Partial reconstitution of DNA large loop repair with purified proteins from Saccharomyces cerevisiae

Small looped mispairs are corrected by DNA mismatch repair. In addition, a distinct process called large loop repair (LLR) corrects heteroduplexes up to several hundred nucleotides in bacteria, yeast and human cells, and in cell-free extracts. Only some LLR protein components are known, however. Previous studies with neutralizing antibodies suggested a role for yeast DNA polymerase δ (Pol δ), RFC and PCNA in LLR repair synthesis. In the current study, biochemical fractionation studies identified FEN1 (Rad27) as another required LLR component. In the presence of purified FEN1, Pol δ, RFC and PCNA, repair occurred on heteroduplexes with loops ranging from 8 to 216 nt. Repair utilized a 5′ nick, with correction directed to the nicked strand, irrespective of which strand contained the loop. In contrast, repair of a G/T mismatch occurred at low levels, suggesting specificity of the reconstituted system for looped mispairs. The presence of RPA enhanced reactivity on some looped substrates, but RPA was not required for activity. Although additional LLR factors remain to be identified, the excision and resynthesis steps of LLR from a 5′ nick can be reconstituted in a purified system with FEN1 and Pol δ, together with PCNA and its loader RFC

Extracellular loops 2 and 3 of the calcitonin receptor selectively modify agonist binding and efficacy.

Class B peptide hormone GPCRs are targets for the treatment of major chronic disease. Peptide ligands of these receptors display biased agonism and this may provide future therapeutic advantage. Recent active structures of the calcitonin (CT) and glucagon-like peptide-1 (GLP-1) receptors reveal distinct engagement of peptides with extracellular loops (ECLs) 2 and 3, and mutagenesis of the GLP-1R has implicated these loops in dynamics of receptor activation. In the current study, we have mutated ECLs 2 and 3 of the human CT receptor (CTR), to interrogate receptor expression, peptide affinity and efficacy. Integration of these data with insights from the CTR and GLP-1R active structures, revealed marked diversity in mechanisms of peptide engagement and receptor activation between the CTR and GLP-1R. While the CTR ECL2 played a key role in conformational propagation linked to Gs/cAMP signalling this was mechanistically distinct from that of GLP-1R ECL2. Moreover, ECL3 was a hotspot for distinct ligand- and pathway- specific effects, and this has implications for the future design of biased agonists of class B GPCRs

Phytochrome structure and GTP-binding proteins

Phytochromes are a family of photoreceptors that regulate plant growth and development. The relative extent of chromophore exposure of the red-absorbing (P\sb{\rm r}) and far-red-absorbing (P\sb{\rm fr}) forms of 124-kDa oat phytochrome and the secondary structure of the phytochrome apoprotein have been investigated using zinc-induced modification of the phytochrome chromophore. Circular dichroism (CD) analyses revealed that upon the addition of a 1:1 molar ratio of zinc ions to phytochrome, a dramatic change in the CD of the P\sb{\rm fr} form is observed, while the CD spectrum of the P\sb{\rm r} form is unaffected. Analysis of the bleached P\sb{\rm fr} CD spectrum reveals that chelation with zinc ions significantly alters the secondary structure of the phytochrome molecule. It is proposed that chelation with zinc ions at the phytochrome chromophore cyclizes the semiextended chromophore, which reduces/eliminates the interactive forces between the chromophore and the polypeptide. As part of testing the hypothesis that phytochrome may interact with signal-transducing GTP-binding proteins, a 24 kDa GTP-binding protein was isolated and characterized from dark grown oat seedlings. The protein bound maximally 0.9 $\pm$ 0.1 mol of GTP$\gamma$S/mol of protein with an apparent K\sb{\rm d} of 32 nM. Competition binding assays indicated that the protein specifically binds GDP and GTP. The 24 kDa protein hydrolyzed GTP with a rate of 0.06 mol P\sb{\rm i}/mol protein/min. In the presence of the P\sb{\rm r} form of phytochrome, the GTPase activity of the protein is enhanced. Nucleoside diphosphate kinase, an enzyme that reportedly co-purifies with a variety of GTP-binding proteins and which has been implicated in cancer metastasis suppression, was also isolated and characterized from oat seedlings. N-terminal sequencing of the Avena protein revealed that 87% of the 23 amino acids sequenced were identical to the human nm23-H2 protein

Partial reconstitution of dna large loop repair with purified proteins from saccharomyces cerevisiae

Small looped mispairs are corrected by DNA mismatch repair. In addition, a distinct process called large loop repair (LLR) corrects heteroduplexes up to several hundred nucleotides in bacteria, yeast and human cells, and in cell-free extracts. Only some LLR protein components are known, however. Previous studies with neutralizing antibodies suggested a role for yeast DNA polymerase (Pol ), RFC and PCNA in LLR repair synthesis. In the current study, biochemical fractionation studies identified FEN1 (Rad27) as another required LLR component. In the presence of purified FEN1, Pol , RFC and PCNA, repair occurred on heteroduplexes with loops ranging from 8 to 216 nt. Repair utilized a 5 nick, with correction directed to the nicked strand, irrespective of which strand contained the loop. In contrast, repair of a G/T mismatch occurred at low levels, suggesting specificity of the reconstituted system for looped mispairs. The presence of RPA enhanced reactivity on some looped substrates, but RPA was not required for activity. Although additional LLR factors remain to be identified, the excision and resynthesis steps of LLR from a 5 nick can be reconstituted in a purified system with FEN1 and Pol , together with PCNA and its loader RFC

DNA polymerase δ, RFC and PCNA are required for repair synthesis of large looped heteroduplexes in Saccharomyces cerevisiae

Small looped mispairs are corrected by DNA mismatch repair (MMR). In addition, a distinct process called large loop repair (LLR) corrects loops up to several hundred nucleotides in extracts of bacteria, yeast or human cells. Although LLR activity can be readily demonstrated, there has been little progress in identifying its protein components. This study identified some of the yeast proteins responsible for DNA repair synthesis during LLR. Polyclonal antisera to either Pol31 or Pol32 subunits of polymerase δ efficiently inhibited LLR in extracts by blocking repair just prior to gap filling. Gap filling was inhibited regardless of whether the loop was retained or removed. These experiments suggest polymerase δ is uniquely required in yeast extracts for LLR-associated synthesis. Similar results were obtained with antisera to the clamp loader proteins Rfc3 and Rfc4, and to PCNA, i.e. LLR was inhibited just prior to gap filling for both loop removal and loop retention. Thus PCNA and RFC seem to act in LLR only during repair synthesis, in contrast to their roles at both pre- and post-excision steps of MMR. These biochemical experiments support the idea that yeast polymerase δ, RFC and PCNA are required for large loop DNA repair synthesis

Synthesis and the Assessment of Adaptation Measures,

The fi nal stage of the CIRCE case-studies integrated assessment involved identi fi cation and evaluation of the effectiveness of local and regional adaptation options in collaboration with stakeholders, and in the context of wider national adaptation policies and strategies. This stage provides a synthesis of both the casestudy work and the wider CIRCE project since it draws on the case-study indicators for present and future periods together with wider CIRCE work on adaptation options, particularly in the thematic areas of agriculture, forestry and ecosystems, and Mediterranean communities. This synthesis and evaluation links impacts and vulnerability with adaptation, and also bene fi ts strongly from the local stakeholder workshops held towards the end of the project. Lessons learnt and key messages from the CIRCE case studies are presented. While the objectives of the CIRCE case studies have generally been achieved, a number of research gaps and needs remain