scholarly journals Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

10.3791/55999 ◽  
2017 ◽  
Author(s):  
Soňa Legartová ◽  
Jana Suchánková ◽  
Jana Krejčí ◽  
Alena Kovaříková ◽  
Eva Bártová
Keyword(s):  
Confocal Microscopy ◽  
Protein Interactions ◽  
Dna Lesions ◽  
Protein Protein Interactions ◽  
Microscopy Techniques

scholarly journals Protein–Protein Interactions in Translesion Synthesis

Molecules ◽  
2021 ◽  
Vol 26 (18) ◽  
pp. 5544
Author(s):  
Radha Charan Dash ◽  
Kyle Hadden
Keyword(s):  
Protein Interactions ◽  
Acquired Resistance ◽  
Translesion Synthesis ◽  
Initial Response ◽  
Structural Features ◽  
Dna Lesions ◽  
Protein Protein Interactions ◽  
Dna Damage Tolerance ◽  
Response To Chemotherapy ◽  
Depth Analysis

Translesion synthesis (TLS) is an error-prone DNA damage tolerance mechanism used by actively replicating cells to copy past DNA lesions and extend the primer strand. TLS ensures that cells continue replication in the presence of damaged DNA bases, albeit at the expense of an increased mutation rate. Recent studies have demonstrated a clear role for TLS in rescuing cancer cells treated with first-line genotoxic agents by allowing them to replicate and survive in the presence of chemotherapy-induced DNA lesions. The importance of TLS in both the initial response to chemotherapy and the long-term development of acquired resistance has allowed it to emerge as an interesting target for small molecule drug discovery. Proper TLS function is a complicated process involving a heteroprotein complex that mediates multiple attachment and switching steps through several protein–protein interactions (PPIs). In this review, we briefly describe the importance of TLS in cancer and provide an in-depth analysis of key TLS PPIs, focusing on key structural features at the PPI interface while also exploring the potential druggability of each key PPI.

scholarly journals Novel Role for the C Terminus of Saccharomyces cerevisiae Rev1 in Mediating Protein-Protein Interactions

2006 ◽  
Vol 26 (21) ◽  
pp. 8173-8182 ◽  
Author(s):  
Sanjay D'Souza ◽  
Graham C. Walker
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Protein Interactions ◽  
Sequence Similarity ◽  
Dna Lesions ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Protein Protein Interactions ◽  
C Terminus ◽  
Negative Effect

ABSTRACT The Saccharomyces cerevisiae REV3/7-encoded polymerase ζ and Rev1 are central to the replicative bypass of DNA lesions, a process called translesion synthesis (TLS). While yeast polymerase ζ extends from distorted DNA structures, Rev1 predominantly incorporates C residues from across a template G and a variety of DNA lesions. Intriguingly, Rev1 catalytic activity does not appear to be required for TLS. Instead, yeast Rev1 is thought to participate in TLS by facilitating protein-protein interactions via an N-terminal BRCT motif. In addition, higher eukaryotic homologs of Rev1 possess a C terminus that interacts with other TLS polymerases. Due to a lack of sequence similarity, the yeast Rev1 C-terminal region, located after the polymerase domain, had initially been thought not to play a role in TLS. Here, we report that elevated levels of the yeast Rev1 C terminus confer a strong dominant-negative effect on viability and induced mutagenesis after DNA damage, highlighting the crucial role that the C terminus plays in DNA damage tolerance. We show that this phenotype requires REV7 and, using immunoprecipitations from crude extracts, demonstrate that, in addition to the polymerase-associated domain, the extreme Rev1 C terminus and the BRCT region of Rev1 mediate interactions with Rev7.

