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The MRN complex (MRX in Saccharomyces cerevisiae, made of Mre11, Rad50 and Nbs1/Xrs2) initiates double-stranded DNA break repair and activates the Tel1/ATM kinase in the DNA damage response. Telomeres counter both outcomes at chromosome ends, partly by keeping MRN-ATM in check. We show that MRX is disabled by telomeric protein Rif2 through an N-terminal motif (MIN, MRN/X-inhibitory motif). MIN executes suppression of Tel1, DNA end-resection and non-homologous end joining by binding the Rad50 N-terminal region. Our data suggest that MIN promotes a transition within MRX that is not conductive for endonuclease activity, DNA-end tethering or Tel1 kinase activation, highlighting an Achilles' heel in MRN, which we propose is also exploited by the RIF2 paralog ORC4 (Origin Recognition Complex 4) in Kluyveromyces lactis and the Schizosaccharomyces pombe telomeric factor Taz1, which is evolutionarily unrelated to Orc4/Rif2. This raises the possibility that analogous mechanisms might be deployed in other eukaryotes as well.

The firing of eukaryotic origins of DNA replication requires CDK and DDK kinase activities. DDK, in particular, is involved in setting the temporal program of origin activation, a conserved feature of eukaryotes. Rif1, originally identified as a telomeric protein, was recently implicated in specifying replication timing in yeast and mammals. We show that this function of Rif1 depends on its interaction with PP1 phosphatases. Mutations of two PP1 docking motifs in Rif1 lead to early replication of telomeres in budding yeast and misregulation of origin firing in fission yeast. Several lines of evidence indicate that Rif1/PP1 counteract DDK activity on the replicative MCM helicase. Our data suggest that the PP1/Rif1 interaction is downregulated by the phosphorylation of Rif1, most likely by CDK/DDK. These findings elucidate the mechanism of action of Rif1 in the control of DNA replication and demonstrate a role of PP1 phosphatases in the regulation of origin firing., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

The conventional DNA polymerase machinery is unable to fully replicate the ends of linear chromosomes. To surmount this problem, nearly all eukaryotes use the telomerase enzyme, a specialized reverse transcriptase that utilizes its own RNA template to add short TG-rich repeats to chromosome ends, thus reversing their gradual erosion occurring at each round of replication. This unique, non-DNA templated mode of telomere replication requires a regulatory mechanism to ensure that telomerase acts at telomeres whose TG tracts are too short, but not at those with long tracts, thus maintaining the protective TG repeat 'cap' at an appropriate average length. The prevailing notion in the field is that telomere length regulation is brought about through a negative feedback mechanism that 'counts' TG repeat-bound protein complexes to generate a signal that regulates telomerase action. This review summarizes experiments leading up to this model and then focuses on more recent experiments, primarily from yeast, that begin to suggest how this 'counting' mechanism might work. The emerging picture is that of a complex interplay between the conventional DNA replication machinery, DNA damage response factors, and a specialized set of proteins that help to recruit and regulate the telomerase enzyme.

The budding yeast Cdc13, Stn1 and Ten1 (CST) proteins are proposed to function as an RPA-like complex at telomeres that protects ('caps') chromosome ends and regulates their elongation by telomerase. We show that Stn1 has a critical function in both processes through the deployment of two separable domains. The N terminus of Stn1 interacts with Ten1 and carries out its essential capping function. The C terminus of Stn1 binds both Cdc13 and Pol12, and we present genetic data indicating that the Stn1-Cdc13 interaction is required to limit continuous telomerase action. Stn1 telomere association, similar to that of Cdc13, peaks during S phase. Significantly, the magnitude of Stn1 telomere binding is independent of telomere TG tract length, suggesting that the negative effect of Stn1 on telomerase action might be regulated by a modification of CST activity or structure in cis at individual telomeres. Genetic analysis suggests that the Tell kinase exerts an effect in parallel with the Stn1 C terminus to counteract its inhibition of telomerase. These data provide new insights into the coordination of telomere capping and telomerase regulation.

