scholarly journals Transcription drives DNA replication initiation and termination in human cells

2018 ◽  
Author(s):  
Yu-Hung Chen ◽  
Sarah Keegan ◽  
Malik Kahli ◽  
Peter Tonzi ◽  
David Fenyö ◽  
...  
Keyword(s):  
Dna Replication ◽  
Rna Polymerase Ii ◽  
Replication Fork ◽  
Human Cells ◽  
Replication Initiation ◽  
Origin Firing ◽  
Dna Replication Initiation ◽  
Origin Activation ◽  
Dna Replication Origins ◽  
The Impact

ABSTRACTThe locations of active DNA replication origins in the human genome, and the determinants of origin activation, remain controversial. Additionally, neither the predominant sites of replication termination nor the impact of transcription on replication-fork mobility have been defined. We demonstrate that replication initiation occurs preferentially in the immediate vicinity of the transcription start site of genes occupied by high levels of RNA polymerase II, ensuring co-directional replication of the most highly transcribed genes. Further, we demonstrate that dormant replication origin firing represents the global activation of pre-existing origins. We also show that DNA replication naturally terminates at the polyadenylation site of transcribed genes. During replication stress, termination is redistributed to gene bodies, generating a global reorientation of replication relative to transcription. Our analysis provides a unified model for the coupling of transcription with replication initiation and termination in human cells.

scholarly journals Activation of the DNA Damage Checkpoint in Mutants Defective in DNA Replication Initiation

2008 ◽  
Vol 19 (10) ◽  
pp. 4374-4382 ◽  
Author(s):  
Ling Yin ◽  
Alexandra Monica Locovei ◽  
Gennaro D'Urso
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Replication Fork ◽  
S Phase ◽  
Replication Initiation ◽  
Dna Damage Checkpoint ◽  
Origin Firing ◽  
Dna Replication Initiation ◽  
Fork Stalling ◽  
S Phase Checkpoint

In the fission yeast, Schizosaccharomyces pombe, blocks to DNA replication elongation trigger the intra-S phase checkpoint that leads to the activation of the Cds1 kinase. Cds1 is required to both prevent premature entry into mitosis and to stabilize paused replication forks. Interestingly, although Cds1 is essential to maintain the viability of mutants defective in DNA replication elongation, mutants defective in DNA replication initiation require the Chk1 kinase. This suggests that defects in DNA replication initiation can lead to activation of the DNA damage checkpoint independent of the intra-S phase checkpoint. This might result from reduced origin firing that leads to an increase in replication fork stalling or replication fork collapse that activates the G2 DNA damage checkpoint. We refer to the Chk1-dependent, Cds1-independent phenotype as the rid phenotype (for replication initiation defective). Chk1 is active in rid mutants, and rid mutant viability is dependent on the DNA damage checkpoint, and surprisingly Mrc1, a protein required for activation of Cds1. Mutations in Mrc1 that prevent activation of Cds1 have no effect on its ability to support rid mutant viability, suggesting that Mrc1 has a checkpoint-independent role in maintaining the viability of mutants defective in DNA replication initiation.

scholarly journals Multiple pathways can bypass the essential role of fission yeast Hsk1 kinase in DNA replication initiation

2011 ◽  
Vol 195 (3) ◽  
pp. 387-401 ◽  
Author(s):  
Seiji Matsumoto ◽  
Motoshi Hayano ◽  
Yutaka Kanoh ◽  
Hisao Masai
Keyword(s):  
Dna Replication ◽  
Fission Yeast ◽  
Replication Fork ◽  
Essential Role ◽  
Replication Initiation ◽  
Origin Firing ◽  
Physiological Conditions ◽  
Dna Replication Initiation ◽  
Initiation Of Dna Replication

