Evolutionary clues to DNA polymerase III beta clamp structural mechanisms

The prokaryotic DNA polymerase III beta homodimeric clamp links the replication complex to DNA during polynucleotide synthesis. This clamp is loaded onto DNA and unloaded by the clamp loader complex, the delta subunit of which by itself can bind to and open the clamp. beta Clamps from diverse bacteria were examined using contrast hierarchical alignment and interaction network (CHAIN) analysis, a statistical approach that categorizes and measures the evolutionary constraints imposed on protein sequences by natural selection. Some constraints are subtle inasmuch as they are unique to certain bacteria. Examination of corresponding molecular interactions within structures of the Escherichia coli beta dimeric and delta-beta complexes reveals that N320, Y323 and R176, which are subject to very strong constraints, form a substructure that may serve as a platform for leveraging and directing delta-induced conformational changes. N320 may play a prominent role, as it is strategically situated between this substructure and regions linked to delta binding and opening of beta's dimeric interface. R176 appears to act as a relay between the delta binding site and the clamp's central hole. Other residues subject to strong constraints are likewise associated with structurally important features. For example, two pairs of interacting residues, R269/E304 and K74/E300, form salt bridges at the dimeric interface, while the C-terminal residues M362, P363, M364 and R365 appear to play key roles in delta binding. Q149 and K198 appear to sense DNA within the clamp's central hole while other residues may relay this information to the delta binding site. Mutagenesis experiments designed to explore possible mechanisms are proposed

Evolutionary clues to eukaryotic DNA clamp-loading mechanisms: analysis of the functional constraints imposed on replication factor C AAA+ ATPases

Ring-shaped sliding clamps encircle DNA and bind to DNA polymerase, thereby preventing it from falling off during DNA replication. In eukaryotes, sliding clamps are loaded onto DNA by the replication factor C (RFC) complex, which consists of five distinct subunits (A–E), each of which contains an AAA+ module composed of a RecA-like α/β ATPase domain followed by a helical domain. AAA+ ATPases mediate chaperone-like protein remodeling. Despite remarkable progress in our understanding of clamp loaders, it is still unclear how recognition of primed DNA by RFC triggers ATP hydrolysis and how hydrolysis leads to conformational changes that can load the clamp onto DNA. While these questions can, of course, only be resolved experimentally, the design of such experiments is itself non-trivial and requires that one first formulate the right hypotheses based on preliminary observations. The functional constraints imposed on protein sequences during evolution are potential sources of information in this regard, inasmuch as these presumably are due to and thus reflect underlying mechanisms. Here, rigorous statistical procedures are used to measure and compare the constraints imposed on various RFC clamp-loader subunits, each of which performs a related but somewhat different, specialized function. Visualization of these constraints, within the context of the RFC structure, provides clues regarding clamp-loader mechanisms—suggesting, for example, that RFC-A possesses a triggering component for DNA-dependent ATP hydrolysis. It also suggests that, starting with RFC-A, four RFC subunits (A–D) are sequentially activated through a propagated switching mechanism in which a conserved arginine swings away from a position that disrupts the catalytic Walker B region and into contact with DNA thread through the center of the RFC/clamp complex. Strong constraints near regions of interaction between subunits and with the clamp likewise provide clues regarding possible coupling of hydrolysis-driven conformational changes to the clamp's release and loading onto DNA

The glycine brace: a component of Rab, Rho, and Ran GTPases associated with hinge regions of guanine- and phosphate-binding loops

<p>Abstract</p> <p>Background</p> <p>Ras-like GTPases function as on-off switches in intracellular signalling pathways and include the Rab, Rho/Rac, Ran, Ras, Arf, Sar and Gα families. How these families have evolutionarily diverged from each other at the sequence level provides clues to underlying mechanisms associated with their functional specialization.</p> <p>Results</p> <p>Bayesian analysis of divergent patterns within a multiple alignment of Ras-like GTPase sequences identifies a structural component, termed here the glycine brace, as the feature that most distinguishes Rab, Rho/Rac, Ran and (to some degree) Ras family GTPases from other Ras-like GTPases. The glycine brace consists of four residues: An aromatic residue that forms a stabilizing CH-π interaction with a conserved glycine at the start of the guanine-binding loop; a second aromatic residue, which is nearly always a tryptophan, that likewise forms stabilizing CH-π and NH-π interactions with a glycine at the start of the phosphate-binding P-loop; and two other residues (typically an aspartate and a serine or threonine) that, together with a conserved buried water molecule, form a network of interactions connecting the two aromatic residues.</p> <p>Conclusion</p> <p>It is proposed that the two glycine residues function as hinges and that the glycine brace influences guanine nucleotide binding and release by interacting with these hinges.</p

Bayesian classification of residues associated with protein functional divergence: Arf and Arf-like GTPases

