PCNA stimulates catalysis by structure-specific nucleases using two distinct mechanisms: substrate targeting and catalytic step

The sliding clamp Proliferating Cell Nuclear Antigen (PCNA) functions as a recruiter and organizer of a wide variety of DNA modifying enzymes including nucleases, helicases, polymerases and glycosylases. The 5′-flap endonuclease Fen-1 is essential for Okazaki fragment processing in eukaryotes and archaea, and is targeted to the replication fork by PCNA. Crenarchaeal XPF, a 3′-flap endonuclease, is also stimulated by PCNA in vitro. Using a novel continuous fluorimetric assay, we demonstrate that PCNA activates these two nucleases by fundamentally different mechanisms. PCNA stimulates Fen-1 by increasing the enzyme's binding affinity for substrates, as suggested previously. However, PCNA activates XPF by increasing the catalytic rate constant by four orders of magnitude without affecting the KM. PCNA may function as a platform upon which XPF exerts force to distort DNA substrates, destabilizing the substrate and/or stabilizing the transition state structure. This suggests that PCNA can function directly in supporting catalysis as an essential cofactor in some circumstances, a new role for a protein that is generally assumed to perform a passive targeting and organizing function in molecular biology. This could provide a mechanism for the exquisite control of nuclease activity targeted to specific circumstances, such as replication forks or damaged DNA with pre-loaded PCNA

Biochemical Characterization of a Structure-Specific Resolving Enzyme from Sulfolobus islandicus Rod-Shaped Virus 2

Sulfolobus islandicus rod shaped virus 2 (SIRV2) infects the archaeon Sulfolobus islandicus at extreme temperature (70°C–80°C) and acidity (pH 3). SIRV2 encodes a Holliday junction resolving enzyme (SIRV2 Hjr) that has been proposed as a key enzyme in SIRV2 genome replication. The molecular mechanism for SIRV2 Hjr four-way junction cleavage bias, minimal requirements for four-way junction cleavage, and substrate specificity were determined. SIRV2 Hjr cleaves four-way DNA junctions with a preference for cleavage of exchange strand pairs, in contrast to host-derived resolving enzymes, suggesting fundamental differences in substrate recognition and cleavage among closely related Sulfolobus resolving enzymes. Unlike other viral resolving enzymes, such as T4 endonuclease VII or T7 endonuclease I, that cleave branched DNA replication intermediates, SIRV2 Hjr cleavage is specific to four-way DNA junctions and inactive on other branched DNA molecules. In addition, a specific interaction was detected between SIRV2 Hjr and the SIRV2 virion body coat protein (SIRV2gp26). Based on this observation, a model is proposed linking SIRV2 Hjr genome resolution to viral particle assembly

Reversion of a fungal genetic code alteration links proteome instability with genomic and phenotypic diversification

Many fungi restructured their proteomes through incorporation of serine (Ser) at thousands of protein sites coded by the leucine (Leu) CUG codon. How these fungi survived this potentially lethal genetic code alteration and its relevance for their biology are not understood. Interestingly, the human pathogen Candida albicans maintains variable Ser and Leu incorporation levels at CUG sites, suggesting that this atypical codon assignment flexibility provided an effective mechanism to alter the genetic code. To test this hypothesis, we have engineered C. albicans strains to misincorporate increasing levels of Leu at protein CUG sites. Tolerance to the misincorporations was very high, and one strain accommodated the complete reversion of CUG identity from Ser back to Leu. Increasing levels of Leu misincorporation decreased growth rate, but production of phenotypic diversity on a phenotypic array probing various metabolic networks, drug resistance, and host immune cell responses was impressive. Genome resequencing revealed an increasing number of genotype changes at polymorphic sites compared with the control strain, and 80% of Leu misincorporation resulted in complete loss of heterozygosity in a large region of chromosome V. The data unveil unanticipated links between gene translational fidelity, proteome instability and variability, genome diversification, and adaptive phenotypic diversity. They also explain the high heterozygosity of the C. albicans genome and open the door to produce microorganisms with genetic code alterations for basic and applied research.We thank Alexander Johnson for providing the C. albicans strains and plasmids and Judith Berman, Csaba Pál, and Dieter Söll for their useful comments and suggestions on the manuscript. The study was funded by the European Union Framework Program 7 (EUFP7) Sybaris Consortium Project 242220 and the Portuguese Science Foundation through Fundo Europeu de Desenvolvimento Regional (FEDER/FCT) Project PTDC/BIA-MIC/099826/2008.publishe

