scholarly journals Molecular mechanisms of eukaryotic origin initiation, replication fork progression, and chromatin maintenance

2020 ◽  
Vol 477 (18) ◽  
pp. 3499-3525
Author(s):  
Zuanning Yuan ◽  
Huilin Li
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Replication Fork ◽  
Dna Methyltransferase ◽  
Molecular Mechanisms ◽  
Methylation Pattern ◽  
Replication Initiation ◽  
Nucleosome Assembly ◽  
Epigenetic Information ◽  
Eukaryotic Origin

Eukaryotic DNA replication is a highly dynamic and tightly regulated process. Replication involves several dozens of replication proteins, including the initiators ORC and Cdc6, replicative CMG helicase, DNA polymerase α-primase, leading-strand DNA polymerase ε, and lagging-strand DNA polymerase δ. These proteins work together in a spatially and temporally controlled manner to synthesize new DNA from the parental DNA templates. During DNA replication, epigenetic information imprinted on DNA and histone proteins is also copied to the daughter DNA to maintain the chromatin status. DNA methyltransferase 1 is primarily responsible for copying the parental DNA methylation pattern into the nascent DNA. Epigenetic information encoded in histones is transferred via a more complex and less well-understood process termed replication-couple nucleosome assembly. Here, we summarize the most recent structural and biochemical insights into DNA replication initiation, replication fork elongation, chromatin assembly and maintenance, and related regulatory mechanisms.

scholarly journals Replication fork dynamics and the DNA damage response

2012 ◽  
Vol 443 (1) ◽  
pp. 13-26 ◽  
Author(s):  
Rebecca M. Jones ◽  
Eva Petermann
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Dna Damage Response ◽  
Replication Fork ◽  
Molecular Mechanisms ◽  
S Phase ◽  
Replication Initiation ◽  
Developmental Defects ◽  
Replication Fork Progression ◽  
Damage Response

Prevention and repair of DNA damage is essential for maintenance of genomic stability and cell survival. DNA replication during S-phase can be a source of DNA damage if endogenous or exogenous stresses impair the progression of replication forks. It has become increasingly clear that DNA-damage-response pathways do not only respond to the presence of damaged DNA, but also modulate DNA replication dynamics to prevent DNA damage formation during S-phase. Such observations may help explain the developmental defects or cancer predisposition caused by mutations in DNA-damage-response genes. The present review focuses on molecular mechanisms by which DNA-damage-response pathways control and promote replication dynamics in vertebrate cells. In particular, DNA damage pathways contribute to proper replication by regulating replication initiation, stabilizing transiently stalled forks, promoting replication restart and facilitating fork movement on difficult-to-replicate templates. If replication fork progression fails to be rescued, this may lead to DNA damage and genomic instability via nuclease processing of aberrant fork structures or incomplete sister chromatid separation during mitosis.

scholarly journals Separable, Ctf4-mediated recruitment of DNA Polymerase α for initiation of DNA synthesis at replication origins and lagging-strand priming during replication elongation

2018 ◽  
Author(s):  
Sarina Y. Porcella ◽  
Natasha C. Koussa ◽  
Colin P. Tang ◽  
Daphne N. Kramer ◽  
Priyanka Srivastava ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Replication Fork ◽  
Replication Initiation ◽  
Replication Origins ◽  
Origin Firing ◽  
Leading Strand ◽  
Strand Initiation ◽  
Lagging Strand

AbstractDuring eukaryotic DNA replication, DNA polymerase alpha/primase (Pol α) initiates synthesis on both the leading and lagging strands. It is unknown whether leading- and lagging-strand priming are mechanistically identical, and whether Pol α associates processively or distributively with the replisome. Here, we titrate cellular levels of Pol α in S. cerevisiae and analyze Okazaki fragments to study both replication initiation and ongoing lagging-strand synthesis in vivo. We observe that both Okazaki fragment initiation and the productive firing of replication origins are sensitive to Pol α abundance, and that both processes are disrupted at similar Pol α concentrations. When the replisome adaptor protein Ctf4 is absent or cannot interact with Pol α, lagging-strand initiation is impaired at Pol α concentrations that still support normal origin firing. Additionally, we observe that activation of the checkpoint becomes essential for viability upon severe depletion of Pol α. Using strains in which the Pol α-Ctf4 interaction is disrupted, we demonstrate that this checkpoint requirement is not solely caused by reduced lagging-strand priming. Our results suggest that Pol α recruitment for replication initiation and ongoing lagging-strand priming are distinctly sensitive to the presence of Ctf4. We propose that the global changes we observe in Okazaki fragment length and origin firing efficiency are consistent with distributive association of Pol α at the replication fork, at least when Pol α is limiting.Author summaryHalf of each eukaryotic genome is replicated continuously as the leading strand, while the other half is synthesized discontinuously as Okazaki fragments on the lagging strand. The bulk of DNA replication is completed by DNA polymerases ε and δ on the leading and lagging strand respectively, while synthesis on each strand is initiated by DNA polymerase α-primase (Pol α). Using the model eukaryote S. cerevisiae, we modulate cellular levels of Pol α and interrogate the impact of this perturbation on both replication initiation on DNA synthesis and cellular viability. We observe that Pol α can associate dynamically at the replication fork for initiation on both strands. Although the initiation of both strands is widely thought to be mechanistically similar, we determine that Ctf4, a hub that connects proteins to the replication fork, stimulates lagging-strand priming to a greater extent than leading-strand initiation. We also find that decreased leading-strand initiation results in a checkpoint response that is necessary for viability when Pol α is limiting. Because the DNA replication machinery is highly conserved from budding yeast to humans, this research provides insights into how DNA replication is accomplished throughout eukaryotes.

