV virions are nonenveloped icosahedral particles with a diameter of 45 nm that contain a circular double-stranded DNA genome of 5.3 kb (1). In BKV and in other polyomaviruses, three genomic areas have been distinguished: (i) a noncoding control region including the origin of viral DNA replication, (ii) the early genes encoding large and small T antigens (TAgs), and (iii) the late genes which code for the capsid proteins VP-1, VP-2, and VP-3 and the agnoprotein (22).BKV DNA replication is similar to that of all other members of the Polyomaviridae family and requires only one viral protein, the multifunctional large TAg, whereas all other replication factors are supplied by the host (13, 14, 28, 39, 47). As the first step, TAg binds to the core origin, which contains the early palindrome, an AT-rich sequence, and the TAg binding site II, which consists of two pairs of G(G/A)GGC pentanucleotides. In the presence of ATP, TAg forms a double hexamer and partially melts the early palindrome (EP) and untwists the AT-rich sequence of the BKV core origin (5, 6, 14). Then the TAg double hexamers bidirectionally unwind the viral replication origin, which requires ATP hydrolysis. In the following process the two hexamers remain associated with each other, with the separated single-stranded DNA (ssDNA) threading through the hexameric channels (14). The viral core origin is sufficient to constitute a functional replication origin, but the presence of auxiliary domains increases its activity 5- to 100-fold in vivo (16, 30). After the viral TAg unwinds the core origin and its flanking sequences, replication protein A (RPA), the main eukaryotic ssDNA-binding protein, covers the resulting stretches of ssDNA, whereas topoisomerase I releases the resulting torsional stress and enhances initiation of DNA replication (5, 7, 43). Then, DNA polymerase α-primase (Pol-prim) is loaded onto this TAg-RPA-topoisomerase 1-DNA complex, yielding a functional initiation complex. In the following step, Pol-prim synthesizes short RNA primers at the origin, and these RNA primers are elongated by the DNA polymerase function of the enzyme complex (9, 35, 47). After a polymerase switch from Pol-prim to DNA polymerase δ (Pol δ) with the help of RPA, replication factor C (RFC), and proliferating cell nuclear antigen (PCNA), processive DNA synthesis is completed by Pol δ in association with PCNA, the sliding clamp, on the leading strand (38, 51, 54, 59). Lagging-strand synthesis is discontinuous, and multiple initiation events catalyzed by Pol-prim must take place. Again, after the elongation of the RNA primers by Pol-prim, DNA synthesis is switched to Pol δ, which then synthesizes the complete Okazaki fragments. The maturation of these Okazaki fragments requires the collaboration of RNase H, PCNA, flap endonuclease 1 (Fen-1), Pol δ, and DNA ligase I to establish a continuous strand also on the lagging strand (9, 19, 20, 51, 55).TAg functions in infected cells rely heavily on specific associations with host proteins; for example, TAg interacts with RPA, Pol-prim, and topoisomerase I to replicate viral DNA. Selective interactions with the host p180 and p48 subunits of Pol-prim were shown to be responsible for species-specific replication of simian virus 40 (SV40) and murine polyomavirus (mPyV) DNAs, respectively (8, 47, 50). The subunits of Pol-prim are highly conserved since 88, 80, 89, and 90% of the amino acids are identical between human and murine p180, p68, p58, and p48, respectively. Biochemical studies have shown that TAg interacts independently with all four subunits of Pol-prim (8, 12, 57). Moreover, the p180, p58, and p48 subunits of Pol-prim also physically bind to RPA (7, 11, 57). RPA and TAg binding sites in the Pol-prim complex are essential for SV40 DNA replication in vitro since the presence of an excess of these purified binding peptides diminishes viral DNA replication in vitro (52, 53). Interestingly, species specificity requires the viral origin of DNA replication, whereas physical protein-protein interactions of purified protein complexes are not host specific in the absence of viral origin DNA (29, 42).Consistent with other polyomaviruses, analyses of BKV TAg-dependent DNA replication recently revealed that BKV DNA cannot be replicated in murine cells and that cell extracts are able to mimic this behavior (28). Furthermore, a BKV DNA replication system with the purified human proteins Pol-prim, RPA, topoisomerase I, and BKV TAg was inhibited by murine extracts, whereas SV40 DNA replication was not. Further investigations revealed that the presence of inhibitory activities (IAs) in extracts from murine cells blocks BKV DNA replication at an early step of TAg-mediated unwinding of the BKV origin of replication. Detailed analyses using the BKV monopolymerase DNA replication system, which we report here, show that Pol-prim functions as a species-specific factor associated with core origin functions. In addition, we reveal that the inhibitory activities in murine extracts, which are associated with origin-flanking sequence-dependent factor(s), regulate BKV DNA replication in murine cell extracts in a Pol-prim-independent manner.     

Statistical modeling of biomedical corpora: mining the Caenorhabditis Genetic Center Bibliography for genes related to life span

Background  

The statistical modeling of biomedical corpora could yield integrated, coarse-to-fine views of biological phenomena that complement discoveries made from analysis of molecular sequence and profiling data. Here, the potential of such modeling is demonstrated by examining the 5,225 free-text items in the Caenorhabditis Genetic Center (CGC) Bibliography using techniques from statistical information retrieval. Items in the CGC biomedical text corpus were modeled using the Latent Dirichlet Allocation (LDA) model. LDA is a hierarchical Bayesian model which represents a document as a random mixture over latent topics; each topic is characterized by a distribution over words.     

