1,720,967 research outputs found
Structural basis for DNA 3'-end processing by human Tyrosyl-DNA phosphodiesterase 1
No full textTyrosyl-DNA phosphodiesterase (Tdp1) is a DNA 3'-end processing enzyme that repairs topoisomerase 1B-induced DNA damage. We use a new tool combining site-specific DNA-protein cross-linking with mass spectrometry to identify Tdp1 interactions with DNA. A conserved phenylalanine (F259) of Tdp1, required for efficient DNA processing in biochemical assays, cross-links to defined positions in DNA substrates. Crystal structures of Tdp1-DNA complexes capture the DNA repair machinery after 3'-end cleavage; these reveal how Tdp1 coordinates the 3'-phosphorylated product of nucleosidase activity and accommodates duplex DNA. A hydrophobic wedge splits the DNA ends, directing the scissile strand through a channel towards the active site. The F259 side-chain stacks against the -3 base pair, delimiting the junction of duplexed and melted DNA, and fixes the scissile strand in the channel. Our results explain why Tdp1 cleavage is non-processive and provide a molecular basis for DNA 3'-end processing by Tdp1.Most of the data files are mass spectrometry datasets which are organised according to the figure panels in the manuscript that they feed into.
## Zip files ##
The .zip files contain .d directories in Bruker format, produced by mass spectrometry equipment; these directories (once unzipped) can be opened using Bruker DataAnalysis, which is commercial software widely used by mass spectrometry research groups.
### Figure 3C + D ###
* For control oligo - Filename: "Hei62-3 trunk H263A xlink_RG6_01_3508.d.zip"
* For -2 5IdU oligo - Filename: "Hei66-4 trunk H263A xlink_RG7_01_3491.d.zip"
* For -3 5IdU oligo - Filename: "Hei77-1 trunk H263A xlink_RG8_01_3495.d.zip"
### Figure 3E ###
* For control oligo - Filename: "Hei62A16_000001.d.zip"
* For -2 5IdU oligo - Filename: "Hei66A16_000001.d.zip"
* For -3 5IdU oligo - Filename: "Hei77A 16_000002.d.zip"
### Figure 3F ###
* For -2 5IdU oligo - Filename: "Hei66A16 iso 740 cid 20 V_000002.d.zip"
* For -3 5IdU oligo - Filename: "Hei77A 16 iso 740 cid 20V_000001.d.zip"
## Image files ##
In addition there are a few image files, again organised by figure panel:
### Figure 3B ###
* Upper panel - Filename: "ff161214 Gels 1-2-3-4.png"
* Lower panel - Filename: "ff161214 Coomassie.JPG"
### Figure 6B ###
* "Figure 6B.png" (image file derived from ff020715.gel)
* "ff020715.gel" (Original ImageQuant file, used for quantitation of data
Structural basis for DNA 3'-end processing by human Tyrosyl-DNA phosphodiesterase 1
No full textMost of the data files are mass spectrometry datasets which are organised according to the figure panels in the manuscript that they feed into.Tyrosyl-DNA phosphodiesterase (Tdp1) is a DNA 3'-end processing enzyme that repairs topoisomerase 1B-induced DNA damage. We use a new tool combining site-specific DNA-protein cross-linking with mass spectrometry to identify Tdp1 interactions with DNA. A conserved phenylalanine (F259) of Tdp1, required for efficient DNA processing in biochemical assays, cross-links to defined positions in DNA substrates. Crystal structures of Tdp1-DNA complexes capture the DNA repair machinery after 3'-end cleavage; these reveal how Tdp1 coordinates the 3'-phosphorylated product of nucleosidase activity and accommodates duplex DNA. A hydrophobic wedge splits the DNA ends, directing the scissile strand through a channel towards the active site. The F259 side-chain stacks against the -3 base pair, delimiting the junction of duplexed and melted DNA, and fixes the scissile strand in the channel. Our results explain why Tdp1 cleavage is non-processive and provide a molecular basis for DNA 3'-end processing by Tdp1.Flett, Fiona J; Interthal, Heidrun; Mackay, Logan. (2017). Structural basis for DNA 3'-end processing by human Tyrosyl-DNA phosphodiesterase 1, [dataset]. University of Edinburgh. School of Biological Sciences. Intitute of Cell Biology. http://dx.doi.org/10.7488/ds/2243
Chemical genetic screen for inhibitors of human telomerase
Get PDFThere remains a pressing need for the development of effective drugs that
meet the clinical needs for cancer treatment, and inhibition of telomere
length maintenance by disrupting human telomerase is a proven and