Implications of DNA replication for eukaryotic gene expression

1991 ◽  
Vol 99 (2) ◽  
pp. 201-206 ◽  
Author(s):  
A.P. Wolffe
Keyword(s):  
Gene Expression ◽  
Dna Replication ◽  
Replication Fork ◽  
Recent Progress ◽  
Gene Activity ◽  
Nucleosome Assembly ◽  
Developmental Processes ◽  
Eukaryotic Gene Expression ◽  
Eukaryotic Gene

DNA replication has a key role in many developmental processes. Recent progress in understanding events at the replication fork suggests mechanisms for both establishing and maintaining programs of eukaryotic gene activity. In this review, I discuss the consequences of replication fork passage for preexisting chromatin structures and describe how the mechanism of nucleosome assembly at the replication fork may facilitate the formation of either transcriptionally active or repressed chromatin.

scholarly journals Nascent chromatin occupancy profiling reveals locus and factor specific chromatin maturation dynamics behind the DNA replication fork

2018 ◽  
Author(s):  
Mónica P. Gutiérrez ◽  
Heather K. MacAlpine ◽  
David M. MacAlpine
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Histone Variants ◽  
Replication Forks ◽  
Nucleosome Assembly ◽  
Genome Wide ◽  
Dna Binding Factors ◽  
Nucleotide Resolution ◽  
Kinetics Of ◽  
Regulatory Landscape

AbstractProper regulation and maintenance of the epigenome is necessary to preserve genome function. However, in every cell division, the epigenetic state is disassembled and then re-assembled in the wake of the DNA replication fork. Chromatin restoration on nascent DNA is a complex and regulated process that includes nucleosome assembly and remodeling, deposition of histone variants, and the re-establishment of transcription factor binding. To study the genome-wide dynamics of chromatin restoration behind the DNA replication fork, we developed Nascent Chromatin Occupancy Profiles (NCOPs) to comprehensively profile nascent and mature chromatin at nucleotide resolution. While nascent chromatin is inherently less organized than mature chromatin, we identified locus specific differences in the kinetics of chromatin maturation that were predicted by the epigenetic landscape, including the histone variant H2A.Z which marked loci with rapid maturation kinetics. The chromatin maturation at origins of DNA replication was dependent on whether the origin underwent initiation or was passively replicated from distal-originating replication forks suggesting distinct chromatin assembly mechanisms between activated and disassembled pre-replicative complexes. Finally, we identified sites that were only occupied transiently by DNA-binding factors following passage of the replication fork which may provide a mechanism for perturbations of the DNA replication program to shape the regulatory landscape of the genome.

scholarly journals Oxidative DNA Damage Modulates DNA Methylation Pattern in Human Breast Cancer 1 (BRCA1) Gene via the Crosstalk between DNA Polymerase β and a de novo DNA Methyltransferase

Cells ◽  
2020 ◽  
Vol 9 (1) ◽  
pp. 225 ◽  
Author(s):  
Zhongliang Jiang ◽  
Yanhao Lai ◽  
Jill M. Beaver ◽  
Pawlos S. Tsegay ◽  
Ming-Lang Zhao ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Dna Methylation ◽  
Dna Damage ◽  
Dna Polymerase ◽  
Dna Methyltransferase ◽  
Oxidative Dna Damage ◽  
De Novo ◽  
Brca1 Gene ◽  
Methylation Pattern ◽  
Dna Polymerase Β

