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Article: Preparing Excitable Cardiac Papillary Muscle and Cardiac Slices for Functional Analyses.

Palmer, Bradley M / Bell, Stephen P

2022 Volume 13, Page(s) 817205

Abstract: While the reductionist approach has been fruitful in understanding the molecular basis of muscle function, intact excitable muscle preparations are still important as experimental model systems. We present here methods that are useful for preparing ... ...

Abstract	While the reductionist approach has been fruitful in understanding the molecular basis of muscle function, intact excitable muscle preparations are still important as experimental model systems. We present here methods that are useful for preparing cardiac papillary muscle and cardiac slices, which represent macroscopic experimental model systems with fully intact intercellular and intracellular structures. The maintenance of these
Language	English
Publishing date	2022-03-03
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2564217-0
ISSN	1664-042X
ISSN	1664-042X
DOI	10.3389/fphys.2022.817205
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Article ; Online: Distinct RPA functions promote eukaryotic DNA replication initiation and elongation.

Pike, Alexandra M / Friend, Caitlin M / Bell, Stephen P

Nucleic acids research

2023 Volume 51, Issue 19, Page(s) 10506–10518

Abstract: Replication protein A (RPA) binds single-stranded DNA (ssDNA) and serves critical functions in eukaryotic DNA replication, the DNA damage response, and DNA repair. During DNA replication, RPA is required for extended origin DNA unwinding and DNA ... ...

Abstract	Replication protein A (RPA) binds single-stranded DNA (ssDNA) and serves critical functions in eukaryotic DNA replication, the DNA damage response, and DNA repair. During DNA replication, RPA is required for extended origin DNA unwinding and DNA synthesis. To determine the requirements for RPA during these processes, we tested ssDNA-binding proteins (SSBs) from different domains of life in reconstituted Saccharomyces cerevisiae origin unwinding and DNA replication reactions. Interestingly, Escherichia coli SSB, but not T4 bacteriophage Gp32, fully substitutes for RPA in promoting origin DNA unwinding. Using RPA mutants, we demonstrated that specific ssDNA-binding properties of RPA are required for origin unwinding but that its protein-interaction domains are dispensable. In contrast, we found that each of these auxiliary RPA domains have distinct functions at the eukaryotic replication fork. The Rfa1 OB-F domain negatively regulates lagging-strand synthesis, while the Rfa2 winged-helix domain stimulates nascent strand initiation. Together, our findings reveal a requirement for specific modes of ssDNA binding in the transition to extensive origin DNA unwinding and identify RPA domains that differentially impact replication fork function.
MeSH term(s)	DNA Replication ; DNA, Single-Stranded/genetics ; DNA, Single-Stranded/metabolism ; DNA-Binding Proteins/metabolism ; Protein Binding ; Replication Protein A/genetics ; Replication Protein A/metabolism ; Saccharomyces cerevisiae/genetics ; Saccharomyces cerevisiae/metabolism ; Bacteriophage T4/metabolism
Chemical Substances	DNA, Single-Stranded ; DNA-Binding Proteins ; Replication Protein A
Language	English
Publishing date	2023-09-18
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	186809-3
ISSN	1362-4962 ; 1362-4954 ; 0301-5610 ; 0305-1048
ISSN (online)	1362-4962 ; 1362-4954
ISSN	0301-5610 ; 0305-1048
DOI	10.1093/nar/gkad765
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Article: Inorganic phosphate accelerates cardiac myofilament relaxation in response to lengthening.

Wakefield, Jane I / Bell, Stephen P / Palmer, Bradley M

Frontiers in physiology

2022 Volume 13, Page(s) 980662

Abstract: Myocardial relaxation in late systole is enhanced by increasing velocities of lengthening. Given that inorganic phosphate (Pi) can rebind to the force-producing myosin enzyme prior to MgADP release and hasten crossbridge detachment, we hypothesized that ... ...

Abstract	Myocardial relaxation in late systole is enhanced by increasing velocities of lengthening. Given that inorganic phosphate (Pi) can rebind to the force-producing myosin enzyme prior to MgADP release and hasten crossbridge detachment, we hypothesized that myocardial relaxation in late systole would be further enhanced by lengthening in the presence of Pi. Wistar rat left ventricular papillary muscles were attached to platinum clips, placed between a force transducer and a length motor at room temperature, and bathed in Krebs solution with 1.8 mM Ca
Language	English
Publishing date	2022-09-12
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2564217-0
ISSN	1664-042X
ISSN	1664-042X
DOI	10.3389/fphys.2022.980662
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Article ; Online: Rethinking origin licensing.

Bell, Stephen P

eLife

2017 Volume 6

Abstract: Human cells that lack a subunit in their origin recognition complex are viable, which suggests the existence of alternative mechanisms to initiate DNA replication. ...