Estimating the rate of evolution of the rate of molecular evolution

A simple model for the evolution of the rate of molecular evolution is presented. With a Bayesian approach, this model can serve as the basis for estimating dates of important evolutionary events even in the absence of the assumption of constant rates among evolutionary lineages. The method can be used in conjunction with any of the widely used models for nucleotide substitution or amino acid replacement. It is illustrated by analyzing a data set of rbcL protein sequences.      

Evaluation of a candidate International Standard for Meningococcal Group C polysaccharide

Meningococcal group C (MenC) plain polysaccharide (PS) and conjugate vaccines are primarily evaluated by physicochemical methods to ensure that batches are consistently manufactured. As different assays are employed to quantify the MenC PS content of final formulations and bulk intermediaries, there is a need for an International MenC PS Standard to calibrate internal references used in the different laboratories. Twelve laboratories from nine different countries participated in a collaborative study to determine the MenC PS content of a candidate International Standard MenC PS preparation (08/214) and to assess its suitability. On the basis of the results from this study the candidate standard 08/214 was established as an International Standard for the quantification of MenC PS content in vaccines and components. It has a content of 1.192 ± 0.192 mg MenC PS/ampoule (expanded uncertainty with coverage factor of k = 2.365 corresponding to a 95% level of confidence), as determined by the resorcinol assays carried out by eight of the participating laboratories. The standard is available from The National Institute of Biological Standards and Control who act as guardians and distributors of the material under the auspices of WHO.     

Ensemble attribute profile clustering: discovering and characterizing groups of genes with similar patterns of biological features

Background  

Ensemble attribute profile clustering is a novel, text-based strategy for analyzing a user-defined list of genes and/or proteins. The strategy exploits annotation data present in gene-centered corpora and utilizes ideas from statistical information retrieval to discover and characterize properties shared by subsets of the list. The practical utility of this method is demonstrated by employing it in a retrospective study of two non-overlapping sets of genes defined by a published investigation as markers for normal human breast luminal epithelial cells and myoepithelial cells.     

Genetic monitoring of natural Drosophila populations in radiation contaminated regions of Belarus

Genetic monitoring of natural Drosophila melanogaster populations inhabiting regions of Belarus with different radiation background (Vetka and Svetilovichi villages), radonuclide-contaminated after the Chernobyl accident, compared with populations from the Berezinsky biosphere reserve (the control area) were conducted. The dominant and recessive lethal mutation levels and genetic structure of the populations were analyzed for frequencies of F- and S-alleles of Adh (alcohol dehydrogenase) of Gpdh (glycerinophosphate dehydrogenase) and Sod (superoxide dismutase) loci. Populations inhabiting the regions with high radiation background exhibited higher frequency of lethal mutations and higher heterozygosity than those from the control area. Moreover, higher frequency of polymorphous Sod locus S-allele was detected in these populations. Apparently, Sod S-alleles are more adaptively valuable under conditions of high radiation background, because as is known, superoxide dismutase is an effective radioprotector at all levels molecular, cellular and organism. Adaptation of populations to stress impacts was analyzed, since 1998. Nonspecific adaptation of natural Drosophila melanogaster populations from Vetka and Svetilovichi villages of Gomel region was reveled. They are higher adapted than the control population from the Berezinsky biosphere reserve to both ionizing radiation effect and to chemical mutagen EMS. After laboratory cultivation within 6-8 generations without irradiation adaptation to radiation in the population from radiocontaminated regions remained. The content of samples from the control natural drosophila population in the laboratory conditions is an environmental stress that led to the formation of nonspecific adaptation within 6-8 generations to unfavorable factors, including ionizing radiation. It should be taken into account that the population adaptation is formed via death of sensitive genotypes at various ontogenesis stages.     

Photoactivated DNA analogs of substrates of the nucleotide excision repair system and their interaction with proteins of NER-competent HeLa cell extract

Photoactivated DNA analogs of nucleotide excision repair (NER) substrates have been created that are 48-mer duplexes containing in internal positions pyrimidine nucleotides with bulky substituents imitating lesions. Fluorochloroazidopyridyl, anthracenyl, and pyrenyl groups introduced using spacer fragments at 4N and 5C positions of dCMP and dUMP were used as model damages. The gel retardation and photo-induced affinity modification techniques were used to study the interaction of modified DNA duplexes with proteins in HeLa cell extracts containing the main components of NER protein complexes. It is shown that the extract proteins selectively bind and form covalent adducts with the model DNA. The efficiency and selectivity of protein modification depend on the structure of used DNA duplex. Apparent molecular masses of extract proteins, undergoing modification, were estimated. Mutual influence of simultaneous presence of extract proteins and recombinant NER protein factors XPC-HR23B, XPA, and RPA on interaction with the model DNA was analyzed. The extract proteins and RPA competed for interaction with photoactive DNA, mutually decreasing the yield of modification products. In this case the presence of extract proteins at particular concentrations tripled the increase in yield of covalent adducts formed by XPC. It is supposed that the XPC subunit interaction with DNA is stimulated by endogenous HR23B present in the extract. Most likely, the mutual effect of XPA and extract proteins stimulating formation of covalent adducts with model DNA is due to the interaction of XPA with endogenous RPA of the extract. A technique based on the use of specific antibodies revealed that RPA present in the extract is a modification target for photoactive DNA imitating NER substrates.