Approaching Protein Barriers: Emerging Mechanisms of Replication Pausing in Eukaryotes

Protein Barrier

During nuclear DNA replication multiprotein replisome machines have to jointly traverse and duplicate the total length of each chromosome during each cell cycle. At certain genomic locations replisomes encounter tight DNA-protein complexes and slow down. This fork pausing is an active process involving recognition of a protein barrier by the approaching replisome via an evolutionarily conserved Fork Pausing/Protection Complex (FPC). Action of the FPC protects forks from collapse at both programmed and accidental protein barriers, thus promoting genome integrity. In addition, FPC stimulates the DNA replication checkpoint and regulates topological transitions near the replication fork. Eukaryotic cells have been proposed to employ physiological programmed fork pausing for various purposes, such as maintaining copy number at repetitive loci, precluding replication-transcription encounters, regulating kinetochore assembly, or controlling gene conversion events during mating-type switching. Here we review the growing number of approaches used to study replication pausing in vivo and in vitro as well as the characterization of additional factors recently reported to modulate fork pausing in different systems. Specifically, we focus on the positive role of topoisomerases in fork pausing. We describe a model where replisome progression is inherently cautious, which ensures general preservation of fork stability and genome integrity but can also carry out specialized functions at certain loci. Furthermore, we highlight classical and novel outstanding questions in the field and propose venues for addressing them. Given how little is known about replisome pausing at protein barriers in human cells more studies are required to address how conserved these mechanisms are.

The Direct Binding of Mrc1, a Checkpoint Mediator, to Mcm6, a Replication Helicase, Is Essential for the Replication Checkpoint against Methyl Methanesulfonate-Induced Stress

10.1128/mcb.01934-08 ◽

2009 ◽

Vol 29 (18) ◽

pp. 5008-5019 ◽

Cited By ~ 43

Author(s):

Makiko Komata ◽

Masashige Bando ◽

Hiroyuki Araki ◽

Katsuhiko Shirahige

Keyword(s):

Amino Acid ◽

Dna Replication ◽

Terminal Region ◽

Methyl Methanesulfonate ◽

Checkpoint Activation ◽

Replication Checkpoint ◽

Hydroxyurea Treatment ◽

Direct Binding

ABSTRACT Mrc1 plays a role in mediating the DNA replication checkpoint. We surveyed replication elongation proteins that interact directly with Mrc1 and identified a replicative helicase, Mcm6, as a specific Mrc1-binding protein. The central portion of Mrc1, containing a conserved coiled-coil region, was found to be essential for interaction with the 168-amino-acid C-terminal region of Mcm6, and introduction of two amino acid substitutions in this C-terminal region abolished the interaction with Mrc1 in vivo. An mcm6 mutant bearing these substitutions showed a severe defect in DNA replication checkpoint activation in response to stress caused by methyl methanesulfonate. Interestingly, the mutant did not show any defect in DNA replication checkpoint activation in response to hydroxyurea treatment. The phenotype of the mcm6 mutant was suppressed when the mutant protein was physically fused with Mrc1. These results strongly suggest for the first time that an Mcm helicase acts as a checkpoint sensor for methyl methanesulfonate-induced DNA damage through direct binding to the replication checkpoint mediator Mrc1.

Stockpiling of Cdc25 during a DNA replication checkpoint arrest in Schizosaccharomyces pombe.

10.1128/mcb.16.1.86 ◽

1996 ◽

Vol 16 (1) ◽

pp. 86-93 ◽

Cited By ~ 45

Author(s):

R Kovelman ◽

P Russell

Keyword(s):

Dna Replication ◽

Schizosaccharomyces Pombe ◽

Replication Checkpoint ◽

Activation Assay ◽

M Phase ◽

High Level ◽

Tyrosyl Phosphorylation ◽

In Cells

The DNA replication checkpoint couples the onset of mitosis with the completion of S phase. It is clear that in the fission yeast Schizosaccharomyces pombe, operation of this checkpoint requires maintenance of the inhibitory tyrosyl phosphorylation of Cdc2. Cdc25 phosphatase induces mitosis by dephosphorylating tyrosine 15 of Cdc2. In this report, Cdc25 is shown to accumulate to a very high level in cells arrested in S. This shows that mechanisms which modulate the abundance of Cdc25 are unconnected to the DNA replication checkpoint. Using a Cdc2/cyclin B activation assay, we found that Cdc25 activity increased approximately 10-fold during transit through M phase. Cdc25 was activated by phosphorylations that were dependent on Cdc2 activity in vivo. Cdc25 activation was suppressed in cells arrested in G1 and S. However, Cdc25 was more highly modified and appeared to be somewhat more active in S than in G1. This finding might be connected to the fact that progression from G1 to S increases the likelihood that constitutive Cdc25 overproduction will cause inappropriate mitosis.