Abstract	Human cells that lack a subunit in their origin recognition complex are viable, which suggests the existence of alternative mechanisms to initiate DNA replication.
MeSH term(s)	Cell Cycle Proteins/genetics ; DNA Replication/genetics ; DNA-Binding Proteins ; Humans ; Minichromosome Maintenance Proteins/genetics ; Origin Recognition Complex/genetics ; Saccharomyces cerevisiae/genetics ; Xenopus Proteins
Chemical Substances	Cell Cycle Proteins ; DNA-Binding Proteins ; Origin Recognition Complex ; Xenopus Proteins ; Minichromosome Maintenance Proteins (EC 3.6.4.12)
Language	English
Publishing date	2017-01-19
Publishing country	England
Document type	Journal Article ; Comment
ZDB-ID	2687154-3
ISSN	2050-084X ; 2050-084X
ISSN (online)	2050-084X
ISSN	2050-084X
DOI	10.7554/eLife.24052
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Article: Changing protein-DNA interactions promote ORC binding site exchange during replication origin licensing.

Zhang, Annie / Friedman, Larry J / Gelles, Jeff / Bell, Stephen P

bioRxiv : the preprint server for biology

2023

Abstract: During origin licensing, the eukaryotic replicative helicase Mcm2-7 forms head-to-head double hexamers to prime origins for bidirectional replication. Recent single-molecule and structural studies revealed that one molecule of the helicase loader ORC can ...

Abstract	During origin licensing, the eukaryotic replicative helicase Mcm2-7 forms head-to-head double hexamers to prime origins for bidirectional replication. Recent single-molecule and structural studies revealed that one molecule of the helicase loader ORC can sequentially load two Mcm2-7 hexamers to ensure proper head-to-head helicase alignment. To perform this task, ORC must release from its initial high-affinity DNA binding site and "flip" to bind a weaker, inverted DNA site. However, the mechanism of this binding-site switch remains unclear. In this study, we used single-molecule Förster resonance energy transfer (sm-FRET) to study the changing interactions between DNA and ORC or Mcm2-7. We found that the loss of DNA bending that occurs during DNA deposition into the Mcm2-7 central channel increases the rate of ORC dissociation from DNA. Further studies revealed temporally-controlled DNA sliding of helicase-loading intermediates, and that the first sliding complex includes ORC, Mcm2-7, and Cdt1. We demonstrate that sequential events of DNA unbending, Cdc6 release, and sliding lead to a stepwise decrease in ORC stability on DNA, facilitating ORC dissociation from its strong binding site during site switching. In addition, the controlled sliding we observed provides insight into how ORC accesses secondary DNA binding sites at different locations relative to the initial binding site. Our study highlights the importance of dynamic protein-DNA interactions in the loading of two oppositely-oriented Mcm2-7 helicases to ensure bidirectional DNA replication. Significance statement: Bidirectional DNA replication, in which two replication forks travel in opposite directions from each origin of replication, is required for complete genome duplication. To prepare for this event, two copies of the Mcm2-7 replicative helicase are loaded at each origin in opposite orientations. Using single-molecule assays, we studied the sequence of changing protein-DNA interactions involved in this process. These stepwise changes gradually reduce the DNA-binding strength of ORC, the primary DNA binding protein involved in this event. This reduced affinity promotes ORC dissociation and rebinding in the opposite orientation on the DNA, facilitating the sequential assembly of two Mcm2-7 molecules in opposite orientations. Our findings identify a coordinated series of events that drive proper DNA replication initiation.
Language	English
Publishing date	2023-06-16
Publishing country	United States
Document type	Preprint
DOI	10.1101/2023.06.16.545300
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Article ; Online: Changing protein-DNA interactions promote ORC binding-site exchange during replication origin licensing.