<p>Abstract</p> <p>Background</p> <p>Certain residues within proteins are highly conserved across very distantly related organisms, yet their (presumably critical) structural or mechanistic roles are completely unknown. To obtain clues regarding such residues within Arf and Arf-like (Arf/Arl) GTPases--which function as on/off switches regulating vesicle trafficking, phospholipid metabolism and cytoskeletal remodeling--I apply a new sampling procedure for comparative sequence analysis, termed multiple category Bayesian Partitioning with Pattern Selection (mcBPPS).</p> <p>Results</p> <p>The mcBPPS sampler classified sequences within the entire P-loop GTPase class into multiple categories by identifying those evolutionarily-divergent residues most likely to be responsible for functional specialization. Here I focus on categories of residues that most distinguish various Arf/Arl GTPases from other GTPases. This identified residues whose specific roles have been previously proposed (and in some cases corroborated experimentally and that thus serve as positive controls), as well as several categories of co-conserved residues whose possible roles are first hinted at here. For example, Arf/Arl/Sar GTPases are most distinguished from other GTPases by a conserved aspartate residue within the phosphate binding loop (P-loop) and by co-conserved residues nearby that, together, can form a network of salt-bridge and hydrogen bond interactions centered on the GTPase active site. Residues corresponding to an N-[VI] motif that is conserved within Arf/Arl GTPases may play a role in the interswitch toggle characteristic of the Arf family, whereas other, co-conserved residues may modulate the flexibility of the guanine binding loop. Arl8 GTPases conserve residues that strikingly diverge from those typically found in other Arf/Arl GTPases and that form structural interactions suggestive of a novel interswitch toggle mechanism.</p> <p>Conclusions</p> <p>This analysis suggests specific mutagenesis experiments to explore mechanisms underlying GTP hydrolysis, nucleotide exchange and interswitch toggling within Arf/Arl GTPases. More generally, it illustrates how the mcBPPS sampler can complement traditional evolutionary analyses by providing an objective, quantitative and statistically rigorous way to explore protein functional-divergence in molecular detail. Because the sampler classifies the input sequences at the same time, it can be used to generate subgroup profiles, in which functionally-divergent categories of residues are annotated automatically.</p> <p>Reviewers</p> <p>This article was reviewed by Frank Eisenhaber, L Aravind and Daniel Gaston (nominated by Eric Bapteste). For the full reviews, go to the Reviewers' comments section.</p

Hypothesis: bacterial clamp loader ATPase activation through DNA-dependent repositioning of the catalytic base and of a trans-acting catalytic threonine

The prokaryotic DNA polymerase III clamp loader complex loads the β clamp onto DNA to link the replication complex to DNA during processive synthesis and unloads it again once synthesis is complete. This minimal complex consists of one δ, one δ′ and three γ subunits, all of which possess an AAA+ module—though only the γ subunit exhibits ATPase activity. Here clues to underlying clamp loader mechanisms are obtained through Bayesian inference of various categories of selective constraints imposed on the γ and δ′ subunits. It is proposed that a conserved histidine is ionized via electron transfer involving structurally adjacent residues within the sensor 1 region of γ's AAA+ module. The resultant positive charge on this histidine inhibits ATPase activity by drawing the negatively charged catalytic base away from the active site. It is also proposed that this arrangement is disrupted upon interaction of DNA with basic residues in γ implicated previously in DNA binding, regarding which a lysine that is near the sensor 1 region and that is highly conserved both in bacterial and in eukaryotic clamp loader ATPases appears to play a critical role. γ ATPases also appear to utilize a trans-acting threonine that is donated by helix 6 of an adjacent γ or δ′ subunit and that assists in the activation of a water molecule for nucleophilic attack on the γ phosphorous atom of ATP. As eukaryotic and archaeal clamp loaders lack most of these key residues, it appears that eubacteria utilize a fundamentally different mechanism for clamp loader activation than do these other organisms

A Model for Detecting Motifs in Biological Sequences

A method for detecting patterns in biological sequences is described that incorporates rigorous statistics for determining significances, and an algebraic system that, in combination with a depth first search procedure, can be used to efficiently search for all patterns up to a specified length. This method includes a context free command language grammar and is formulated using a mathematical model amendable to additions enhancements, The method was implemented and verified by detection of various types of patterns in protein sequences

Gapped alignment of protein sequence motifs through Monte Carlo optimization of a hidden Markov model

BACKGROUND: Certain protein families are highly conserved across distantly related organisms and belong to large and functionally diverse superfamilies. The patterns of conservation present in these protein sequences presumably are due to selective constraints maintaining important but unknown structural mechanisms with some constraints specific to each family and others shared by a larger subset or by the entire superfamily. To exploit these patterns as a source of functional information, we recently devised a statistically based approach called contrast hierarchical alignment and interaction network (CHAIN) analysis, which infers the strengths of various categories of selective constraints from co-conserved patterns in a multiple alignment. The power of this approach strongly depends on the quality of the multiple alignments, which thus motivated development of theoretical concepts and strategies to improve alignment of conserved motifs within large sets of distantly related sequences. RESULTS: Here we describe a hidden Markov model (HMM), an algebraic system, and Markov chain Monte Carlo (MCMC) sampling strategies for alignment of multiple sequence motifs. The MCMC sampling strategies are useful both for alignment optimization and for adjusting position specific background amino acid frequencies for alignment uncertainties. Associated statistical formulations provide an objective measure of alignment quality as well as automatic gap penalty optimization. Improved alignments obtained in this way are compared with PSI-BLAST based alignments within the context of CHAIN analysis of three protein families: G(iα )subunits, prolyl oligopeptidases, and transitional endoplasmic reticulum (p97) AAA+ ATPases. CONCLUSION: While not entirely replacing PSI-BLAST based alignments, which likewise may be optimized for CHAIN analysis using this approach, these motif-based methods often more accurately align very distantly related sequences and thus can provide a better measure of selective constraints. In some instances, these new approaches also provide a better understanding of family-specific constraints, as we illustrate for p97 ATPases. Programs implementing these procedures and supplementary information are available from the authors