Cdc18p can block mitosis by two independent mechanisms

Journal of Cell Science ◽

10.1242/jcs.111.20.3101 ◽

1998 ◽

Vol 111 (20) ◽

pp. 3101-3108 ◽

Cited By ~ 2

Author(s):

E. Greenwood ◽

H. Nishitani ◽

P. Nurse

Keyword(s):

Dna Replication ◽

S Phase ◽

Checkpoint Control ◽

Replication Checkpoint ◽

Mitotic Kinase ◽

C Terminus ◽

N Terminus ◽

Checkpoint Genes

The DNA replication checkpoint is required to maintain the integrity of the genome, inhibiting mitosis until S phase has been successfully completed. The checkpoint preventing premature mitosis in Schizosaccharomyces pombe relies on phosphorylation of the tyrosine-15 residue on cdc2p to prevent its activation and hence mitosis. The cdc18 gene is essential for both generating the DNA replication checkpoint and the initiation of S phase, thus providing a key role for the overall control and coordination of the cell cycle. We show that the C terminus of the protein is capable of both initiating DNA replication and the checkpoint function of cdc18p. The C terminus of cdc18p acts upstream of the DNA replication checkpoint genes rad1, rad3, rad9, rad17, hus1 and cut5 and requires the wee1p/mik1p tyrosine kinases to block mitosis. The N terminus of cdc18p can also block mitosis but does so in the absence of the DNA replication checkpoint genes and the wee1p/mik1p kinases therefore acting downstream of these genes. Because the N terminus of cdc18p associates with cdc2p in vivo, we suggest that by binding the cdc2p/cdc13p mitotic kinase directly, it exerts an effect independently of the normal checkpoint control, probably in an unphysiological manner.

G-Quadruplexes at Telomeres: Friend or Foe?

Molecules ◽

10.3390/molecules25163686 ◽

2020 ◽

Vol 25 (16) ◽

pp. 3686 ◽

Cited By ~ 3

Author(s):

Tracy M. Bryan

Keyword(s):

Tandem Repeats ◽

Genome Instability ◽

Protein Complexes ◽

Telomere Maintenance ◽

Telomeric Dna ◽

Positive Role ◽

Telomere Biology ◽

Linear Chromosomes

Telomeres are DNA-protein complexes that cap and protect the ends of linear chromosomes. In almost all species, telomeric DNA has a G/C strand bias, and the short tandem repeats of the G-rich strand have the capacity to form into secondary structures in vitro, such as four-stranded G-quadruplexes. This has long prompted speculation that G-quadruplexes play a positive role in telomere biology, resulting in selection for G-rich tandem telomere repeats during evolution. There is some evidence that G-quadruplexes at telomeres may play a protective capping role, at least in yeast, and that they may positively affect telomere maintenance by either the enzyme telomerase or by recombination-based mechanisms. On the other hand, G-quadruplex formation in telomeric DNA, as elsewhere in the genome, can form an impediment to DNA replication and a source of genome instability. This review summarizes recent evidence for the in vivo existence of G-quadruplexes at telomeres, with a focus on human telomeres, and highlights some of the many unanswered questions regarding the location, form, and functions of these structures.

The absence of a DNA replication checkpoint in porcine zygotes

Zygote ◽

10.1017/s0967199406003583 ◽

2006 ◽

Vol 14 (1) ◽

pp. 33-37 ◽

Cited By ~ 2

Author(s):

Irena Vackova ◽

Radomir Kren ◽

Pasqualino Loi ◽

Vladimír Krylov ◽

Josef Fulka

Keyword(s):

Dna Replication ◽

Intracytoplasmic Sperm Injection ◽

Sperm Head ◽

Replication Checkpoint ◽

Dna Replication Checkpoint

It has been demonstrated that in the zygotes of some mammals a unique checkpoint controls the onset of DNA replication. Thus, DNA replication begins in the maternal pronucleus only after the paternal pronucleus is fully formed. In our experiments we have investigated whether this checkpoint also operates in porcine zygotes produced either by in vitro fertilization (IVF) or by intracytoplasmic sperm injection (ICSI). Our results show that the onset of DNA replication occurs in the maternal pronucleus even in the presence of an intact sperm head in zygotes produced by ICSI, as well as in polyspermic eggs where some sperm heads are intact or male pronuclei are not yet fully developed. We conclude that in porcine zygotes there is an absence of the DNA replication checkpoint that is typical for some other mammals.

