scholarly journals Transcription shapes DNA replication initiation to preserve genome integrity

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Yang Liu ◽  
Chen Ai ◽  
Tingting Gan ◽  
Jinchun Wu ◽  
Yongpeng Jiang ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Mammalian Cells ◽  
Origin Recognition Complex ◽  
Replication Initiation ◽  
Open Chromatin ◽  
Dna Replication Initiation ◽  
Early Replication

Abstract Background Early DNA replication occurs within actively transcribed chromatin compartments in mammalian cells, raising the immediate question of how early DNA replication coordinates with transcription to avoid collisions and DNA damage. Results We develop a high-throughput nucleoside analog incorporation sequencing assay and identify thousands of early replication initiation zones in both mouse and human cells. The identified early replication initiation zones fall in open chromatin compartments and are mutually exclusive with transcription elongation. Of note, early replication initiation zones are mainly located in non-transcribed regions adjacent to transcribed regions. Mechanistically, we find that RNA polymerase II actively redistributes the chromatin-bound mini-chromosome maintenance complex (MCM), but not the origin recognition complex (ORC), to actively restrict early DNA replication initiation outside of transcribed regions. In support of this finding, we detect apparent MCM accumulation and DNA replication initiation in transcribed regions due to anchoring of nuclease-dead Cas9 at transcribed genes, which stalls RNA polymerase II. Finally, we find that the orchestration of early DNA replication initiation by transcription efficiently prevents gross DNA damage. Conclusion RNA polymerase II redistributes MCM complexes, but not the ORC, to prevent early DNA replication from initiating within transcribed regions. This RNA polymerase II-driven MCM redistribution spatially separates transcription and early DNA replication events and avoids the transcription-replication initiation collision, thereby providing a critical regulatory mechanism to preserve genome stability.

scholarly journals How MCM loading and spreading specify eukaryotic DNA replication initiation sites

F1000Research ◽  
2016 ◽  
Vol 5 ◽  
pp. 2063 ◽  
Author(s):  
Olivier Hyrien
Keyword(s):  
Dna Replication ◽  
Mammalian Cells ◽  
Origin Recognition Complex ◽  
Cell Types ◽  
S Phase ◽  
Replication Initiation ◽  
Replication Process ◽  
Dna Replication Initiation ◽  
Eukaryotic Dna ◽  
Transcription Complexes

DNA replication origins strikingly differ between eukaryotic species and cell types. Origins are localized and can be highly efficient in budding yeast, are randomly located in early fly and frog embryos, which do not transcribe their genomes, and are clustered in broad (10-100 kb) non-transcribed zones, frequently abutting transcribed genes, in mammalian cells. Nonetheless, in all cases, origins are established during the G1-phase of the cell cycle by the loading of double hexamers of the Mcm 2-7 proteins (MCM DHs), the core of the replicative helicase. MCM DH activation in S-phase leads to origin unwinding, polymerase recruitment, and initiation of bidirectional DNA synthesis. Although MCM DHs are initially loaded at sites defined by the binding of the origin recognition complex (ORC), they ultimately bind chromatin in much greater numbers than ORC and only a fraction are activated in any one S-phase. Data suggest that the multiplicity and functional redundancy of MCM DHs provide robustness to the replication process and affect replication time and that MCM DHs can slide along the DNA and spread over large distances around the ORC. Recent studies further show that MCM DHs are displaced along the DNA by collision with transcription complexes but remain functional for initiation after displacement. Therefore, eukaryotic DNA replication relies on intrinsically mobile and flexible origins, a strategy fundamentally different from bacteria but conserved from yeast to human. These properties of MCM DHs likely contribute to the establishment of broad, intergenic replication initiation zones in higher eukaryotes.

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 Camptothecin-Induced Replication Stress Affects DNA Replication Profiling by E/L Repli-Seq

2021 ◽  
pp. 1-8
Author(s):  
Takuya Hayakawa ◽  
Rino Suzuki ◽  
Kazuhiro Kagotani ◽  
Katsuzumi Okumura ◽  
Shin-ichiro Takebayashi
Keyword(s):  
Dna Replication ◽  
Mammalian Cells ◽  
Topoisomerase I ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Replication Timing ◽  
Replication Stress ◽  
Replication Initiation ◽  
Domain Structures ◽  
Early Replication

E/L Repli-seq is a powerful tool for detecting cell type-specific replication landscapes in mammalian cells, but its potential to monitor DNA replication under replication stress awaits better understanding. Here, we used E/L Repli-seq to examine the temporal order of DNA replication in human retinal pigment epithelium cells treated with the topoisomerase I inhibitor camptothecin. We found that the replication profiles by E/L Repli-seq exhibit characteristic patterns after replication-stress induction, including the loss of specific initiation zones within individual early replication timing domains. We also observed global disappearance of the replication timing domain structures in the profiles, which can be explained by checkpoint-dependent suppression of replication initiation. Thus, our results demonstrate the effectiveness of E/L Repli-seq at identifying cells with replication-stress-induced altered DNA replication programs.