Telomere function is mediated by the assembly of a protein complex on an array of telomeric DNA (TG) repeats synthesized by the telomerase enzyme. Telomerase action at chromosome ends is finely tuned by the telomeric complex so that a constant average number of repeats is maintained. This is achieved through a negative feedback process that is sensitive to TG tract length, but whose underlying mechanism is unknown. We show that short telomeres, which are preferential substrates for telomerase, display increased association with the enzyme in the S phase of the cell cycle, when telomerase acts. In addition, we provide support for a molecular mechanism by which this key step of telomerase recruitment is regulated by TG tract length.

The maintenance of an appropriate number of telomere repeats by telomerase is essential for proper chromosome protection. The action of telomerase at the telomere terminus is regulated by opposing activities that either recruit/activate the enzyme at shorter telomeres or inhibit it at longer ones, thus achieving a stable average telomere length. To elucidate the mechanistic details of telomerase regulation we engineered specific chromosome ends in yeast so that a single telomere could be suddenly shortened and, as a consequence of its reduced length, elongated by telomerase. We show that shortened telomeres replicate early in S phase, unlike normal-length telomeres, due to the early firing of origins of DNA replication in subtelomeric regions. Early telomere replication correlates with increased telomere length and telomerase activity. These data reveal an epigenetic effect of telomere length on the activity of nearby replication origins and an unanticipated link between telomere replication timing and telomerase action.

We have analyzed in detail the structure of RAP1-UAS(RPG) complexes in Saccharomyces cerevisiae cells using multi-hit KMnO(4), UV and micrococcal nuclease high-resolution footprinting. Three copies of the Rap1 protein are bound to the promoter simultaneously in exponentially growing cells, as shown by KMnO(4) multi-hit footprinting analysis, causing extended and diagnostic changes in the DNA structure of the region containing the UAS(RPG). Amino acid starvation does not cause loss of Rap1p from the complex; however, in vivo UV-footprinting reveals the occurrence of structural modifications of the complex. Moreover, low-resolution micrococcal nuclease digestion shows that the chromatin of the entire region is devoid of positioned nucleosomes but is susceptible to changes in accessibility to the nuclease upon amino acid starvation. The implications of these results for the mechanism of Rap1p action are discussed., ((c) 2002 Elsevier Science Ltd.)

"Avanzi 3-Grano 23" (G23) is an old variety of Triticum aestivum from the mountain areas of Lunigiana (north Tuscany, Italy), where traditional farming communities have contributed to its success and on-farm conservation. G23 flour, traditionally used for typical food products, is characterized by particular nutritional and sensory traits but has technological properties which limit its suitability for breadmaking. The aim of this work was to evaluate how to promote the use of G23 through the optimization of bread formulation by leveraging both flour blending and the leavening system. During the preliminary test, three different mixes of G23 flour and a strong flour (C) were tested in terms of their leavening power as a function of leavening agent (baker's yeast or sourdough). The selected M2 flour, composed of G23:C (1:1 w/w), was used for further breadmaking trials and 100% C flour was utilized as a control. The sourdough bread obtained with the M2 flour (SB-M2) showed an improved sensory profile compared with the related control (SB-C). Furthermore, SB-M2 exhibited the best aromatic (high content in aldehydes, pyrazines and carboxylic acids) and phytochemical profile (total polyphenols and flavonoids content and antioxidant activity). In contrast, the use of baker's yeast, although optimal from the point of view of breadmaking, did not result in the same levels of aromatic complexity because it tends to standardize the product without valorizing the sensory and nutritional qualities of the flour. In conclusion, in the experimental conditions adopted, this old wheat variety appears to be suitable for the production of sourdough bakery products. [ABSTRACT FROM AUTHOR]

