scholarly journals Top1 and Top2 promote replication fork arrest at a programmed pause site

2020 ◽  
Vol 34 (1-2) ◽  
pp. 1-3 ◽  
Author(s):  
Mélanie V. Larcher ◽  
Philippe Pasero
Keyword(s):  
Replication Fork ◽  
Replication Fork Arrest ◽  
Pause Site

Replication forks pause at yeast centromeres

1992 ◽  
Vol 12 (9) ◽  
pp. 4056-4066
Author(s):  
S A Greenfeder ◽  
C S Newlon
Keyword(s):  
Gel Electrophoresis ◽  
Replication Fork ◽  
S Phase ◽  
Core Structure ◽  
Replication Forks ◽  
Agarose Gel Electrophoresis ◽  
Unusual Structure ◽  
Dna Complex ◽  
Derivatives Of ◽  
Pause Site

The 120 bp of yeast centromeric DNA is tightly complexed with protein to form a nuclease-resistant core structure 200 to 240 bp in size. We have used two-dimensional agarose gel electrophoresis to analyze the replication of the chromosomal copies of yeast CEN1, CEN3, and CEN4 and determine the fate of replication forks that encounter the protein-DNA complex at the centromere. We have shown that replication fork pause sites are coincident with each of these centromeres and therefore probably with all yeast centromeres. We have analyzed the replication of plasmids containing mutant derivatives of CEN3 to determine whether the replication fork pause site is a result of an unusual structure adopted by centromere DNA or a result of the protein-DNA complex formed at the centromere. The mutant centromere derivatives varied in function as well as the ability to form the nuclease-resistant core structure. The data obtained from analysis of these derivatives indicate that the ability to cause replication forks to pause correlates with the ability to form the nuclease-resistant core structure and not with the presence or absence of a particular DNA sequence. Our findings further suggest that the centromere protein-DNA complex is present during S phase when replication forks encounter the centromere and therefore may be present throughout the cell cycle.

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 Lamin A/C Depletion Enhances DNA Damage-Induced Stalled Replication Fork Arrest

2013 ◽  
Vol 33 (16) ◽  
pp. 3390-3390
Author(s):  
Mayank Singh ◽  
Clayton R. Hunt ◽  
Raj K. Pandita ◽  
Rakesh Kumar ◽  
Chin-Rang Yang ◽  
...  
Keyword(s):  
Dna Damage ◽  
Replication Fork ◽  
Lamin A ◽  
Replication Fork Arrest

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 Phosphorylation of CMG helicase and Tof1 is required for programmed fork arrest

2016 ◽  
Vol 113 (26) ◽  
pp. E3639-E3648 ◽  
Author(s):  
Deepak Bastia ◽  
Pankaj Srivastava ◽  
Shamsu Zaman ◽  
Malay Choudhury ◽  
Bidyut K. Mohanty ◽  
...  
Keyword(s):  
Replication Fork ◽  
General Mechanism ◽  
Nonhistone Protein ◽  
In Vivo Experiments ◽  
Vitro Binding ◽  
Fork Protection Complex ◽  
Replication Fork Arrest ◽  
Replication Factors

Several important physiological transactions, including control of replicative life span (RLS), prevention of collision between replication and transcription, and cellular differentiation, require programmed replication fork arrest (PFA). However, a general mechanism of PFA has remained elusive. We previously showed that the Tof1–Csm3 fork protection complex is essential for PFA by antagonizing the Rrm3 helicase that displaces nonhistone protein barriers that impede fork progression. Here we show that mutations of Dbf4-dependent kinase (DDK) of Saccharomyces cerevisiae, but not other DNA replication factors, greatly reduced PFA at replication fork barriers in the spacer regions of the ribosomal DNA array. A key target of DDK is the mini chromosome maintenance (Mcm) 2–7 complex, which is known to require phosphorylation by DDK to form an active CMG [Cdc45 (cell division cycle gene 45), Mcm2–7, GINS (Go, Ichi, Ni, and San)] helicase. In vivo experiments showed that mutational inactivation of DDK caused release of Tof1 from the chromatin fractions. In vitro binding experiments confirmed that CMG and/or Mcm2–7 had to be phosphorylated for binding to phospho-Tof1–Csm3 but not to its dephosphorylated form. Suppressor mutations that bypass the requirement for Mcm2–7 phosphorylation by DDK restored PFA in the absence of the kinase. Retention of Tof1 in the chromatin fraction and PFA in vivo was promoted by the suppressor mcm5-bob1, which bypassed DDK requirement, indicating that under this condition a kinase other than DDK catalyzed the phosphorylation of Tof1. We propose that phosphorylation regulates the recruitment and retention of Tof1–Csm3 by the replisome and that this complex antagonizes the Rrm3 helicase, thereby promoting PFA, by preserving the integrity of the Fob1–Ter complex.

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