Tyrosine Phosphorylation of Cdc2 Is Required for the Replication Checkpoint in Schizosaccharomyces pombe

10.1128/mcb.18.7.3782 ◽

1998 ◽

Vol 18 (7) ◽

pp. 3782-3787 ◽

Cited By ~ 68

Author(s):

Nicholas Rhind ◽

Paul Russell

Keyword(s):

Dna Replication ◽

Schizosaccharomyces Pombe ◽

Biochemical Study ◽

Replication Checkpoint ◽

Hydroxyurea Treatment ◽

Activated State ◽

Cdc2 Activity ◽

In Cells

ABSTRACT The DNA replication checkpoint inhibits mitosis in cells that are unable to replicate their DNA, as when nucleotide biosynthesis is inhibited by hydroxyurea. In the fission yeastSchizosaccharomyces pombe, genetic evidence suggests that this checkpoint involves the inhibition of Cdc2 activity through the phosphorylation of tyrosine-15. On the contrary, a recent biochemical study indicated that Cdc2 is in an activated state during a replication checkpoint, suggesting that phosphorylation of Cdc2 on tyrosine-15 is not part of the replication checkpoint mechanism. We have undertaken biochemical and genetic studies to resolve this controversy. We report that the DNA replication checkpoint in S. pombe is abrogated in cells that carry the allele cdc2-Y15F, expressing an unphosphorylatable form of Cdc2. Furthermore, Cdc2 isolated from replication checkpoint-arrested cells can be activated in vitro by Cdc25, the tyrosine phosphatase responsible for dephosphorylating Cdc2 in vivo, to the same extent as Cdc2 isolated from cdc25ts-blocked cells, indicating that hydroxyurea treatment causes Cdc2 activity to be maintained at a low level that is insufficient to induce mitosis. These studies show that inhibitory tyrosine-15 phosphorylation of Cdc2 is essential for the DNA replication checkpoint and suggests that Cdc25, and/or one or both of Wee1 and Mik1, the tyrosine kinases that phosphorylate Cdc2, are regulated by the replication checkpoint.

The Schizosaccharomyces pombe cdt2+ Gene, a Target of G1-S Phase-Specific Transcription Factor Complex DSC1, Is Required for Mitotic and Premeiotic DNA Replication

Genetics ◽

10.1093/genetics/164.3.881 ◽

2003 ◽

Vol 164 (3) ◽

pp. 881-893 ◽

Cited By ~ 1

Author(s):

Shu-hei Yoshida ◽

Hiba Al-Amodi ◽

Taro Nakamura ◽

Christopher J McInerny ◽

Chikashi Shimoda

Keyword(s):

Dna Replication ◽

Schizosaccharomyces Pombe ◽

Mitotic Cell ◽

Life Cycles ◽

S Phase ◽

Growth Defect ◽

Replication Checkpoint ◽

Synthetically Lethal

Abstract We have defined five sev genes by genetic analysis of Schizosaccharomyces pombe mutants, which are defective in both proliferation and sporulation. sev1+/cdt2+ was transcribed during the G1-S phase of the mitotic cell cycle, as well as during the premeiotic S phase. The mitotic expression of cdt2+ was regulated by the MCB-DSC1 system. A mutant of a component of DSC1 affected cdt2+ expression in vivo, and a cdt2+ promoter fragment containing MCB motifs bound DSC1 in vitro. Cdt2 protein also accumulated in S phase and localized to the nucleus. cdt2 null mutants grew slowly at 30° and were unable to grow at 19°. These cdt2 mutants were also medially sensitive to hydroxyurea, camptothecin, and 4-nitroquinoline-1-oxide and were synthetically lethal in combination with DNA replication checkpoint mutations. Flow cytometry analysis and pulsed-field gel electrophoresis revealed that S-phase progression was severely retarded in cdt2 mutants, especially at low temperatures. Under sporulation conditions, premeiotic DNA replication was impaired with meiosis I blocked. Furthermore, overexpression of suc22+, a ribonucleotide reductase gene, fully complemented the sporulation defect of cdt2 mutants and alleviated their growth defect at 19°. These observations suggest that cdt2+ plays an important role in DNA replication in both the mitotic and the meiotic life cycles of fission yeast.

The DNA Replication Checkpoint Directly Regulates MBF-Dependent G1/S Transcription

10.1128/mcb.00596-08 ◽

2008 ◽

Vol 28 (19) ◽

pp. 5977-5985 ◽

Cited By ~ 37

Author(s):

Chaitali Dutta ◽

Prasanta K. Patel ◽

Adam Rosebrock ◽

Anna Oliva ◽

Janet Leatherwood ◽

...