As is widely accepted, the quality decay of freshly baked bread that affects product shelf life is the result of a complex multifactorial process that involves physical staling, together with microbiological, chemical and sensorial spoilage. In this context, this paper provides a critical review of the recent literature about the main factors affecting shelf life of bread during post‐baking. An overview of the recent findings about the mechanism of bread staling is firstly provided. Afterwards, the effect on staling induced by baker's yeasts and sourdough as well as by the extra ingredients commonly utilized for bread fortification is also addressed and discussed. As inclusion/exclusion criteria, only papers dealing with wheat bread and not with long‐life bread or gluten‐free bakery products are taken into consideration. Despite recent developments in international scientific literature, the whole mechanism that induces bread staling is far from being completely understood and the best analytical methods to be adopted to measure and/or describe in depth this process appear still debated. In this topic, the effects induced on bread shelf life by the use of biological leavening agents (baker's yeasts and sourdough) as well as by some extra ingredients included in the bread recipe have been individuated as two key issues to be addressed and discussed in terms of their influence on the kinetics of bread staling. © 2020 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

This research aimed to explore the feasibility of fortifying bread with cooked Vitelotte potato powder and Citrus albedo, comparing the use of baker's yeast or sourdough as leavening agents. Breads obtained were thus subjected to physico-chemical and sensory characterizations. The replacement of part of the wheat flour with purple potato and albedo determined a significant enhancement of the phenolic profile and antioxidant status of fortified breads, as well as a longer shelf life. Thanks to its acidity and antimicrobial activity, sourdough improved the levels of health-promoting compounds and stability. Both the fortification and the leavening agent deeply affected the organoleptic, expression, and the aroma profile, of the fortified bread. Interestingly, albedo addition, despite its effectiveness in boosting the phenolic profile, determined a higher perception of aftertaste and bitterness, irrespective of the leavening agent. Based on these results, the use of purple potatoes and Citrus albedo, if properly formulated, could represent a valuable strategy for the development of high-quality products, with longer shelf-life. [ABSTRACT FROM AUTHOR]

The essential Saccharomyces cerevisiae regulatory protein Rap1 contains two tandem Myb-like DNA binding sub-domains that interact with two defined DNA “hemisites”, separated by a trinucleotide linker sequence. We have mapped the thermodynamically defined DNA-binding site of Rap1 by a primer extension method coupled with electrophoretic separation of bound and unbound DNAs. Relative to published consensus sequences, we detect binding interactions that extend 3 bp beyond the 5′-end of the putative DNA-binding site. This new site of interaction is located where the DNA minor groove faces the protein, and may account for the major DNA bending induced by Rap1p that previous studies have mapped to a site immediately upstream of the consensus binding site. In addition, we show that a minimal DNA-binding site made of one single consensus hemisite, preceded or followed by a spacer trinucleotide that interacts with the unstructured protein linker between the two Rap1p DNA binding domains, is able to bind the protein, although at lower affinity. These findings may explain the observed in vivo binding properties of Rap1p at many promoters that lack canonical binding sites. [Copyright &y& Elsevier]

We have analyzed in detail the structure of RAP1–UASRPG complexes in Saccharomyces cerevisiae cells using multi-hit KMnO4, UV and micrococcal nuclease high-resolution footprinting. Three copies of the Rap1 protein are bound to the promoter simultaneously in exponentially growing cells, as shown by KMnO4 multi-hit footprinting analysis, causing extended and diagnostic changes in the DNA structure of the region containing the UASRPG. Amino acid starvation does not cause loss of Rap1p from the complex; however, in vivo UV-footprinting reveals the occurrence of structural modifications of the complex. Moreover, low-resolution micrococcal nuclease digestion shows that the chromatin of the entire region is devoid of positioned nucleosomes but is susceptible to changes in accessibility to the nuclease upon amino acid starvation. The implications of these results for the mechanism of Rap1p action are discussed. [Copyright &y& Elsevier]