Prediction of bitter pit in apples through Mg2+ infiltration and ethephon application on fruits

Estudou-se o efeito da infiltração de magnésio e cálcio no desenvolvimento de sintomas de bitter pit de maçãs 'Gala'. Examinou-se, também, a eficiência da infiltração de magnésio e da aceleração da maturação de maçãs 'Royal Gala' como métodos de previsão da ocorrência de bitter pit após o armazenamento. No primeiro experimento, maçãs 'Gala' foram infiltradas com MgCl2; CaCl2 ou MgCl2 mais CaCl2. No segundo experimento, maçãs 'Royal Gala' foram infiltradas com MgCl2; ou imersas em solução contendo ethephon por cinco minutos. Como controle, frutos de cada cultivar foram armazenados em atmosfera controlada por seis meses mais 18 dias a 20&deg;C. Frutos infiltrados com MgCl2 apresentaram significativo acréscimo na incidência e severidade de bitter pit, proporcional à concentração de MgCl2 na solução. Frutos infiltrados com CaCl2 não apresentaram sintomas de bitter pit. A aceleração da maturação de maçãs 'Royal Gala' na colheita foi efetiva na previsão de bitter pit após o armazenamento.The effect of magnesium and calcium infiltration on bitter pit symptoms development in Gala apples was studied. It was also examined the efficiency of magnesium infiltration and the ripening hastening of Royal Gala apples as a predictive methods of bitter pit occurrence after storage. In the first experiment, Gala apples were infiltrated with MgCl2; CaCl2; or MgCl2 plus CaCl2. In the second experiment Royal Gala apples were infiltrated with MgCl2; or immersed into solution containing ethephon during five minutes. As a control a sample of each cultivar was stored in controlled atmosphere during 6 months plus eighteen days at 20&deg;C. Fruits infiltrated with MgCl2 showed increased incidence and severity of bitter pit proportional to the MgCl2 concentration in the solution. Infiltration with CaCl2 prevents bitter pit-like symptoms. The ripening hastening of Royal Gala apples at harvest was effective on the bitter pit prediction after storage

Water influx through the wetted surface of a sweet cherry fruit: Evidence for an associated solute efflux

Sweet cherries are susceptible to rain-cracking. The fruit skin is permeable to water, but also to solutes. The objectives of this study were to (1) establish whether a solute efflux occurs when a sweet cherry fruit is incubated in water; (2) identify the solutes involved; (3) identify the mechanism(s) of efflux; and (4) quantify any changes in solute efflux occurring during development and storage. Solute efflux was gravimetrically measured in wetted fruit as the increasing dry mass of the bathing solution, and anthocyanin efflux was measured spectrophotometrically. Solute and anthocyanin effluxes from a wetted fruit and water influx increased with time. All fluxes were higher for the cracked than for the non-cracked fruit. The effluxes of osmolytes and anthocyanins were positively correlated. Solute efflux depended on the stage of development and on the cultivar. In ‘Regina’, the solute efflux was lowest during stage II (25 days after full bloom (DAFB)), highest for mid-stage III (55 DAFB), and slightly lower at maturity (77 DAFB). In contrast with ‘Regina’, solute efflux in ‘Burlat’ increased continuously towards maturity, being 4.8-fold higher than in ‘Regina’. Results showed that solute efflux occurred from wetted fruit. The gravimetrically determined water uptake represents a net mass change—the result of an influx minus a solute efflux

Measurement of Water Vapor Condensation on Apple Surfaces during Controlled Atmosphere Storage

Apples are stored at temperatures close to 0 °C and high relative humidity (up to 95%) under controlled atmosphere conditions. Under these conditions, the cyclic operation of the refrigeration machine and the associated temperature fluctuations can lead to localized undershoots of the dew point on fruit surfaces. The primary question for the present study was to prove that such condensation processes can be measured under practical conditions during apple storage. Using the example of a measuring point in the upper apple layer of a large bin in the supply air area, this evidence was provided. Using two independent measuring methods, a wetness sensor attached to the apple surface and determination of climatic conditions near the fruit, the phases of condensation, namely active condensation and evaporation, were measured over three weeks as a function of the operating time of the cooling system components (refrigeration machine, fans, defrosting regime). The system for measurement and continuous data acquisition in the case of an airtight CA-storage room is presented and the influence of the operation of the cooling system components in relation to condensation phenomena was evaluated. Depending on the set point specifications for ventilation and defrost control, condensed water was present on the apple surface between 33.4% and 100% of the duration of the varying cooling/re-warming cycles