Hybrid Detectors Improved Time-Lapse Confocal Microscopy of PML and 53BP1 Nuclear Body Colocalization in DNA Lesions

2013 ◽  
Vol 19 (2) ◽  
pp. 360-369 ◽  
Author(s):  
Veronika Foltánková ◽  
Pavel Matula ◽  
Dmitry Sorokin ◽  
Stanislav Kozubek ◽  
Eva Bártová
Keyword(s):  
Confocal Microscopy ◽  
Protein Interactions ◽  
Laser Scanning ◽  
Nuclear Body ◽  
Dna Lesions ◽  
Nuclear Bodies ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Γ Irradiation ◽  
Pml Nuclear Bodies

AbstractWe used hybrid detectors (HyDs) to monitor the trajectories and interactions of promyelocytic leukemia (GFP-PML) nuclear bodies (NBs) and mCherry-53BP1-positive DNA lesions. 53BP1 protein accumulates in NBs that occur spontaneously in the genome or in γ-irradiation-induced foci. When we induced local DNA damage by ultraviolet irradiation, we also observed accumulation of 53BP1 proteins into discrete bodies, instead of the expected dispersed pattern. In comparison with photomultiplier tubes, which are used for standard analysis by confocal laser scanning microscopy, HyDs significantly eliminated photobleaching of GFP and mCherry fluorochromes during image acquisition. The low laser intensities used for HyD-based confocal analysis enabled us to observe NBs for the longer time periods, necessary for studies of the trajectories and interactions of PML and 53BP1 NBs. To further characterize protein interactions, we used resonance scanning and a novel bioinformatics approach to register and analyze the movements of individual PML and 53BP1 NBs. The combination of improved HyD-based confocal microscopy with a tailored bioinformatics approach enabled us to reveal damage-specific properties of PML and 53BP1 NBs.

scholarly journals Structural basis of the XPB–Bax1 complex as a dynamic helicase–nuclease machinery for DNA repair

2020 ◽  
Vol 48 (11) ◽  
pp. 6326-6339 ◽  
Author(s):  
Kevin DuPrez ◽  
Feng He ◽  
Zhenhang Chen ◽  
Eduardo Hilario ◽  
Li Fan
Keyword(s):  
Dna Repair ◽  
Protein Interactions ◽  
Active Sites ◽  
Atp Hydrolysis ◽  
Excision Repair ◽  
Dna Lesions ◽  
Structural Basis ◽  
Protein Protein Interactions ◽  
Nucleotide Excision ◽  
First Time

Abstract Nucleotide excision repair (NER) is a major DNA repair pathway for a variety of DNA lesions. XPB plays a key role in DNA opening at damage sites and coordinating damage incision by nucleases. XPB is conserved from archaea to human. In archaea, XPB is associated with a nuclease Bax1. Here we report crystal structures of XPB in complex with Bax1 from Archaeoglobus fulgidus (Af) and Sulfolobus tokodaii (St). These structures reveal for the first time four domains in Bax1, which interacts with XPB mainly through its N-terminal domain. A Cas2-like domain likely helps to position Bax1 at the forked DNA allowing the nuclease domain to incise one arm of the fork. Bax1 exists in monomer or homodimer but forms a heterodimer exclusively with XPB. StBax1 keeps StXPB in a closed conformation and stimulates ATP hydrolysis by XPB while AfBax1 maintains AfXPB in the open conformation and reduces its ATPase activity. Bax1 contains two distinguished nuclease active sites to presumably incise DNA damage. Our results demonstrate that protein-protein interactions regulate the activities of XPB ATPase and Bax1 nuclease. These structures provide a platform to understand the XPB-nuclease interactions important for the coordination of DNA unwinding and damage incision in eukaryotic NER.

Protein-protein interactions of the p53 family with the Bcl-2 family in hepatocellular carcinoma

2011 ◽  
Vol 49 (08) ◽  
Author(s):  
LC König ◽  
M Meinhard ◽  
C Sandig ◽  
MH Bender ◽  
A Lovas ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Protein Interactions ◽  
Protein Protein Interactions ◽  
P53 Family

Partial Purification of a Specific Plasminogen Activator from Porcine Parotid Glands

1974 ◽  
Vol 31 (03) ◽  
pp. 403-414 ◽  
Author(s):  
Terence Cartwright
Keyword(s):  
Nucleic Acid ◽  
Salivary Glands ◽  
Plasminogen Activator ◽  
Protein Interactions ◽  
Partial Purification ◽  
Protein Protein Interactions ◽  
Parotid Glands ◽  
Two Stage ◽  
Preliminary Characterization ◽  
Specific Inhibitors

SummaryA method is described for the extraction with buffers of near physiological pH of a plasminogen activator from porcine salivary glands. Substantial purification of the activator was achieved although this was to some extent complicated by concomitant extraction of nucleic acid from the glands. Preliminary characterization experiments using specific inhibitors suggested that the activator functioned by a similar mechanism to that proposed for urokinase, but with some important kinetic differences in two-stage assay systems. The lack of reactivity of the pig gland enzyme in these systems might be related to the tendency to protein-protein interactions observed with this material.

