scholarly journals The "enemies within": regions of the genome that are inherently difficult to replicate

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 666 ◽  
Author(s):  
Rahul Bhowmick ◽  
Ian D Hickson
Keyword(s):  
Dna Repair ◽  
Dna Replication ◽  
Genome Organization ◽  
Molecular Basis ◽  
Mammalian Cells ◽  
Fragile Sites ◽  
Chromosome Fragility ◽  
Valuable Role ◽  
Eukaryotic Genomes

An unusual feature of many eukaryotic genomes is the presence of regions that appear intrinsically difficult to copy during the process of DNA replication. Curiously, the location of these difficult-to-replicate regions is often conserved between species, implying a valuable role in some aspect of genome organization or maintenance. The most prominent class of these regions in mammalian cells is defined as chromosome fragile sites, which acquired their name because of a propensity to form visible gaps/breaks on otherwise-condensed chromosomes in mitosis. This fragility is particularly apparent following perturbation of DNA replication—a phenomenon often referred to as “replication stress”. Here, we review recent data on the molecular basis for chromosome fragility and the role of fragile sites in the etiology of cancer. In particular, we highlight how studies on fragile sites have provided unexpected insights into how the DNA repair machinery assists in the completion of DNA replication.

Role of yeast Rth1 nuclease and its homologs in mutation avoidance, DNA repair, and DNA replication

1998 ◽  
Vol 34 (1) ◽  
pp. 21-29 ◽  
Author(s):  
Robert E. Johnson ◽  
Gopala K. Kovvali ◽  
Louise Prakash ◽  
S. Prakash
Keyword(s):  
Dna Repair ◽  
Dna Replication ◽  
Mutation Avoidance

scholarly journals The Protective Role of Dormant Origins in Response to Replicative Stress

2018 ◽  
Vol 19 (11) ◽  
pp. 3569 ◽  
Author(s):  
Lilas Courtot ◽  
Jean-Sébastien Hoffmann ◽  
Valérie Bergoglio
Keyword(s):  
Dna Replication ◽  
Mammalian Cells ◽  
Genome Stability ◽  
Replication Timing ◽  
Protective Role ◽  
Entire Genome ◽  
Replicative Stress ◽  
A Cell ◽  
The Many

Genome stability requires tight regulation of DNA replication to ensure that the entire genome of the cell is duplicated once and only once per cell cycle. In mammalian cells, origin activation is controlled in space and time by a cell-specific and robust program called replication timing. About 100,000 potential replication origins form on the chromatin in the gap 1 (G1) phase but only 20–30% of them are active during the DNA replication of a given cell in the synthesis (S) phase. When the progress of replication forks is slowed by exogenous or endogenous impediments, the cell must activate some of the inactive or “dormant” origins to complete replication on time. Thus, the many origins that may be activated are probably key to protect the genome against replication stress. This review aims to discuss the role of these dormant origins as safeguards of the human genome during replicative stress.

scholarly journals Molecular Basis of Substrate Recognition of Endonuclease Q from the Euryarchaeon Pyrococcus furiosus

2019 ◽  
Vol 202 (2) ◽  
Author(s):  
Miyako Shiraishi ◽  
Shigenori Iwai
Keyword(s):  
Dna Repair ◽  
Bacillus Subtilis ◽  
Substrate Specificity ◽  
Molecular Basis ◽  
Pyrococcus Furiosus ◽  
Substrate Recognition ◽  
Base Flipping ◽  
Content Type ◽  
Cleavage Activity

