PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-8 (8)
 

Clipboard (0)
None

Select a Filter Below

Journals
Year of Publication
Document Types
1.  Experimental and computational analysis of DNA unwinding and polymerization kinetics 
DNA unwinding and polymerization are complex processes involving many intermediate species in the reactions. Our understanding of these processes is limited because the rates of the reactions or the existence of intermediate species is not apparent without specially designed experimental techniques and data analysis procedures. In this chapter we describe how pre-steady state single-turnover measurements analyzed by model-based methods can be used for estimating the elementary rate constants. Using the hexameric helicase and the DNA polymerase from bacteriophage T7 as model systems, we provide stepwise procedures for measuring the kinetics of the reactions they catalyze based on radioactivity and fluorescence. We also describe analysis of the experimental measurements using publicly available models and software gfit (http://gfit.sf.net).
doi:10.1007/978-1-60327-355-8_5
PMCID: PMC3787510  PMID: 20225142
Hexameric helicase; Replication; DNA unwinding; T7 bacteriophage; DNA polymerase; DNA synthesis; Strand displacement; Primer extension; gfit; global regression analysis
2.  Haplotype Association Mapping Identifies a Candidate Gene Region in Mice Infected With Staphylococcus aureus 
G3: Genes|Genomes|Genetics  2012;2(6):693-700.
Exposure to Staphylococcus aureus has a variety of outcomes, from asymptomatic colonization to fatal infection. Strong evidence suggests that host genetics play an important role in susceptibility, but the specific host genetic factors involved are not known. The availability of genome-wide single nucleotide polymorphism (SNP) data for inbred Mus musculus strains means that haplotype association mapping can be used to identify candidate susceptibility genes. We applied haplotype association mapping to Perlegen SNP data and kidney bacterial counts from Staphylococcus aureus-infected mice from 13 inbred strains and detected an associated block on chromosome 7. Strong experimental evidence supports the result: a separate study demonstrated the presence of a susceptibility locus on chromosome 7 using consomic mice. The associated block contains no genes, but lies within the gene cluster of the 26-member extended kallikrein gene family, whose members have well-recognized roles in the generation of antimicrobial peptides and the regulation of inflammation. Efficient mixed-model association (EMMA) testing of all SNPs with two alleles and located within the gene cluster boundaries finds two significant associations: one of the three polymorphisms defining the associated block and one in the gene closest to the block, Klk1b11. In addition, we find that 7 of the 26 kallikrein genes are differentially expressed between susceptible and resistant mice, including the Klk1b11 gene. These genes represent a promising set of candidate genes influencing susceptibility to Staphylococcus aureus.
doi:10.1534/g3.112.002501
PMCID: PMC3362298  PMID: 22690378
host genetic susceptibility; infectious disease; kallikrein gene family
3.  Mechanism of ATP-driven PCNA clamp loading by S. cerevisiae RFC 
Journal of molecular biology  2009;388(3):431-442.
Circular clamps tether polymerases to DNA, serving as essential processivity factors in genome replication, and function in other critical cellular processes as well. Clamp loaders catalyze clamp assembly onto DNA, and the question of how these proteins construct a topological link between a clamp and DNA remains open, especially the mechanism by which ATP is utilized for the task. Here we describe pre-steady state analysis of ATP hydrolysis, PCNA clamp opening and DNA binding by S. cerevisiae RFC, and present the first kinetic model of a eukaryotic clamp loading reaction validated by global data analysis. ATP binding to multiple RFC subunits initiates a slow conformational change in the clamp loader, enabling it to bind and open PCNA, and bind DNA as well. PCNA opening locks RFC into an active state, and the resulting RFC•ATP•PCNA(open) intermediate is ready for entry of DNA into the clamp. DNA binding commits RFC to ATP hydrolysis, which is followed by PCNA closure and PCNA•DNA release. This model enables quantitative understanding of the multi-step mechanism of a eukaryotic clamp loader, and furthermore facilitates comparative analysis of loaders from diverse organisms.
doi:10.1016/j.jmb.2009.03.014
PMCID: PMC2700029  PMID: 19285992
Processive DNA replication; RFC clamp loader; PCNA clamp; ATPase kinetics
4.  Model-Based Global Analysis of Heterogeneous Experimental Data Using gfit 
Summary
Regression analysis is indispensible for quantitative understanding of biological systems and for developing accurate computational models. By applying regression analysis, one can validate models and quantify components of the system, including ones that cannot be observed directly. Global (simultaneous) analysis of all experimental data available for the system produces the most informative results. To quantify components of a complex system, the dataset needs to contain experiments of different types performed under a broad range of conditions. However, heterogeneity of such datasets complicates implementation of the global analysis. Computational models continuously evolve to include new knowledge and to account for novel experimental data, creating the demand for flexible and efficient analysis procedures. To address these problems, we have developed gfit software to globally analyze many types of experiments, to validate computational models, and to extract maximum information from the available experimental data.