Target-Templated de novo Design of Macrocyclic D-/L-Peptides: Inhibitors of the PD-1/PD-L1 Interaction

2020 ◽  
Author(s):  
Salvador Guardiola ◽  
Monica Varese ◽  
Xavier Roig ◽  
Jesús Garcia ◽  
Ernest Giralt
Keyword(s):  
Protein Interactions ◽  
Cyclic Peptides ◽  
General Framework ◽  
Large Scale ◽  
De Novo ◽  
Inhibitory Effect ◽  
Original Text ◽  
Protein Protein Interactions ◽  
Retraction Notice ◽  
Pharmaceutical Properties

<p>NOTE: This preprint has been retracted by consensus from all authors. See the retraction notice in place above; the original text can be found under "Version 1", accessible from the version selector above.</p><p><br></p><p>------------------------------------------------------------------------</p><p><br></p><p>Peptides, together with antibodies, are among the most potent biochemical tools to modulate challenging protein-protein interactions. However, current structure-based methods are largely limited to natural peptides and are not suitable for designing target-specific binders with improved pharmaceutical properties, such as macrocyclic peptides. Here we report a general framework that leverages the computational power of Rosetta for large-scale backbone sampling and energy scoring, followed by side-chain composition, to design heterochiral cyclic peptides that bind to a protein surface of interest. To showcase the applicability of our approach, we identified two peptides (PD-<i>i</i>3 and PD-<i>i</i>6) that target PD-1, a key immune checkpoint, and work as protein ligand decoys. A comprehensive biophysical evaluation confirmed their binding mechanism to PD-1 and their inhibitory effect on the PD-1/PD-L1 interaction. Finally, elucidation of their solution structures by NMR served as validation of our <i>de novo </i>design approach. We anticipate that our results will provide a general framework for designing target-specific drug-like peptides.<i></i></p>

Target-Templated de novo Design of Macrocyclic D-/L-Peptides: Inhibitors of the PD-1/PD-L1 Interaction

2020 ◽  
Author(s):  
Salvador Guardiola ◽  
Monica Varese ◽  
Xavier Roig ◽  
Jesús Garcia ◽  
Ernest Giralt
Keyword(s):  
Protein Interactions ◽  
Cyclic Peptides ◽  
General Framework ◽  
Large Scale ◽  
De Novo ◽  
Inhibitory Effect ◽  
Original Text ◽  
Protein Protein Interactions ◽  
Retraction Notice ◽  
Pharmaceutical Properties

<p>NOTE: This preprint has been retracted by consensus from all authors. See the retraction notice in place above; the original text can be found under "Version 1", accessible from the version selector above.</p><p><br></p><p>------------------------------------------------------------------------</p><p><br></p><p>Peptides, together with antibodies, are among the most potent biochemical tools to modulate challenging protein-protein interactions. However, current structure-based methods are largely limited to natural peptides and are not suitable for designing target-specific binders with improved pharmaceutical properties, such as macrocyclic peptides. Here we report a general framework that leverages the computational power of Rosetta for large-scale backbone sampling and energy scoring, followed by side-chain composition, to design heterochiral cyclic peptides that bind to a protein surface of interest. To showcase the applicability of our approach, we identified two peptides (PD-<i>i</i>3 and PD-<i>i</i>6) that target PD-1, a key immune checkpoint, and work as protein ligand decoys. A comprehensive biophysical evaluation confirmed their binding mechanism to PD-1 and their inhibitory effect on the PD-1/PD-L1 interaction. Finally, elucidation of their solution structures by NMR served as validation of our <i>de novo </i>design approach. We anticipate that our results will provide a general framework for designing target-specific drug-like peptides.<i></i></p>

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