ABSTRACT Endonuclease Q (EndoQ), a DNA repair endonuclease, was originally identified in the hyperthermophilic euryarchaeon Pyrococcus furiosus in 2015. EndoQ initiates DNA repair by generating a nick on DNA strands containing deaminated bases and an abasic site. Although EndoQ is thought to be important for maintaining genome integrity in certain bacteria and archaea, the underlying mechanism catalyzed by EndoQ remains unclear. Here, we provide insights into the molecular basis of substrate recognition by EndoQ from P. furiosus (PfuEndoQ) using biochemical approaches. Our results of the substrate specificity range and the kinetic properties of PfuEndoQ demonstrate that PfuEndoQ prefers the imide structure in nucleobases along with the discovery of its cleavage activity toward 5,6-dihydrouracil, 5-hydroxyuracil, 5-hydroxycytosine, and uridine in DNA. The combined results for EndoQ substrate binding and cleavage activity analyses indicated that PfuEndoQ flips the target base from the DNA duplex, and the cleavage activity is highly dependent on spontaneous base flipping of the target base. Furthermore, we find that PfuEndoQ has a relatively relaxed substrate specificity; therefore, the role of EndoQ in restriction modification systems was explored. The activity of the EndoQ homolog from Bacillus subtilis was found not to be inhibited by the uracil glycosylase inhibitor from B. subtilis bacteriophage PBS1, whose genome is completely replaced by uracil instead of thymine. Our findings suggest that EndoQ not only has additional functions in DNA repair but also could act as an antiviral enzyme in organisms with EndoQ. IMPORTANCE Endonuclease Q (EndoQ) is a lesion-specific DNA repair enzyme present in certain bacteria and archaea. To date, it remains unclear how EndoQ recognizes damaged bases. Understanding the mechanism of substrate recognition by EndoQ is important to grasp genome maintenance systems in organisms with EndoQ. Here, we find that EndoQ from the euryarchaeon Pyrococcus furiosus recognizes the imide structure in nucleobases by base flipping, and the cleavage activity is enhanced by the base pair instability of the target base, along with the discovery of its cleavage activity toward 5,6-dihydrouracil, 5-hydroxyuracil, 5-hydroxycytosine, and uridine in DNA. Furthermore, a potential role of EndoQ in Bacillus subtilis as an antiviral enzyme by digesting viral genome is demonstrated.

scholarly journals Ribonucleotide reduction is a cytosolic process in mammalian cells independently of DNA damage

2008 ◽  
Vol 105 (46) ◽  
pp. 17801-17806 ◽  
Author(s):  
Giovanna Pontarin ◽  
Artur Fijolek ◽  
Paola Pizzo ◽  
Paola Ferraro ◽  
Chiara Rampazzo ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Mitochondrial Dna ◽  
Dna Replication ◽  
Ribonucleotide Reductase ◽  
Mammalian Cells ◽  
S Phase ◽  
Mt Dna ◽  
Intact Cells ◽  
Mitochondrial Dna Depletion

Ribonucleotide reductase provides deoxynucleotides for nuclear and mitochondrial (mt) DNA replication and repair. The mammalian enzyme consists of a catalytic (R1) and a radical-generating (R2 or p53R2) subunit. During S-phase, a R1/R2 complex is the major provider of deoxynucleotides. p53R2 is induced by p53 after DNA damage and was proposed to supply deoxynucleotides for DNA repair after translocating from the cytosol to the cell nucleus. Similarly R1 and R2 were claimed to move to the nucleus during S-phase to provide deoxynucleotides for DNA replication. These models suggest translocation of ribonucleotide reductase subunits as a regulatory mechanism. In quiescent cells that are devoid of R2, R1/p53R2 synthesizes deoxynucleotides also in the absence of DNA damage. Mutations in human p53R2 cause severe mitochondrial DNA depletion demonstrating a vital function for p53R2 different from DNA repair and cast doubt on a nuclear localization of the protein. Here we use three independent methods to localize R1, R2, and p53R2 in fibroblasts during cell proliferation and after DNA damage: Western blotting after separation of cytosol and nuclei; immunofluorescence in intact cells; and transfection with proteins carrying fluorescent tags. We thoroughly validate each method, especially the specificity of antibodies. We find in all cases that ribonucleotide reductase resides in the cytosol suggesting that the deoxynucleotides produced by the enzyme diffuse into the nucleus or are transported into mitochondria and supporting a primary function of p53R2 for mitochondrial DNA replication.

scholarly journals Functional correlation of H3K9me2 and nuclear compartment formation

2020 ◽  
Author(s):  
Kei Fukuda ◽  
Chikako Shimura ◽  
Hisashi Miura ◽  
Akie Tanigawa ◽  
Takehiro Suzuki ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Genome Organization ◽  
Mammalian Cells ◽  
Embryonic Stem ◽  
Epigenetic Mark ◽  
3 Dimensional ◽  
3D Genome ◽  
Functional Correlation ◽  
Genome Wide

