DNA Replication Fork Arrest by the Bacillus subtilis RTP–DNA Complex Involves a Mechanism that Is Independent of the Affinity of RTP–DNA Binding

2006 ◽  
Vol 361 (1) ◽  
pp. 1-6 ◽  
Author(s):  
Iain G. Duggin
Keyword(s):  
Bacillus Subtilis ◽  
Dna Replication ◽  
Dna Binding ◽  
Replication Fork ◽  
Replication Fork Arrest ◽  
Dna Complex

scholarly journals Delineation of the Ancestral Tus-Dependent Replication Fork Trap

2021 ◽  
Author(s):  
Casey Toft ◽  
Morgane Moreau ◽  
Jiri Perutka ◽  
Savitri Mandapati ◽  
Peter Enyeart ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Replication Fork ◽  
Wide Distribution ◽  
Replication Forks ◽  
Genome Wide ◽  
Replication Fork Arrest

In Escherichia coli, DNA replication termination is orchestrated by two clusters of Ter sites forming a DNA replication fork trap when bound by Tus proteins. The formation of a ‘locked’ Tus-Ter complex is essential for halting incoming DNA replication forks. However, the absence of replication fork arrest at some Ter sites raised questions about their significance. In this study, we examined the genome-wide distribution of Tus and found that only the six innermost Ter sites (TerA-E and G) were significantly bound by Tus. We also found that a single ectopic insertion of TerB in its non-permissive orientation could not be achieved, advocating against a need for ‘back-up’ Ter sites. Finally, examination of the genomes of a variety of Enterobacterales revealed a new replication fork trap architecture mostly found outside the Enterobacteriaceae family. Taken together, our data enabled the delineation of a narrow ancestral Tus-dependent DNA replication fork trap consisting of only two Ter sites.

scholarly journals Dual Functions of Single-stranded DNA-binding Protein in Helicase Loading at the Bacteriophage T4 DNA Replication Fork

2004 ◽  
Vol 279 (18) ◽  
pp. 19035-19045 ◽  
Author(s):  
Yujie Ma ◽  
Tongsheng Wang ◽  
Jana L. Villemain ◽  
David P. Giedroc ◽  
Scott W. Morrical
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Replication Fork ◽  
Binding Protein ◽  
Bacteriophage T4 ◽  
Dna Binding Protein ◽  
Single Stranded Dna ◽  
Helicase Loading ◽  
Dual Functions

scholarly journals Enhanced H2AX Phosphorylation, DNA Replication Fork Arrest, and Cell Death in the Absence of Chk1

2010 ◽  
Vol 21 (5) ◽  
pp. 739-752 ◽  
Author(s):  
Mary E. Gagou ◽  
Pedro Zuazua-Villar ◽  
Mark Meuth
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Replication Fork ◽  
Brdu Incorporation ◽  
H2ax Phosphorylation ◽  
Direct Role ◽  
Γh2ax Foci ◽  
Dna Replication Stress ◽  
Sw480 Cells ◽  
Replication Fork Arrest

H2AX phosphorylation at serine 139 (γH2AX) is a sensitive indicator of both DNA damage and DNA replication stress. Here we show that γH2AX formation is greatly enhanced in response to replication inhibitors but not ionizing radiation in HCT116 or SW480 cells depleted of Chk1. Although H2AX phosphorylation precedes the induction of apoptosis in such cells, our results suggest that cells containing γH2AX are not committed to death. γH2AX foci in these cells largely colocalize with RPA foci and their formation is dependent upon the essential replication helicase cofactor Cdc45, suggesting that H2AX phosphorylation occurs at sites of stalled forks. However Chk1-depleted cells released from replication inhibitors retain γH2AX foci and do not appear to resume replicative DNA synthesis. BrdU incorporation only occurs in a minority of Chk1-depleted cells containing γH2AX foci after release from thymidine arrest and, in cells incorporating BrdU, DNA synthesis does not occur at sites of γH2AX foci. Furthermore activated ATM and Chk2 persist in these cells. We propose that the γH2AX foci in Chk1-depleted cells may represent sites of persistent replication fork damage or abandonment that are unable to resume DNA synthesis but do not play a direct role in the Chk1 suppressed death pathway.

scholarly journals Nuclease dead Cas9 is a programmable roadblock for DNA replication

2018 ◽  
Author(s):  
Kelsey Whinn ◽  
Gurleen Kaur ◽  
Jacob S. Lewis ◽  
Grant Schauer ◽  
Stefan Müller ◽  
...  
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Replication Fork ◽  
Molecular Machines ◽  
Replication Forks ◽  
Chromosomal Dna ◽  
Single Molecule Level ◽  
Replication Fork Arrest

DNA replication occurs on chromosomal DNA while processes such as DNA repair, recombination and transcription continue. However, we have limited experimental tools to study the consequences of collisions between DNA-bound molecular machines. Here, we repurpose a catalytically inactivated Cas9 (dCas9) construct fused to the photo-stable dL5 protein fluoromodule as a novel, targetable protein-DNA roadblock for studying replication fork arrest at the single-molecule level in vitro as well as in vivo. We find that the specifically bound dCas9–guideRNA complex arrests viral, bacterial and eukaryotic replication forks in vitro.

scholarly journals TheEscherichia coliGTPase ObgE modulates hydroxyl radical levels in response to DNA replication fork arrest

FEBS Journal ◽  
2012 ◽  
Vol 279 (19) ◽  
pp. 3692-3704 ◽  
Author(s):  
Cyrielle I. Kint ◽  
Natalie Verstraeten ◽  
Inez Wens ◽  
Veerle R. Liebens ◽  
Johan Hofkens ◽  
...  
Keyword(s):  
Dna Replication ◽  
Hydroxyl Radical ◽  
Replication Fork ◽  
Replication Fork Arrest

Site-directed mutants of RTP of Bacillus subtilis and the mechanism of replication fork arrest 1 1Edited by M. Gottesman

1999 ◽  
Vol 286 (5) ◽  
pp. 1325-1335 ◽  
Author(s):  
I.G Duggin ◽  
P.A Andersen ◽  
M.T Smith ◽  
J.A Wilce ◽  
G.F King ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Replication Fork ◽  
Replication Fork Arrest

scholarly journals Nuclease dead Cas9 is a programmable roadblock for DNA replication

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Kelsey S. Whinn ◽  
Gurleen Kaur ◽  
Jacob S. Lewis ◽  
Grant D. Schauer ◽  
Stefan H. Mueller ◽  
...  
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Molecular Machines ◽  
Enzymatic Activities ◽  
Replication Forks ◽  
Dna Substrates ◽  
Replication Fork Arrest

Abstract Limited experimental tools are available to study the consequences of collisions between DNA-bound molecular machines. Here, we repurpose a catalytically inactivated Cas9 (dCas9) construct as a generic, novel, targetable protein–DNA roadblock for studying mechanisms underlying enzymatic activities on DNA substrates in vitro. We illustrate the broad utility of this tool by demonstrating replication fork arrest by the specifically bound dCas9–guideRNA complex to arrest viral, bacterial and eukaryotic replication forks in vitro.

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