doi:10.1007/978-1-59745-525-1_12
PMCID: PMC2850822  PMID: 19399438
Regression analysis; Computational model; Curve fitting; MATLAB; Computer simulation; Least-squares
5.  Multiplexed RNA trafficking in oligodendrocytes and neurons 
Biochimica et biophysica acta  2008;1779(8):453-458.
Summary
In oligodendrocytes and neurons genetic information is transmitted from nucleus to dendrites in the form of RNA granules. Here we describe how transport of multiple different RNA molecules in individual granules is analogous to the process of multiplexing in telecommunications. In both cases multiple messages are combined into a composite signal for transmission on a single carrier. Multiplexing provides a mechanism to coordinate local expression of ensembles of genes in myelin in oligodendrocytes and at synapses in neurons.
doi:10.1016/j.bbagrm.2008.04.002
PMCID: PMC2584806  PMID: 18442491
HnRNP A2; myelin; RNA granules; synapse; TOG
6.  Multiplexed Dendritic Targeting of α Calcium Calmodulin-dependent Protein Kinase II, Neurogranin, and Activity-regulated Cytoskeleton-associated Protein RNAs by the A2 Pathway 
Molecular Biology of the Cell  2008;19(5):2311-2327.
In neurons, many different RNAs are targeted to dendrites where local expression of the encoded proteins mediates synaptic plasticity during learning and memory. It is not known whether each RNA follows a separate trafficking pathway or whether multiple RNAs are targeted to dendrites by the same pathway. Here, we show that RNAs encoding α calcium calmodulin-dependent protein kinase II, neurogranin, and activity-regulated cytoskeleton-associated protein are coassembled into the same RNA granules and targeted to dendrites by the same cis/trans-determinants (heterogeneous nuclear ribonucleoprotein [hnRNP] A2 response element and hnRNP A2) that mediate dendritic targeting of myelin basic protein RNA by the A2 pathway in oligodendrocytes. Multiplexed dendritic targeting of different RNAs by the same pathway represents a new organizing principle for coordinating gene expression at the synapse.
doi:10.1091/mbc.E07-09-0914
PMCID: PMC2366844  PMID: 18305102
7.  DNA synthesis by a polymerase provides the driving force to accelerate DNA unwinding by a helicase 
Nature  2005;435(7040):370-373.
Helicases are molecular motors that use the energy of NTP hydrolysis to translocate along a nucleic acid strand and catalyze reactions such as DNA unwinding. The ring-shaped helicase1 of bacteriophage T7 translocates along single stranded (ss) DNA at a speed of 130 base per second2. However, T7 helicase slows down nearly 10-fold when unwinding the strands of duplex DNA3. Here we report that T7 DNA polymerase, unable to catalyze strand displacement DNA synthesis by itself, can increase the unwinding rate to 114 base pairs per second, bringing the helicase to similar speeds as along ssDNA. The helicase-rate stimulation depends upon the DNA synthesis rate and does not rely on specific interactions between the helicase and the polymerase. Efficient duplex DNA synthesis is achieved only by the combined action of the helicase and polymerase. The DNA polymerase depends on the unwinding activity of the helicase that provides ssDNA template. The rapid trapping of the ssDNA bases by the DNA synthesis activity of the polymerase in turn drives the helicase to move forward through duplex DNA at speeds similar to those observed along ssDNA.
doi:10.1038/nature03615
PMCID: PMC1563444  PMID: 15902262
8.  SIGNIFICANT PROPORTIONS OF NUCLEAR TRANSPORT PROTEINS WITH REDUCED INTRACELLULAR MOBILITIES RESOLVED BY FLUORESCENCE CORRELATION SPECTROSCOPY 
Journal of molecular biology  2006;365(1):50-65.
Nuclear transport requires freely diffusing nuclear transport proteins to facilitate movement of cargo molecules through the nuclear pore. We analyzed dynamic properties of importin α, importin β, Ran and NTF2 in nucleus, cytoplasm and at the nuclear pore of neuroblastoma cells using fluorescence correlation spectroscopy. Mobile components were quantified by global fitting of autocorrelation data from multiple cells. Immobile components were quantified by analysis of photobleaching kinetics. Wild type Ran was compared to various mutant Ran proteins to identify components representing GTP or GDP forms of Ran. Untreated cells were compared to cells treated with nocodazole or latrunculin to identify components associated with cytoskeletal elements. The results indicate that freely diffusing importin α, importin β, Ran and NTF2 are in dynamic equilibrium with larger pools associated with immobile binding partners such as microtubules in the cytoplasm. These findings suggest that formation of freely diffusing nuclear transport intermediates is in competition with binding to immobile partners. Variation in concentrations of freely diffusing nuclear transport intermediates among cells indicates that the nuclear transport system is sufficiently robust to function over a wide range of conditions.
doi:10.1016/j.jmb.2006.09.08
PMCID: PMC1831836  PMID: 17056062
importin; Ran; NTF2; fluorescence correlation spectroscopy; nuclear pore complex

Results 1-8 (8)