Cdc7/Hsk1 is a conserved kinase required for initiation of DNA replication that potentially regulates timing and locations of replication origin firing. Here, we show that viability of fission yeast hsk1Δ cells can be restored by loss of mrc1, which is required for maintenance of replication fork integrity, by cds1Δ, or by a checkpoint-deficient mutant of mrc1. In these mutants, normally inactive origins are activated in the presence of hydroxyurea and binding of Cdc45 to MCM is stimulated. mrc1Δ bypasses hsk1Δ more efficiently because of its checkpoint-independent inhibitory functions. Unexpectedly, hsk1Δ is viable at 37°C. More DNA is synthesized, and some dormant origins fire in the presence of hydroxyurea at 37°C. Furthermore, hsk1Δ bypass strains grow poorly at 25°C compared with higher temperatures. Our results show that Hsk1 functions for DNA replication can be bypassed by different genetic backgrounds as well as under varied physiological conditions, providing additional evidence for plasticity of the replication program in eukaryotes.

scholarly journals Human RECQ1 and RECQ4 Helicases Play Distinct Roles in DNA Replication Initiation

2010 ◽  
Vol 30 (6) ◽  
pp. 1382-1396 ◽  
Author(s):  
Saravanabhavan Thangavel ◽  
Ramiro Mendoza-Maldonado ◽  
Erika Tissino ◽  
Julia M. Sidorova ◽  
Jinhu Yin ◽  
...  
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Replication Initiation ◽  
Replication Origins ◽  
Origin Firing ◽  
Recq Helicases ◽  
Dna Replication Initiation ◽  
Replication Fork Progression ◽  
A Cell

ABSTRACT Cellular and biochemical studies support a role for all five human RecQ helicases in DNA replication; however, their specific functions during this process are unclear. Here we investigate the in vivo association of the five human RecQ helicases with three well-characterized human replication origins. We show that only RECQ1 (also called RECQL or RECQL1) and RECQ4 (also called RECQL4) associate with replication origins in a cell cycle-regulated fashion in unperturbed cells. RECQ4 is recruited to origins at late G1, after ORC and MCM complex assembly, while RECQ1 and additional RECQ4 are loaded at origins at the onset of S phase, when licensed origins begin firing. Both proteins are lost from origins after DNA replication initiation, indicating either disassembly or tracking with the newly formed replisome. Nascent-origin DNA synthesis and the frequency of origin firing are reduced after RECQ1 depletion and, to a greater extent, after RECQ4 depletion. Depletion of RECQ1, though not that of RECQ4, also suppresses replication fork rates in otherwise unperturbed cells. These results indicate that RECQ1 and RECQ4 are integral components of the human replication complex and play distinct roles in DNA replication initiation and replication fork progression in vivo.

scholarly journals DNA Replication Origins Fire Stochastically in Fission Yeast

2006 ◽  
Vol 17 (1) ◽  
pp. 308-316 ◽  
Author(s):  
Prasanta K. Patel ◽  
Benoit Arcangioli ◽  
Stephen P. Baker ◽  
Aaron Bensimon ◽  
Nicholas Rhind
Keyword(s):  
Dna Replication ◽  
Fission Yeast ◽  
Replication Initiation ◽  
Epigenetic Mechanism ◽  
Replication Origins ◽  
Origin Firing ◽  
Cell Cycles ◽  
Initiation Of Replication ◽  
Dna Combing ◽  
Dna Replication Origins

DNA replication initiates at discrete origins along eukaryotic chromosomes. However, in most organisms, origin firing is not efficient; a specific origin will fire in some but not all cell cycles. This observation raises the question of how individual origins are selected to fire and whether origin firing is globally coordinated to ensure an even distribution of replication initiation across the genome. We have addressed these questions by determining the location of firing origins on individual fission yeast DNA molecules using DNA combing. We show that the firing of replication origins is stochastic, leading to a random distribution of replication initiation. Furthermore, origin firing is independent between cell cycles; there is no epigenetic mechanism causing an origin that fires in one cell cycle to preferentially fire in the next. Thus, the fission yeast strategy for the initiation of replication is different from models of eukaryotic replication that propose coordinated origin firing.

scholarly journals Topologically associating domain boundaries are enriched in early firing origins and restrict replication fork progression