PCNA activates the Holliday junction resolving enzyme Hjc

The resolving enzyme Hjc, which cleaves Holliday junctions with a high degree of structural specificity, is conserved in all archaea. Like RuvC in Escherichia coli, Hjc functions in the related processes of homologous recombination and double-strand break repair. In bacteria, the RuvAB complex binds Holliday junctions and catalyses ATP-dependent branch migration, but the equivalent proteins in archaea and eukarya are unknown. Here, we demonstrate that Hjc from Sulfolobus solfataricus forms a physical interaction with the sliding clamp PCNA via a C-terminal PCNA-interacting peptide (PIP) motif in Hjc. PCNA stimulates the Holliday junction cleavage activity of Hjc in vitro, and deletion of the PIP motif abrogates this effect. This is the first report of a functional interaction between a sliding clamp and a junction-resolving enzyme, and raises the possibility that PCNA could recruit a variety of different proteins to act on Holliday junctions in vivo. (c) 2006 Elsevier Ltd. All rights reserved.</p

Equal rates of repair of DNA photoproducts in transcribed and non-transcribed strands in <em>Sulfolobus solfataricus</em>

The nucleotide excision repair (NER) pathway removes bulky lesions such as photoproducts from DNA. In both bacteria and eukarya, lesions located in transcribed strands are repaired significantly faster than those located in non-transcribed strands due to damage signalling by stalled RNA polymerase molecules: a phenomenon known as transcription-coupled repair (TCR). TCR requires a mechanism for coupling the detection of stalled RNA polymerase molecules to the NER pathway, provided in bacteria by the Mfd protein. In the third domain of life, archaea, the pathway of NER is not well defined, there are no Mfd homologues and the existence of TCR has not been investigated. In this report we looked at rates of removal of photoproducts in three different operons of the crenarchaeon Sulfolobus solfataricus following UV irradiation. We found no evidence for significantly faster repair in the transcribed strands of these three operons. The rate of global genome repair in S. solfataricus is relatively rapid, and this may obviate the requirement for a specialized TCR pathway. Significantly faster repair kinetics were observed in the presence of visible light, consistent with the presence of a gene for photolyase in the genome of S. solfataricus.</p

Economic benefits of biodiversity exceed costs of conservation at an African rainforest reserve

Economic research on biodiversity conservation has focused on the costs of conservation reserves and the benefits of intact ecosystems; however, no study has simultaneously considered the costs and benefits of species diversity, a fundamental component of biodiversity. We quantified the costs and benefits of avian biodiversity at a rainforest reserve in Uganda through a combination of economic surveys of tourists, spatial land-use analyses, and species-area relationships. Our results show that revising entrance fees and redistributing ecotourism revenues would protect 114 of 143 forest bird species (80%) under current market conditions. This total would increase to 131 species (≈90%) if entrance fees were optimized to capture the tourist's willingness to pay for forest visits and the chance of seeing increased numbers of bird species. In contrast, the cost of purchasing agricultural land for ecological rehabilitation of the avian habitat would be economically prohibitive. These results suggest that local biodiversity markets could play a positive role in tropical conservation strategies if the appropriate institutions for redistribution can be developed

Stellar Population Astrophysics (SPA) with TNG Atmospheric parameters of members of 16 unstudied open clusters

Thanks to modern understanding of stellar evolution, we can accurately measure the age of Open Clusters (OCs). Given their position, they are ideal tracers of the Galactic disc. Gaia data release 2, besides providing precise parallaxes, led to the detection of many new clusters, opening a new era for the study of the Galactic disc. However, detailed information on the chemical abundance for OCs is necessary to accurately date them and to efficiently use them to probe the evolution of the disc.Mapping and exploring the Milky Way structure %to combine accurate chemical information of OCs is the main aim of the Stellar Population Astrophysics (SPA) project. Part of this work involves the use of OCs and the derivation of their precise and accurate chemical composition.We analyze here a sample of OCs located within about 2 kpc from the Sun, with ages from about 50 Myr to a few Gyr.We used HARPS-N at the Telescopio Nazionale Gaileo and collected very high-resolution spectra (R = 115\,000) of 40 red giant/red clump stars in 18 OCs (16 never or scarcely studied plus two comparison clusters). We measured their radial velocities and derived the stellar parameters.We discussed the relationship between metallicity and Galactocentric distance, adding literature data to our results to enlarge the sample and taking also age into account. We compared the result of observational data with that from chemo-dynamical models. These models generally reproduce the metallicity gradient well. However, at young ages we found a large dispersion in metallicity, not reproduced by models. Several possible explanations are explored, including uncertainties in the derived metallicity. We confirm the difficulties in determining parameters for young stars (age < 200 Myr), due to a combination of intrinsic factors which atmospheric models can not easily reproduce and which affect the parameters uncertaintyComment: 21 pages 9 figures Astronomy & Astrophysic