Formation of noncanonical DNA structures mediated by human ORC4, a protein component of the origin recognition complex

Many genomic sequences, DNA replication origins included, contain specific structural motifs prone to alternative base pairing. Structural rearrangements of DNA require specific environmental conditions and could be favored by chemical agents or proteins. To improve our understanding of alternative conformations of origins and the manner in which they form, we have investigated the effect of DNA-binding, AAA+ protein human ORC4 on single-stranded origin DNA or various oligonucleotides. Here we demonstrate that human ORC4 stimulated formation of inter- and intramolecular T.A.T triplexes and created novel structures, such as homoadenine duplexes. Adenine-based structures were held together by Hoogsteen hydrogen bonds, as demonstrated on 7-deaza-dAMP- or dAMP-containing substrates, and characterized by increased thermal stability. Adenine pairing occurred only in the presence of human ORC4, in a neutral buffer supplemented with ATP and Mg (2+) ions. The protein mutant that could not bind ATP was inactive in this reaction. Since the action of human ORC4 could be biologically important, its potential impact on DNA replication is discussed.     

9 Beyond biogeography: large-scale patterns of distribution and abundance of intertidal marine algae along the us west coast

This research was directed to gather a better understanding about the unicellular alga Chlamydomonas acidophila, one of the 6 algal species found in the Berkeley Pit Lake. Berkeley Pit Lake is a flodded, abandoned pit mine with a pH of 2.7 and high metal concentrations. It has been found that the effective concentrations of metals that limit the growth of C. acidophila by 50% were 9.024 mg/L for Cu2+ and 75.4 mg/L for Zn 2+. We have been able to grow C. acidophila from Berkeley Pit samples at high densities in medium containing 15.36 mg/L Cu2+ and 83.65 mg/L Zn2+. Moreover, this species is able to grow in nutrified Berkeley Pit water, which contains approximately 110 mg/L Cu2+ and 323 mg/L of Zn2+. The hypothesis is that the species found in Berkeley Pit Lake represents a genetic strain adapted to high metal concentrations environments. A comparison between the American Type Culture Collection strain of C. acidophila and the strain collected from Berkeley Pit was made. Growth rate of the two strains in Bold Basil Medium, Modified Acid Medium and Berkeley Pit nutrified water were calculated and compared. Moreover, preliminary investigations of the genome of C.acidophila from the Berkeley Pit Lake were initiated.     

The CFTR M470V gene variant as a potential modifier of COPD severity: study of Serbian population

Chronic obstructive pulmonary disease (COPD) is a complex disease influenced by genetic and environmental factors. Cystic fibrosis transmembrane conductance regulator (CFTR) protein is an important component of the lung tissue homeostasis, involved in the regulation of the rate of mucociliary clearance. As it is known that certain CFTR variants have consequences on the function of CFTR protein, the aim of this study was to examine the possible role of F508del, M470V, Tn locus, and R75Q variants in COPD development and modulation. Total number of 86 COPD patients and 102 control subjects were included in the study. Possible association between COPD susceptibility, severity, and onset of the disease and allele or genotype of four analyzed CFTR variants was examined. No associations were detected between COPD development, onset of the disease and tested CFTR alleles and genotypes. However, VV470 genotype was associated with mild/moderate COPD stages in comparison to severe/very severe ones (OR = 0.29, 95%CI = 0.11-0.80, p = 0.016). Our study showed that patients with VV470 genotype had a 3.4-fold decreased risk for the appearance of severe/very severe COPD symptoms, and the obtained results indicate that this genotype may have a protective role. These results also suggest the importance of studying CFTR gene as a modifier of this disease.     