tractable target for suppression of cancer cell growth. In response to the lack
of currently available small molecules with efficacy against human
telomerase, we developed a genetically and chemically tractable cell-based
system in which S. cerevisiae is used to streamline the search for novel human
telomerase inhibitors. Our results confirmed that yeast cell growth was
rapidly inhibited upon induction of functional human telomerase at the
telomere. This inducible growth arrest was used as a read-out for a high-throughput
chemical screen for human telomerase inhibitors based on their
ability to restore growth in the yeast system. From a library consisting of
small, bioactive and cell-permeable compounds of diverse structure, we
identified three novel “drug-like” compounds that inhibited the activity of
native and recombinant telomerase complexes in vitro. “Validation assays”
also confirmed the novel inhibitors were free of uncharacterized adverse
effects against yeast and human cell models, thus confirming the specificity
of these novel inhibitors against human telomerase target. This surrogate
yeast model has therefore proven to be a cost-effective alternative to
accelerate the search for human telomerase inhibitors, which we hope will
serve to streamline the identification of further lead compounds effective
against human cancer
Spatial regulation of microtubule-associated proteins by 14-3-3 in oocytes
Get PDFOocytes have a large volume and yet must assemble a bipolar meiotic spindle specifically around the chromosomes. Recently, 14-3-3 was discovered to spatially regulate the spindle assembly factor Kinesin-14/Ncd in fly oocytes. 14-3-3 is also known to spatially regulate another kinesin, Mklp1/Pavarotti, in mitosis. I hypothesise that other meiotic targets of this regulatory pathway may also exist. To test this, I established a new method to cosediment microtubules and associated proteins (MAPs) from fly ovaries. I discovered that over fifty proteins significantly changed in their microtubule-binding activity when 14-3-3 was inhibited in this assay. As well as the two previously reported 14-3-3 interactors, I identified candidate proteins to test for 14-3-3 regulation, including the CPC (chromosomal passenger complex) components Borealin and Incenp, and seven candidates not previously known to have a role in oocyte meiosis.
By expressing shRNA against the seven candidates without known meiotic function, I identified five which produced spindle or oogenesis defects, suggesting that they may have a role in spindle assembly in oocytes. Slender Lobes protein localised to the spindle in live imaging, and RNAi of slender lobes led to spindle pole focusing defects.
Similarly, the microtubule depolymeriser Stathmin may be required for spindle bipolarity.
Borealin is already known to have a role in targeting CPC at the spindle. I identified a candidate 14-3-3 binding site in Borealin with similarity to those of Ncd and Pavarotti. In vitro pulldowns showed that 14-3-3 binds this region of Borealin in a phosphorylation-dependent manner, and that this binding is inhibited by Aurora B phosphorylation, in keeping with the proposed model for 14-3-3 regulation of Ncd.
Additionally, I found that this region of Borealin is competent to bind microtubules in vitro, independent of its previously reported microtubule-binding site or interaction with other members of the CPC.
Finally, a mutation of this binding site caused the decreased localisation of Borealin to the spindle and centromeres, and chromosome biorientation defects in oocytes.
Overall, I propose that 14-3-3 is an important regulator of spindle assembly in oocytes, by spatially regulating microtubule binding of multiple spindle MAPs
Replication arrest and bypass at Tus/ter complexes in the terminus of the Escherichia coli chromosome
Get PDFGenome replication is frequently challenged by obstacles that can result from DNA
damage, topological stress or tightly bound proteins. Replication fork stalling at DNA-bound
proteins can lead to collapse of the fork and promote mutation and genomic instability, a
hallmark of cancer cells. Interaction between replisomes and naturally occurring barriers can
provide important information for understanding genome instability mechanisms.