Zhang, Annie / Friedman, Larry J / Gelles, Jeff / Bell, Stephen P

Proceedings of the National Academy of Sciences of the United States of America

2023 Volume 120, Issue 30, Page(s) e2305556120

Abstract	During origin licensing, the eukaryotic replicative helicase Mcm2-7 forms head-to-head double hexamers to prime origins for bidirectional replication. Recent single-molecule and structural studies revealed that one molecule of the helicase loader ORC (origin recognition complex) can sequentially load two Mcm2-7 hexamers to ensure proper head-to-head helicase alignment. To perform this task, ORC must release from its initial high-affinity DNA-binding site and "flip" to bind a weaker, inverted DNA site. However, the mechanism of this binding-site switch remains unclear. In this study, we used single-molecule Förster resonance energy transfer to study the changing interactions between DNA and ORC or Mcm2-7. We found that the loss of DNA bending that occurs during DNA deposition into the Mcm2-7 central channel increases the rate of ORC dissociation from DNA. Further studies revealed temporally controlled DNA sliding of helicase-loading intermediates and that the first sliding complex includes ORC, Mcm2-7, and Cdt1. We demonstrate that sequential events of DNA unbending, Cdc6 release, and sliding lead to a stepwise decrease in ORC stability on DNA, facilitating ORC dissociation from its strong binding site during site switching. In addition, the controlled sliding we observed provides insight into how ORC accesses secondary DNA-binding sites at different locations relative to the initial binding site. Our study highlights the importance of dynamic protein-DNA interactions in the loading of two oppositely oriented Mcm2-7 helicases to ensure bidirectional DNA replication.
MeSH term(s)	DNA Replication ; Replication Origin ; Saccharomyces cerevisiae/genetics ; Saccharomyces cerevisiae/metabolism ; Saccharomyces cerevisiae Proteins/metabolism ; Minichromosome Maintenance Proteins/metabolism ; DNA/genetics ; DNA/metabolism ; Binding Sites ; Cell Cycle Proteins/metabolism ; Origin Recognition Complex/genetics ; Origin Recognition Complex/metabolism
Chemical Substances	Saccharomyces cerevisiae Proteins ; Minichromosome Maintenance Proteins (EC 3.6.4.12) ; DNA (9007-49-2) ; Cell Cycle Proteins ; Origin Recognition Complex
Language	English
Publishing date	2023-07-18
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	209104-5
ISSN	1091-6490 ; 0027-8424
ISSN (online)	1091-6490
ISSN	0027-8424
DOI	10.1073/pnas.2305556120
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Article ; Online: Regulation of replication origin licensing by ORC phosphorylation reveals a two-step mechanism for Mcm2-7 ring closing.

Amasino, Audra L / Gupta, Shalini / Friedman, Larry J / Gelles, Jeff / Bell, Stephen P

Proceedings of the National Academy of Sciences of the United States of America

2023 Volume 120, Issue 29, Page(s) e2221484120

Abstract: Eukaryotic DNA replication must occur exactly once per cell cycle to maintain cell ploidy. This outcome is ensured by temporally separating replicative helicase loading (G1 phase) and activation (S phase). In budding yeast, helicase loading is prevented ... ...

Abstract	Eukaryotic DNA replication must occur exactly once per cell cycle to maintain cell ploidy. This outcome is ensured by temporally separating replicative helicase loading (G1 phase) and activation (S phase). In budding yeast, helicase loading is prevented outside of G1 by cyclin-dependent kinase (CDK) phosphorylation of three helicase-loading proteins: Cdc6, the Mcm2-7 helicase, and the origin recognition complex (ORC). CDK inhibition of Cdc6 and Mcm2-7 is well understood. Here we use single-molecule assays for multiple events during origin licensing to determine how CDK phosphorylation of ORC suppresses helicase loading. We find that phosphorylated ORC recruits a first Mcm2-7 to origins but prevents second Mcm2-7 recruitment. The phosphorylation of the Orc6, but not of the Orc2 subunit, increases the fraction of first Mcm2-7 recruitment events that are unsuccessful due to the rapid and simultaneous release of the helicase and its associated Cdt1 helicase-loading protein. Real-time monitoring of first Mcm2-7 ring closing reveals that either Orc2 or Orc6 phosphorylation prevents Mcm2-7 from stably encircling origin DNA. Consequently, we assessed formation of the MO complex, an intermediate that requires the closed-ring form of Mcm2-7. We found that ORC phosphorylation fully inhibits MO complex formation and we provide evidence that this event is required for stable closing of the first Mcm2-7. Our studies show that multiple steps of helicase loading are impacted by ORC phosphorylation and reveal that closing of the first Mcm2-7 ring is a two-step process started by Cdt1 release and completed by MO complex formation.
MeSH term(s)	Origin Recognition Complex/genetics ; Origin Recognition Complex/metabolism ; Phosphorylation ; Replication Origin ; Saccharomyces cerevisiae/metabolism ; Saccharomyces cerevisiae Proteins/genetics ; Saccharomyces cerevisiae Proteins/metabolism ; Minichromosome Maintenance Proteins/metabolism ; Cell Cycle Proteins/metabolism ; DNA Replication ; Cyclin-Dependent Kinases/metabolism
Chemical Substances	Origin Recognition Complex ; Saccharomyces cerevisiae Proteins ; Minichromosome Maintenance Proteins (EC 3.6.4.12) ; Cell Cycle Proteins ; Cyclin-Dependent Kinases (EC 2.7.11.22)
Language	English
Publishing date	2023-07-10
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	209104-5
ISSN	1091-6490 ; 0027-8424
ISSN (online)	1091-6490
ISSN	0027-8424
DOI	10.1073/pnas.2221484120
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Article: Regulation of replication origin licensing by ORC phosphorylation reveals a two-step mechanism for Mcm2-7 ring closing.