DNA damage and base excision repair (BER) are actively involved in the modulation of DNA methylation and demethylation. However, the underlying molecular mechanisms remain unclear. In this study, we seek to understand the mechanisms by exploring the effects of oxidative DNA damage on the DNA methylation pattern of the tumor suppressor breast cancer 1 (BRCA1) gene in the human embryonic kidney (HEK) HEK293H cells. We found that oxidative DNA damage simultaneously induced DNA demethylation and generation of new methylation sites at the CpGs located at the promoter and transcribed regions of the gene ranging from −189 to +27 in human cells. We demonstrated that DNA damage-induced demethylation was mediated by nucleotide misincorporation by DNA polymerase β (pol β). Surprisingly, we found that the generation of new DNA methylation sites was mediated by coordination between pol β and the de novo DNA methyltransferase, DNA methyltransferase 3b (DNMT3b), through the interaction between the two enzymes in the promoter and encoding regions of the BRCA1 gene. Our study provides the first evidence that oxidative DNA damage can cause dynamic changes in DNA methylation in the BRCA1 gene through the crosstalk between BER and de novo DNA methylation.

scholarly journals The Role of Metabolites in the Link between DNA Replication and Central Carbon Metabolism in Escherichia coli

Genes ◽  
2020 ◽  
Vol 11 (4) ◽  
pp. 447
Author(s):  
Klaudyna Krause ◽  
Monika Maciąg-Dorszyńska ◽  
Anna Wosinski ◽  
Lidia Gaffke ◽  
Joanna Morcinek-Orłowska ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Protein Interactions ◽  
Carbon Metabolism ◽  
Molecular Mechanisms ◽  
Central Carbon Metabolism ◽  
Replication Initiation ◽  
Cell Physiology ◽  
E Coli ◽  
Central Carbon

A direct link between DNA replication regulation and central carbon metabolism (CCM) has been previously demonstrated in Bacillus subtilis and Escherichia coli, as effects of certain mutations in genes coding for replication proteins could be specifically suppressed by particular mutations in genes encoding CCM enzymes. However, specific molecular mechanism(s) of this link remained unknown. In this report, we demonstrate that various CCM metabolites can suppress the effects of mutations in different replication genes of E. coli on bacterial growth, cell morphology, and nucleoid localization. This provides evidence that the CCM-replication link is mediated by metabolites rather than direct protein-protein interactions. On the other hand, action of metabolites on DNA replication appears indirect rather than based on direct influence on the replication machinery, as rate of DNA synthesis could not be corrected by metabolites in short-term experiments. This corroborates the recent discovery that in B. subtilis, there are multiple links connecting CCM to DNA replication initiation and elongation. Therefore, one may suggest that although different in detail, the molecular mechanisms of CCM-dependent regulation of DNA replication are similar in E. coli and B. subtilis, making this regulation an important and common constituent of the control of cell physiology in bacteria.

scholarly journals Escherichia coli Cells with Increased Levels of DnaA and Deficient in Recombinational Repair Have Decreased Viability

2003 ◽  
Vol 185 (2) ◽  
pp. 630-644 ◽  
Author(s):  
Aline V. Grigorian ◽  
Rachel B. Lustig ◽  
Elena C. Guzmán ◽  
Joseph M. Mahaffy ◽  
Judith W. Zyskind
Keyword(s):  
Escherichia Coli ◽  
Dna Polymerase ◽  
Replication Fork ◽  
Replication Initiation ◽  
Recombinational Repair ◽  
Replication Forks ◽  
Dna Replication Initiation ◽  
Escherichia Coli Cells ◽  
Repair Pathways ◽  
Stalled Replication Forks

ABSTRACT The dnaA operon of Escherichia coli contains the genes dnaA, dnaN, and recF encoding DnaA, β clamp of DNA polymerase III holoenzyme, and RecF. When the DnaA concentration is raised, an increase in the number of DNA replication initiation events but a reduction in replication fork velocity occurs. Because DnaA is autoregulated, these results might be due to the inhibition of dnaN and recF expression. To test this, we examined the effects of increasing the intracellular concentrations of DnaA, β clamp, and RecF, together and separately, on initiation, the rate of fork movement, and cell viability. The increased expression of one or more of the dnaA operon proteins had detrimental effects on the cell, except in the case of RecF expression. A shorter C period was not observed with increased expression of the β clamp; in fact, many chromosomes did not complete replication in runout experiments. Increased expression of DnaA alone resulted in stalled replication forks, filamentation, and a decrease in viability. When the three proteins of the dnaA operon were simultaneously overexpressed, highly filamentous cells were observed (>50 μm) with extremely low viability and, in runout experiments, most chromosomes had not completed replication. The possibility that recombinational repair was responsible for the survival of cells overexpressing DnaA was tested by using mutants in different recombinational repair pathways. The absence of RecA, RecB, RecC, or the proteins in the RuvABC complex caused an additional ∼100-fold drop in viability in cells with increased levels of DnaA, indicating a requirement for recombinational repair in these 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.

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