Human initiation protein Orc4 prefers triple stranded DNA

In higher eukaryotes mechanism of DNA replication origin recognition and binding by origin recognition complex (ORC) is still unknown. Origin transfer studies have shown that origin sites are genetically determined, containing functionally interchangeable modules. One of such modules from the human lamin B2 origin of replication has the ability to adopt unorthodox structure partly composed of intramolecular triplex. Sequences involved in triplex formation coincide with ORC binding sites both in vitro and in vivo. To explore potential significance of unorthodox DNA structures in origin recognition by ORC, we tested DNA binding properties of human ORC subunit 4 (HsOrc4) which has independent DNA binding activity in vitro and similar binding characteristics as ORC holocomplex. Our results demonstrated that DNA binding activity of HsOrc4 depends on length and structure of DNA with triplex being the protein’s preferred binding target. Such feature could play part in origin selection through directing ORC to DNA sequence prone to adopt unorthodox structure.     

Risk-aware limited lookahead control for dynamic resource provisioning in enterprise computing systems

Utility or on-demand computing, a provisioning model where a service provider makes computing infrastructure available to customers as needed, is becoming increasingly common in enterprise computing systems. Realizing this model requires making dynamic, and sometimes risky, resource provisioning and allocation decisions in an uncertain operating environment to maximize revenue while reducing operating cost. This paper develops an optimization framework wherein the resource provisioning problem is posed as one of sequential decision making under uncertainty and solved using a limited lookahead control scheme. The proposed approach accounts for the switching costs incurred during resource provisioning and explicitly encodes risk in the optimization problem. Simulations using workload traces from the Soccer World Cup 1998 web site show that a computing system managed by our controller generates up to 20% more profit than a system without dynamic control while incurring low control overhead.

Power and performance management of virtualized computing environments via lookahead control

There is growing incentive to reduce the power consumed by large-scale data centers that host online services such as banking, retail commerce, and gaming. Virtualization is a promising approach to consolidating multiple online services onto a smaller number of computing resources. A virtualized server environment allows computing resources to be shared among multiple performance-isolated platforms called virtual machines. By dynamically provisioning virtual machines, consolidating the workload, and turning servers on and off as needed, data center operators can maintain the desired quality-of-service (QoS) while achieving higher server utilization and energy efficiency. We implement and validate a dynamic resource provisioning framework for virtualized server environments wherein the provisioning problem is posed as one of sequential optimization under uncertainty and solved using a lookahead control scheme. The proposed approach accounts for the switching costs incurred while provisioning virtual machines and explicitly encodes the corresponding risk in the optimization problem. Experiments using the Trade6 enterprise application show that a server cluster managed by the controller conserves, on average, 22% of the power required by a system without dynamic control while still maintaining QoS goals. Finally, we use trace-based simulations to analyze controller performance on server clusters larger than our testbed, and show how concepts from approximation theory can be used to further reduce the computational burden of controlling large systems.

Noncanonical DNA elements in the lamin B2 origin of DNA replication

DNA replication origins of eukaryotes lack linear replicator elements but contain short (dT)(n) (dA)(n) sequences that could build mutually equivalent unorthodox structures. Here we report that the lamin B2 origin of DNA replication adopts an alternative form characterized by unpaired regions CTTTTTTTTTTCC/GGAAAAAAAAAAG (3900-3912) and CCTTTTTTTTC/GAAAAAAAAGG (4141-4151). Both unpaired regions are resistant to DNase and except in central parts of their homopyrimidine strands are sensitive to single strand-specific chemicals. Interactions that protect central pyrimidines probably stabilize the bubble-like areas. Because DNA fragments containing either one or both bubbles migrate in TBM (89 mm Tris base, 89 mm boric acid, and 2 mm MgCl(2)) PAGE even faster than expected from their linear size, interacting regions are expected to belong to the same molecule. In an origin fragment containing a single bubble, free homopyrimidine strand can only interact with Hoogsteen hydrogen bonding surfaces from a complementary double stranded sequence. Indeed, this origin fragment reacts with triplex preferring antibody. In competition binding experiments control double stranded DNA or single stranded (dT)(40) do not affect origin-antibody interaction, whereas TAT and GGC triplexes exert competitive effect. Because the chosen fragment does not contain potential GGC forming sequences, these experiments confirm that the lamin B2 origin adopts a structure partly composed of intramolecular TAT triads.