The simplicity of the Escherichia coli chromosome replication is ideal for studies of
complex interactions between replication forks and replication barriers. E. coli carries a single
chromosome that encodes a single origin of bidirectional replication, oriC, and a region
diametrically opposite to oriC where replication terminates. The terminus region encodes four
23 bp ter sites, terA and terD on right replichore and terC and terB on the left. A ter sequence
bound by Tus protein acts as a polar (unidirectional) natural barrier to fork progression. The
Tus/ter system allows replisomes to enter the terminus, but will arrest their progress into the
opposite replichore, that is, towards oriC.
In this work two dimensional native-native gel electrophoresis was utilized to detect
stalled replication forks at naturally occurring Tus/ter barriers in the E. coli chromosome
terminus. The majority of arrested replication forks were found to accumulate at the first
Tus/ter barrier on the left replichore, Tus/terC. Notably fewer arrested forks were detected at
terA, terB and terD. The strength of ter sites was shown to be independent of the location in
the terminus, whereas the sequence of ter sites was critical. The terB sequence forms the
strongest terminator and restricts frequent replication fork bypass observed at terC. This
correlates with the published data on the strength of nucleoprotein barriers formed by ter
sequences observed in vitro. The presence of a strong terminator on each replichore helps the Tus/ter system prevent unwanted replication to escape the terminus. In the situation where
additional rounds of replication were initiated in the terminus in the absence of the RecG
helicase, most of replication forks were able to bypass Tus/terC barrier and were arrested at
Tus/terB.
Previous studies, in the Michel laboratory, revealed that the UvrD helicase can promote
the bypass of a synthetically introduced Tus/terB replication fork barrier in the middle of the
right replichore, but only as a consequence of RecA-mediated homologous recombination.
The work presented in this thesis shows that, even in the absence of RecA-mediated
homologous recombination, UvrD could promote the bypass of the naturally occurring
Tus/terC (soft) barrier in the chromosome terminus. However, UvrD was unable to promote
fork movement through stronger Tus/terB and Tus/terA barriers in the terminus. The terC and
terB nucleotide sequences differ in three separate segments. I have shown that, one of the
segments outside of the conserved region plays a critical role in the UvrD-dependent
replication fork bypass of the Tus/terC barrier. My results suggest a distinct role of the UvrD
helicase in the alleviation of replication fork stalling at the naturally occurring Tus/terC barrier
in the chromosomal terminus
Quantitative detection of low abundance gene expression products in individual E. coli cells
Get PDFStochastic fluctuations in mRNA and protein copy number between cells are
inevitable during the process gene expression, even when cells carry identical
chromosomes. Such fluctuations are able to impact the phenotypic fate of the cell,
and are known to have greater impact when the copy number of the molecule
involved is low. Additionally, up to 50% of proteins in Escherichia coli are present
in the cell at a level of 10 molecules per cell or fewer (Taniguchi et al. 2010). As
such, quantification of low copy number gene expression products and their
distribution in cellular populations is key in understanding the process of gene
expression. Currently, there are few techniques that allow investigation with the
single cell and single molecule resolution required to study low copy number gene
expression products. This work presents a novel method for protein quantification at
the single molecule level, Quantitative HaloTag-TMR labelling, and uses the
technique to quantify the absolute numbers of the low copy number RecB, RecC and
RecD subunits of the bacterial DNA repair enzyme RecBCD, finding each subunit is
present at between two and eight molecules per cell with mean numbers per cell of
4.9, 4.7 and 4.5 respectively. Additionally single molecule mRNA FISH was used to
quantify the mRNA levels of recB and recD within cells, with means of 0.21 and
0.31 mRNA per cell being observed respectively. Finally this work presents a new
method for use detecting both mRNA and protein simultaneously in individual cells
by combining the HaloTag and FISH protocols to give HaloFISH. This work
introduces two novel techniques that allow for single cell examination of gene
expression, and investigates RecBCD expression at the single molecule level
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
- …