2020 ◽  
Author(s):  
Emilia Puig Lombardi ◽  
Madalena Tarsounas
Keyword(s):  
Binding Sites ◽  
Replication Fork ◽  
Replication Timing ◽  
Replication Initiation ◽  
Ctcf Binding ◽  
Origin Firing ◽  
Replication Fork Progression ◽  
Fork Stalling ◽  
Genomic Regions ◽  
The Impact

ABSTRACTTopologically associating domains (TADs) are units of the genome architecture defined by binding sites for the CTCF transcription factor and cohesin-mediated loop extrusion. Genomic regions containing DNA replication initiation sites have been mapped in the proximity of TAD boundaries. However, the factors that determine this positioning have not been identified. Moreover, the impact of TADs on the directionality of replication fork progression remains unknown. Here we use EdU-seq technology to map origin firing sites at 10 kb resolution and to monitor replication fork progression after restart from hydroxyurea arrest. We show that origins firing in early/mid S-phase within TAD boundaries map to two distinct peaks flanking the centre of the boundary, which is occupied by CTCF and cohesin. When transcription is inhibited chemically or deregulated by oncogene overexpression, replication origins become repositioned to the centre of the TAD. Furthermore, we demonstrate the strikingly asymmetric fork progression initiating from origins located within TAD boundaries. Divergent CTCF binding sites and neighbouring TADs with different replication timing (RT) cause fork stalling in regions external to the TAD. Thus, our work assigns for the first time a role to transcription within TAD boundaries in promoting replication origin firing and demonstrates how genomic regions adjacent to the TAD boundaries could restrict replication progression.

scholarly journals Investigating the role of Rts1 in DNA replication initiation

2018 ◽  
Vol 3 ◽  
pp. 23 ◽  
Author(s):  
Ana B.A. Wallis ◽  
Conrad A. Nieduszynski
Keyword(s):  
Dna Replication ◽  
Temperature Sensitivity ◽  
Protein Phosphatase ◽  
Dna Helicase ◽  
Replication Initiation ◽  
Replication Origins ◽  
Origin Firing ◽  
Replication Factor ◽  
Dna Replication Initiation ◽  
Genomic Screen

Background: Understanding DNA replication initiation is essential to understand the mis-regulation of replication seen in cancer and other human disorders. DNA replication initiates from DNA replication origins. In eukaryotes, replication is dependent on cell cycle kinases which function during S phase. Dbf4-dependent kinase (DDK) and cyclin-dependent kinase (CDK) act to phosphorylate the DNA helicase (composed of mini chromosome maintenance proteins: Mcm2-7) and firing factors to activate replication origins. It has recently been found that Rif1 can oppose DDK phosphorylation. Rif1 can recruit protein phosphatase 1 (PP1) to dephosphorylate MCM and restricts origin firing. In this study, we investigate a potential role for another phosphatase, protein phosphatase 2A (PP2A), in regulating DNA replication initiation. The PP2A regulatory subunit Rts1 was previously identified in a large-scale genomic screen to have a genetic interaction with ORC2 (a DNA replication licensing factor). Deletion of RTS1 synthetically rescued the temperature-sensitive (ts-) phenotype of ORC2 mutants. Methods: We deleted RTS1 in multiple ts-replication factor Saccharomyces cerevisiae strains, including ORC2.  Dilution series assays were carried out to compare qualitatively the growth of double mutant ∆rts1 ts-replication factor strains relative to the respective single mutant strains.   Results: No synthetic rescue of temperature-sensitivity was observed. Instead we found an additive phenotype, indicating gene products function in separate biological processes. These findings are in agreement with a recent genomic screen which found that RTS1 deletion in several ts-replication factor strains led to increased temperature-sensitivity. Conclusions: We find no evidence that Rts1 is involved in the dephosphorylation of DNA replication initiation factors.

Major DNA replication initiation sites in thec-myc locus in human cells

2000 ◽  
Vol 78 (3) ◽  
pp. 442-457 ◽  
Author(s):  
Liang Tao ◽  
Zhifeng Dong ◽  
Michael Leffak ◽  
Maria Zannis-Hadjopoulos ◽  
Gerald Price
Keyword(s):  
Dna Replication ◽  
Human Cells ◽  
Replication Initiation ◽  
Dna Replication Initiation
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