Amasino, Audra / Gupta, Shalini / Friedman, Larry J / Gelles, Jeff / Bell, Stephen P

bioRxiv : the preprint server for biology

2023

Abstract	Eukaryotic DNA replication must occur exactly once per cell cycle to maintain cell ploidy. This outcome is ensured by temporally separating replicative helicase loading (G1 phase) and activation (S phase). In budding yeast, helicase loading is prevented outside of G1 by cyclin-dependent kinase (CDK) phosphorylation of three helicase-loading proteins: Cdc6, the Mcm2-7 helicase, and the origin recognition complex (ORC). CDK inhibition of Cdc6 and Mcm2-7 are well understood. Here we use single-molecule assays for multiple events during origin licensing to determine how CDK phosphorylation of ORC suppresses helicase loading. We find that phosphorylated ORC recruits a first Mcm2-7 to origins but prevents second Mcm2-7 recruitment. Phosphorylation of the Orc6, but not of the Orc2 subunit, increases the fraction of first Mcm2-7 recruitment events that are unsuccessful due to the rapid and simultaneous release of the helicase and its associated Cdt1 helicase-loading protein. Real-time monitoring of first Mcm2-7 ring closing reveals that either Orc2 or Orc6 phosphorylation prevents Mcm2-7 from stably encircling origin DNA. Consequently, we assessed formation of the MO complex, an intermediate that requires the closed-ring form of Mcm2-7. We found that ORC phosphorylation fully inhibits MO-complex formation and provide evidence that this event is required for stable closing of the first Mcm2-7. Our studies show that multiple steps of helicase loading are impacted by ORC phosphorylation and reveal that closing of the first Mcm2-7 ring is a two-step process started by Cdt1 release and completed by MO-complex formation. Significance statement: Each time a eukaryotic cell divides (by mitosis) it must duplicate its chromosomal DNA exactly once to ensure that one full copy is passed to each resulting cell. Both under-replication or over-replication result in genome instability and disease or cell death. A key mechanism to prevent over-replication is the temporal separation of loading of the replicative DNA helicase at origins of replication and activation of these same helicases during the cell division cycle. Here we define the mechanism by which phosphorylation of the primary DNA binding protein involved in these events inhibits helicase loading. Our studies identify multiple steps of inhibition and provide new insights into the mechanism of helicase loading in the uninhibited condition.
Language	English
Publishing date	2023-01-02
Publishing country	United States
Document type	Preprint
DOI	10.1101/2023.01.02.522488
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Article ; Online: Selective carbon-hydrogen bond hydroxylation using an engineered cytochrome P450 peroxygenase.

Akter, Jinia / Stockdale, Tegan P / Child, Stella A / Lee, Joel H Z / De Voss, James J / Bell, Stephen G

Journal of inorganic biochemistry

2023 Volume 244, Page(s) 112209

Abstract: The cytochrome P450 enzyme CYP102A1 (P450BM3) is a versatile monooxygenase enzyme which has been adapted and engineered for multiple applications in chemical synthesis. Mutation of threonine 268 to glutamate (Thr268Glu) converted the heme domain of this ... ...

Abstract	The cytochrome P450 enzyme CYP102A1 (P450BM3) is a versatile monooxygenase enzyme which has been adapted and engineered for multiple applications in chemical synthesis. Mutation of threonine 268 to glutamate (Thr268Glu) converted the heme domain of this enzyme into a H
MeSH term(s)	Hydroxylation ; Hydrogen Peroxide ; Hydrogen Bonding ; Cytochrome P-450 Enzyme System/metabolism ; Oxidation-Reduction ; Fatty Acids/chemistry ; Heme
Chemical Substances	peroxygenase (EC 1.14.-) ; Hydrogen Peroxide (BBX060AN9V) ; Cytochrome P-450 Enzyme System (9035-51-2) ; Fatty Acids ; Heme (42VZT0U6YR)
Language	English
Publishing date	2023-04-06
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	162843-4
ISSN	1873-3344 ; 0162-0134
ISSN (online)	1873-3344
ISSN	0162-0134
DOI	10.1016/j.jinorgbio.2023.112209
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Article ; Online: Rethinking origin licensing

Stephen P Bell

eLife, Vol

2017 Volume 6

Abstract: Human cells that lack a subunit in their origin recognition complex are viable, which suggests the existence of alternative mechanisms to initiate DNA replication. ...

Abstract	Human cells that lack a subunit in their origin recognition complex are viable, which suggests the existence of alternative mechanisms to initiate DNA replication.
Keywords	DNA replication ; CDC6 ; ORC ; MCM2-7 ; CDT1 ; Medicine ; R ; Science ; Q ; Biology (General) ; QH301-705.5
Language	English
Publishing date	2017-01-01T00:00:00Z
Publisher	eLife Sciences Publications Ltd
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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