scholarly journals Genomic Study of Replication Initiation in Human Chromosomes Reveals the Influence of Transcription Regulation and Chromatin Structure on Origin Selection

2010 ◽  
Vol 21 (3) ◽  
pp. 393-404 ◽  
Author(s):  
Neerja Karnani ◽  
Christopher M. Taylor ◽  
Ankit Malhotra ◽  
Anindya Dutta
Keyword(s):  
Rna Polymerase Ii ◽  
Chromatin Structure ◽  
Binding Sites ◽  
Transcription Initiation ◽  
Origin Recognition Complex ◽  
Replication Initiation ◽  
Open Chromatin ◽  
Lambda Exonuclease ◽  
Genomic Study ◽  
Exonuclease Digestion

DNA replication in metazoans initiates from multiple chromosomal loci called origins. Currently, there are two methods to purify origin-centered nascent strands: lambda exonuclease digestion and anti-bromodeoxyuridine immunoprecipitation. Because both methods have unique strengths and limitations, we purified nascent strands by both methods, hybridized them independently to tiling arrays (1% genome) and compared the data to have an accurate view of genome-wide origin distribution. By this criterion, we identified 150 new origins that were reproducible across the methods. Examination of a subset of these origins by chromatin immunoprecipitation against origin recognition complex (ORC) subunits 2 and 3 showed 93% of initiation peaks to localize at/within 1 kb of ORC binding sites. Correlation of origins with functional elements of the genome revealed origin activity to be significantly enriched around transcription start sites (TSSs). Consistent with proximity to TSSs, we found a third of initiation events to occur at or near the RNA polymerase II binding sites. Interestingly, ∼50% of the early origin activity was localized within 5 kb of transcription regulatory factor binding region clusters. The chromatin signatures around the origins were enriched in H3K4-(di- and tri)-methylation and H3 acetylation modifications on histones. Affinity of origins for open chromatin was also reiterated by their proximity to DNAse I-hypersensitive sites. Replication initiation peaks were AT rich, and >50% of the origins mapped to evolutionarily conserved regions of the genome. In summary, these findings indicate that replication initiation is influenced by transcription initiation and regulation as well as chromatin structure.

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 Nuclear Organization of DNA Replication Initiation Proteins in Mammalian Cells

2002 ◽  
Vol 277 (12) ◽  
pp. 10354-10361 ◽  
Author(s):  
Masatoshi Fujita ◽  
Yukio Ishimi ◽  
Hiromu Nakamura ◽  
Tohru Kiyono ◽  
Tatsuya Tsurumi
Keyword(s):  
Dna Replication ◽  
Mammalian Cells ◽  
Nuclear Organization ◽  
Replication Initiation ◽  
Dna Replication Initiation ◽  
Replication Initiation Proteins

scholarly journals Wwp2-Mediated Ubiquitination of the RNA Polymerase II Large Subunit in Mouse Embryonic Pluripotent Stem Cells

2007 ◽  
Vol 27 (15) ◽  
pp. 5296-5305 ◽  
Author(s):  
Hui Li ◽  
Zhihong Zhang ◽  
Beibei Wang ◽  
Junmei Zhang ◽  
Yingming Zhao ◽  
...  
Keyword(s):  
Dna Damage ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Mammalian Cells ◽  
Large Subunit ◽  
Terminal Domain ◽  
Ubiquitin E3 Ligase ◽  
Lysine Residues

ABSTRACT Ubiquitination and the degradation of the large subunit of RNA polymerase II, Rpb1, is not only involved in DNA damage-induced arrest but also in other transcription-obstructing events. However, the ubiquitin ligases responsible for DNA damage-independent processes in mammalian cells remain to be identified. Here, we identified Wwp2, a mouse HECT domain ubiquitin E3 ligase, as a novel ubiquitin ligase of Rpb1. We found that Wwp2 specifically interacted with mouse Rpb1 and targeted it for ubiquitination both in vitro and in vivo. Interestingly, the interaction with and ubiquitination of Rpb1 was dependent neither on its phosphorylation state nor on DNA damage. However, the enzymatic activity of Wwp2 was absolutely required for its ubiquitin modification of Rpb1. Furthermore, our study indicates that the interaction between Wwp2 and Rpb1 was mediated through WW domain of Wwp2 and C-terminal domain of Rpb1, respectively. Strikingly, downregulation of Wwp2 expression compromised Rpb1 ubiquitination and elevated its intracellular steady-state protein level significantly. Importantly, we identified six lysine residues in the C-terminal domain of Rpb1 as ubiquitin acceptor sites mediated by Wwp2. These results indicate that Wwp2 plays an important role in regulating expression of Rpb1 in normal physiological conditions.

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