The role of the Mcm2 subunit in regulating the activities of the Mcm2-7 helicase

The transmission of genetic information from parental to daughter cells requires the faithful duplication of an organism’s genome. Uncontrolled DNA replication can result in proliferative diseases, such as cancer. DNA replication requires a single-stranded DNA template to be produced from duplex DNA. In eukaryotes, DNA unwinding for replication is performed by the heterohexameric replicative helicase complex comprised of the minichromosome maintenance proteins 2 through 7. Each of the Mcm2-7 subunits likely has a unique role in DNA binding and unwinding by the Mcm2-7 complex. The present study examines the role of the Saccharomyces cerevisiae Mcm2 subunit in regulating the activities of Mcm complexes. Using in vitro assays for DNA unwinding and DNA binding with E. coli-purified Mcm subunits, this work demonstrates that Mcm2 requires nucleotide to actively regulate DNA binding and unwinding by Mcm complexes. These studies define Mcm2 as an active regulatory subunit within the Mcm2-7 complex. Mcm2-7 is also targeted by various kinases that presumably modulate its activities. The Dbf4-dependent kinase (DDK) comprised of the Cdc7 catalytic subunit and Dbf4 regulatory subunit is one such kinase. Here, the residues of Mcm2 targeted for phosphorylation by DDK were mapped using recombinant proteins and verified in cells. The effects of phosphomimetic substitutions at these positions on the activities of the Mcm2-7 complex were examined. Interestingly, the ATPase activity of Mcm2 of the phosphomimetic Mcm2 is lower compared to wild type. A version of Mcm2-7 containing the phopshomimetic mutant of Mcm2 had a higher affinity for DNA, which in turn inhibited DNA unwinding by the complex. The biological function of phosphorylation of Mcm2 by DDK in budding yeast was also examined using cells containing a version of mcm2 that cannot be phosphorylated by DDK. This mutation rendered the cells sensitive to agents that cause DNA base damage. Additionally, the mutant allele interacted with genes involved in the DNA damage checkpoint as determined by synthetic genetic array analysis. In sum, a model in which DDK-dependent phosphorylation of Mcm2 regulates its ATPase activity to slow replication forks in the cell’s response to DNA damage is proposed

Contouring Trivariate Data

In many applications, the data consist of discrete 3-D points at which one or more parameters are given. To display contours, the data are represented by a continuous function which is evaluated at any point as needed for contouring. Contouring results are presented which are applicable both to artitrarily spaced data and to data which lie on a topologically rectangular three dimensional grid. the contours are assumed to be described by 3-D display lists for viewing on a dynamic color graphics device; that is, they will not simply be projected into 2-D and viewed as a static image on a frame buffer. Dynamic viewing of color contours may be essential to the proper interpretation of results. The gridded data lie on a topologically rectangular grid although two or more nodes may be the same point. Parametric tensor product methods may be used to fit the gridded data and to generate the contours. Rectangular elements are convenient but not necessary. For example, there are other methods which are effective for contouring over tetrahedral elements

Incorporating salal berry (Gaultheria shallon) and blackcurrant (Ribes nigrum) pomace in yogurt for the development of a beverage with antidiabetic properties

This work is part of the Strategic Research Programme 2016–2021 and is funded by the Scottish Government's Rural and Environment Science and Analytical Services Division (RESAS).Peer reviewedPublisher PD

The forgotten pirates: Iraqi and Iranian pirates in the Arabian gulf who have never been reported to the international maritime bureau

If piracy attacks are unreported, a misleading impression is given of piracy situations in regions where there could be serious consequences for ships traveling in waterways on the assumption that they are piracy-free waters. However, sometimes not reporting piracy attacks could help to contain piracy before it expands, because reporting can lead to the media over-focusing on piratical incidents, and armed guards being deployed on ships, which causes pirates to use heavier arms and escalates the level of conflict. Piracy that took place during 2003–2012 in the north and the center of the Arabian Gulf has never been reported to the International Maritime Bureau. The present article examines this case of unreporting, and discusses its causes and consequences

