Chl1 helicase controls replication fork progression by regulating dNTP pools

Eukaryotic cells have evolved a replication stress response that helps to overcome stalled/collapsed replication forks and ensure proper DNA replication. The replication checkpoint protein Mrc1 plays important roles in these processes, although its functional interactions are not fully understood. Here, we show that MRC1 negatively interacts with CHL1, which encodes the helicase protein Chl1, suggesting distinct roles for these factors during the replication stress response. Indeed, whereas Mrc1 is known to facilitate the restart of stalled replication forks, we uncovered that Chl1 controls replication fork rate under replication stress conditions. Chl1 loss leads to increased RNR1 gene expression and dNTP levels at the onset of S phase likely without activating the DNA damage response. This in turn impairs the formation of RPA-coated ssDNA and subsequent checkpoint activation. Thus, the Chl1 helicase affects RPA-dependent checkpoint activation in response to replication fork arrest by ensuring proper intracellular dNTP levels, thereby controlling replication fork progression under replication stress conditions.

Propofol Regulates the Proliferation and Migration of Lung Cancer Cells Through Atr Signaling Pathway

Aims: Here we aim to investigate the regulation of propofol on DNA damage caused by replication fork arrest in esophageal squamous cell carcinoma cells.Methods: A549 and NCI-H460 cells were treated with propofol and hydroxyurea (HU) in vitro. CCK-8 assay was used to examine cell proliferation. Transwell assay was employed to investigate cell migration and invasion abilities. Western blotting was carried out to study the activities of ATR signals. Laser confocal microscopy was utilized to study the formation of p-RPA32 foci.Results: Propofol treatment promoted the apoptosis and suppresses the proliferation, migration and invasion, possibly by increasing the sensitivity of A549 and NCI-H460 cells against DNA damage. Propofol treatment enhanced the sensitivity of A549 and NCI-H460 cells to damages caused by replication fork arrest, as well as the activity of ATR signaling pathway. Propofol regulated the sensitivity of A549 and NCI-H460 cells to DNA replication damage by affecting the level of H3K27me3.Conclusions: The present study demonstrates that propofol up-regulates the expression of H3K27me3 in lung cancer cells, promotes the recruitment of exonuclease MUS81 in stagnant replication fork, induces apoptosis caused by DNA damage, and thus inhibits the proliferation and metastasis of tumor cells.

Delineation of the Ancestral Tus-Dependent Replication Fork Trap

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.

Delineation of the Ancestral Tus-Dependent Replication Fork Trap

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.

Defining the prototypical DNA replication fork trap in bacteria

In Escherichia coli, DNA replication termination is orchestrated by two opposite 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. 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 ectopic insertion of a TerB sequence in its non-permissive orientation could not be achieved, advocating against the necessity for 'back-up' Ter sites due to the inefficient formation of a 'locked' Tus-Ter complex. Finally, examination of the genomes of a variety of Enterobacterales revealed two major types of replication fork traps including a prototypical architecture consisting of two unique Ter sequences in opposite orientation.

The S-Phase Cyclin Clb5 Promotes rRNA Gene (rDNA) Stability by Maintaining Replication Initiation Efficiency in rDNA

ABSTRACT Regulation of replication origins is important for complete duplication of the genome, but the effect of origin activation on the cellular response to replication stress is poorly understood. The budding yeast rRNA gene (rDNA) forms tandem repeats and undergoes replication fork arrest at the replication fork barrier (RFB), inducing DNA double-strand breaks (DSBs) and genome instability accompanied by copy number alterations. Here, we demonstrate that the S-phase cyclin Clb5 promotes rDNA stability. Absence of Clb5 led to reduced efficiency of replication initiation in rDNA but had little effect on the number of replication forks arrested at the RFB, suggesting that arrival of the converging fork is delayed and forks are more stably arrested at the RFB. Deletion of CLB5 affected neither DSB formation nor its repair at the RFB but led to homologous recombination-dependent rDNA instability. Therefore, arrested forks at the RFB may be subject to DSB-independent, recombination-dependent rDNA instability. The rDNA instability in clb5Δ was not completely suppressed by the absence of Fob1, which is responsible for fork arrest at the RFB. Thus, Clb5 establishes the proper interval for active replication origins and shortens the travel distance for DNA polymerases, which may reduce Fob1-independent DNA damage.

The S-phase Cyclin Clb5 Promotes rDNA Stability by Maintaining Replication Initiation Efficiency in rDNA

ABSTRACTRegulation of replication origins is important for complete duplication of the genome, but the effect of origin activation on the cellular response to replication stress is poorly understood. The budding yeast ribosomal RNA gene (rDNA) forms tandem repeats and undergoes replication fork arrest at the replication fork barrier (RFB), inducing DNA double-strand breaks (DSBs) and genome instability accompanied by copy number alterations. Here we demonstrate that the S-phase cyclin Clb5 promotes rDNA stability. Absence of Clb5 led to reduced efficiency of replication initiation in rDNA but had little effect on the amount of replication forks arrested at the RFB, suggesting that arrival of the converging fork is delayed and forks are more stably arrested at the RFB. Deletion of CLB5 affected neither DSB formation nor its repair at the RFB, but led to an accumulation of recombination intermediates. Therefore, arrested forks at the RFB may be subject to DSB-independent, recombination-dependent rDNA instability. The rDNA instability in clb5Δ was not completely suppressed by the absence of Fob1, which is responsible for fork arrest at the RFB. Thus, Clb5 establishes the proper interval for active replication origins and shortens the travel distance for DNA polymerases, which may reduce Fob1-independent DNA damage.

Active Replication Checkpoint Drives Genome Instability in Fission Yeast mcm4 Mutant

ABSTRACT Upon replication fork arrest, the replication checkpoint kinase Cds1 is stimulated to preserve genome integrity. Robust activation of Cds1 in response to hydroxyurea prevents the endonuclease Mus81 from cleaving the stalled replication fork inappropriately. However, we find that the response is different in temperature-sensitive mcm4 mutants, affecting a subunit of the MCM replicative helicase. We show that Cds1 inhibition of Mus81 promotes genomic instability and allows mcm4-dg cells to evade cell cycle arrest. Cds1 regulation of Mus81 activity also contributes to the formation of the replication stress-induced DNA damage markers replication protein A (RPA) and Ku. These results identify a surprising role for Cds1 in driving DNA damage and disrupted chromosomal segregation under certain conditions of replication stress.

Nuclease dead Cas9 is a programmable roadblock for DNA replication

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.