Keyword(s):

Transcription Factor ◽

Dna Replication ◽

Budding Yeast ◽

Replication Stress ◽

Phosphorylation Sites ◽

Transcriptional Program ◽

Replication Checkpoint ◽

Regulatory Similarity

ABSTRACT The DNA replication checkpoint transcriptionally upregulates genes that allow cells to adapt to and survive replication stress. Our results show that, in the fission yeast Schizosaccharomyces pombe, the replication checkpoint regulates the entire G1/S transcriptional program by directly regulating MBF, the G1/S transcription factor. Instead of initiating a checkpoint-specific transcriptional program, the replication checkpoint targets MBF to maintain the normal G1/S transcriptional program during replication stress. We propose a mechanism for this regulation, based on in vitro phosphorylation of the Cdc10 subunit of MBF by the Cds1 replication-checkpoint kinase. Replacement of two potential phosphorylation sites with phosphomimetic amino acids suffices to promote the checkpoint transcriptional program, suggesting that Cds1 phosphorylation directly regulates MBF-dependent transcription. The conservation of MBF between fission and budding yeast, and recent results implicating MBF as a target of the budding yeast replication checkpoint, suggests that checkpoint regulation of the MBF transcription factor is a conserved strategy for coping with replication stress. Furthermore, the structural and regulatory similarity between MBF and E2F, the metazoan G1/S transcription factor, suggests that this checkpoint mechanism may be broadly conserved among eukaryotes.

Sp1 trans-activates the murine H+-K+-ATPase α2-subunit gene

AJP Renal Physiology ◽

10.1152/ajprenal.00039.2009 ◽

2009 ◽

Vol 297 (1) ◽

pp. F63-F70 ◽

Cited By ~ 3

Author(s):

Zhiyuan Yu ◽

Mei Li ◽

Dongyu Zhang ◽

William Xu ◽

Bruce C. Kone

Keyword(s):

Transcriptional Control ◽

Protein Complexes ◽

Collecting Duct ◽

Point Mutations ◽

Distal Colon ◽

Proximal Promoter ◽

Mobility Shift ◽

Positive Role

The H+-K+-ATPase α2 (HKα2) gene of the renal collecting duct and distal colon plays a central role in potassium and acid-base homeostasis, yet its transcriptional control remains poorly characterized. We previously demonstrated that the proximal 177 bp of its 5′-flanking region confers basal transcriptional activity in murine inner medullary collecting duct (mIMCD3) cells and that NF-κB and CREB-1 bind this region to alter transcription. In the present study, we sought to determine whether the −144/−135 Sp element influences basal HKα2 gene transcription in these cells. Electrophoretic mobility shift and supershift assays using probes for −154/−127 revealed Sp1-containing DNA-protein complexes in nuclear extracts of mIMCD3 cells. Chromatin immunoprecipitation (ChIP) assays demonstrated that Sp1, but not Sp3, binds to this promoter region of the HKα2 gene in mIMCD3 cells in vivo. HKα2 minimal promoter-luciferase constructs with point mutations in the −144/−135 Sp element exhibited much lower activity than the wild-type promoter in transient transfection assays. Overexpression of Sp1, but not Sp3, trans-activated an HKα2 proximal promoter-luciferase construct in mIMCD3 cells as well as in SL2 insect cells, which lack Sp factors. Conversely, small interfering RNA knockdown of Sp1 inhibited endogenous HKα2 mRNA expression, and binding of Sp1 to chromatin associated with the proximal HKα2 promoter without altering the binding or regulatory influence of NF-κB p65 or CREB-1 on the proximal HKα2 promoter. We conclude that Sp1 plays an important and positive role in controlling basal HKα2 gene expression in mIMCD3 cells in vivo and in vitro.

Mcm2-7 Is an Active Player in the DNA Replication Checkpoint Signaling Cascade via Proposed Modulation of Its DNA Gate

10.1128/mcb.01357-14 ◽

2015 ◽

Vol 35 (12) ◽

pp. 2131-2143 ◽

Cited By ~ 11

Author(s):

Feng-Ling Tsai ◽

Sriram Vijayraghavan ◽

Joseph Prinz ◽

Heather K. MacAlpine ◽

David M. MacAlpine ◽

...

Keyword(s):

Dna Replication ◽

Conformational Change ◽

Replication Checkpoint ◽

Checkpoint Signaling ◽

Kinase Activation ◽

Checkpoint Response ◽

Active Player ◽

Replication Factors

The DNA replication checkpoint (DRC) monitors and responds to stalled replication forks to prevent genomic instability. How core replication factors integrate into this phosphorylation cascade is incompletely understood. Here, through analysis of a uniquemcmallele targeting a specific ATPase active site (mcm2DENQ), we show that the Mcm2-7 replicative helicase has a novel DRC function as part of the signal transduction cascade. This allele exhibits normal downstream mediator (Mrc1) phosphorylation, implying DRC sensor kinase activation. However, the mutant also exhibits defective effector kinase (Rad53) activation and classic DRC phenotypes. Our previousin vitroanalysis showed that themcm2DENQmutation prevents a specific conformational change in the Mcm2-7 hexamer. We infer that this conformational change is required for its DRC role and propose that it allosterically facilitates Rad53 activation to ensure a replication-specific checkpoint response.