The effects of oligomerization on Saccharomyces cerevisiae Mcm4/6/7 function

<p>Abstract</p> <p>Background</p> <p>Minichromosome maintenance proteins (Mcm) 2, 3, 4, 5, 6 and 7 are related by sequence and form a variety of complexes that unwind DNA, including Mcm4/6/7. A Mcm4/6/7 trimer forms one half of the Mcm2-7 hexameric ring and can be thought of as the catalytic core of Mcm2-7, the replicative helicase in eukaryotic cells. Oligomeric analysis of Mcm4/6/7 suggests that it forms a hexamer containing two Mcm4/6/7 trimers, however, under certain conditions trimeric Mcm4/6/7 has also been observed. The functional significance of the different Mcm4/6/7 oligomeric states has not been assessed. The results of such an assessment would have implications for studies of both Mcm4/6/7 and Mcm2-7.</p> <p>Results</p> <p>Here, we show that <it>Saccharomyces cerevisiae </it>Mcm4/6/7 reconstituted from individual subunits exists in an equilibrium of oligomeric forms in which smaller oligomers predominate in the absence of ATP. In addition, we found that ATP, which is required for Mcm4/6/7 activity, shifts the equilibrium towards larger oligomers, likely hexamers of Mcm4/6/7. ATPγS and to a lesser extent ADP also shift the equilibrium towards hexamers. Study of Mcm4/6/7 complexes containing mutations that interfere with the formation of inter-subunit ATP sites (arginine finger mutants) indicates that full activity of Mcm4/6/7 requires all of its ATP sites, which are formed in a hexamer and not a trimer. In keeping with this observation, Mcm4/6/7 binds DNA as a hexamer.</p> <p>Conclusions</p> <p>The minimal functional unit of Mcm4/6/7 is a hexamer. One of the roles of ATP binding by Mcm4/6/7 may be to stabilize formation of hexamers.</p

Experimental Study of Wake / Flap Interaction Noise and the Reduction of Flap Side Edge Noise

The effects of the interaction of a wake with a half-span flap on radiated noise are examined. The incident wake is generated by bars of various widths and lengths or by a simplified landing gear model. Single microphone and phased array measurements are used to isolate the effects of the wake interaction on the noise radiating from the flap side edge and flap cove regions. The effects on noise of the wake generator's geometry and relative placement with respect to the flap are assessed. Placement of the wake generators upstream of the flap side edge is shown to lead to the reduction of flap side edge noise by introducing a velocity deficit and likely altering the instabilities in the flap side edge vortex system. Significant reduction in flap side edge noise is achieved with a bar positioned directly upstream of the flap side edge. The noise reduction benefit is seen to improve with increased bar width, length and proximity to the flap edge. Positioning of the landing gear model upstream of the flap side edge also leads to decreased flap side edge noise. In addition, flap cove noise levels are significantly lower than when the landing gear is positioned upstream of the flap mid-span. The impact of the local flow velocity on the noise radiating directly from the landing gear is discussed. The effects of the landing gear side-braces on flap side edge, flap cove and landing gear noise are shown

Towards a classification of Australian Aboriginal stone arrangements : an investigation of methodological problems with a gazetteer of selected sites

A classification of Australian Aboriginal stone arrangements is fundamental to the understanding of their function and social significance for both Australian and world prehistory. The implications of certain problems with the archaeological data for a classification of arrangements, such as dating and inadequate reporting, are discussed. Possible principles governing the mode of construction, design and location of arrangements are investigated, and the criteria for classification suggested. A two-tier classification is proposed. On the first level, the technological and morphological characteristics of discrete stone arrangements are organised into classes. On the second level, the combination of arrangement classes at any one site defines site types. 144 sites in four regions in New South Wales are classified. Comparisons are made between classes and site types within each region and across regions. Existing opinions about the distribution of so-called 'simple' and 'complex' types are challenged. An investigation of the relationship between classes or site types, and other kinds of archaeological sites, such as rock art, reveals no perfect correlations either within one region or across regions. It is proposed that any governing principles are more likely to have operated at a local level, reflecting such factors as local topography, beliefs and traditions, and population density, rather than at a universal level. The significance of a classification of stone arrangements for studies on culture areas, and on complex Aboriginal hunter-gathering is discussed. Further research is proposed with regard to the former. The construction and location of many arrangements is regarded as evidence for a considerable investment of time and energy in non-subsistence activities. It is suggested that these stone arrangements are associated with the archaeological evidence identified by Australian and overseas researchers, for an increasingly more complex stage in the evolution of hunter-gatherers, in which ceremonial and ritual requirements were paramount