AbstractBackgroundHistone H3 lysine 9 dimethylation (H3K9me2) is a highly conserved silencing epigenetic mark. Chromatin marked with H3K9me2 forms large domains in mammalian cells and correlates well with lamina-associated domains and the B compartment. However, the role of H3K9me2 in 3-dimensional (3D) genome organization remains unclear.ResultsWe investigated the genome-wide H3K9me2 distribution, the transcriptome and 3D genome organization in mouse embryonic stem cells (mESCs) upon the inhibition or depletion of H3K9 methyltransferases (MTases) G9a/GLP, SETDB1, and SUV39H1/2. We found that H3K9me2 is regulated by these five MTases; however, H3K9me2 and transcription in the A and B compartments were largely regulated by different sets of the MTases: H3K9me2 in the A compartments were mainly regulated by G9a/GLP and SETDB1, while H3K9me2 in the B compartments were regulated by all five H3K9 MTases. Furthermore, decreased H3K9me2 correlated with the changes to the more active compartmental state that accompanied transcriptional activation.ConclusionOur data showed that H3K9me2 domain formation is functionally linked to 3D genome organization.

scholarly journals K-fiber bundles in the mitotic spindle are mechanically reinforced by Kif15

2020 ◽  
Author(s):  
Marcus A Begley ◽  
April L Solon ◽  
Elizabeth Mae Davis ◽  
Michael Grant Sherrill ◽  
Ryoma Ohi ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Molecular Basis ◽  
Mammalian Cells ◽  
Fiber Bundles ◽  
Similar Magnitude ◽  
Microtubule Binding ◽  
Mechanical Integrity ◽  
Physical Response

AbstractThe mitotic spindle, a self-constructed microtubule-based machine, segregates chromosomes into two eventual daughter nuclei. In mammalian cells, microtubule bundles called kinetochore-fibers (k-fibers) anchor chromosomes within the spindle. Chromosome segregation thus depends on the mechanical integrity of k-fibers. Here, we investigate the physical and molecular basis of k-fiber bundle cohesion. We sever k-fibers using laser ablation, thereby detaching them from poles and testing the contribution of pole-localized force generation to k-fiber cohesion. We then measure the physical response of the remaining kinetochore-bound segments of the k-fibers. We observe that microtubules within ablated k-fibers often, but not always, splay apart from their minus-ends. Furthermore, we find that minus-end clustering forces induced in response to ablation seem at least partially responsible for k-fiber splaying. We also investigate the role of the putative k-fiber-binding kinesin-12 Kif15. We find that pharmacological inhibition of Kif15 microtubule binding reduces k-fiber mechanical integrity. In contrast, inhibition of its motor activity but not its microtubule binding does not greatly affect splaying. Altogether, the data suggest that forces holding k-fibers together are of similar magnitude to other spindle forces, and that Kif15, acting as a microtubule crosslinker, helps fortify and repair k-fibers. This feature of Kif15 may help support robust k-fiber function and prevent chromosome segregation errors.

scholarly journals Cohesin-Mediated Genome Architecture Does Not Define DNA Replication Timing Domains

Genes ◽  
2019 ◽  
Vol 10 (3) ◽  
pp. 196 ◽  
Author(s):  
Phoebe Oldach ◽  
Conrad A. Nieduszynski
Keyword(s):  
Dna Replication ◽  
Genome Organization ◽  
Mammalian Cells ◽  
Replication Timing ◽  
S Phase ◽  
Genome Architecture ◽  
3D Genome ◽  
Synchronized Cells ◽  
Dna Replication Timing ◽  
Phase Entry

3D genome organization is strongly predictive of DNA replication timing in mammalian cells. This work tested the extent to which loop-based genome architecture acts as a regulatory unit of replication timing by using an auxin-inducible system for acute cohesin ablation. Cohesin ablation in a population of cells in asynchronous culture was shown not to disrupt patterns of replication timing as assayed by replication sequencing (RepliSeq) or BrdU-focus microscopy. Furthermore, cohesin ablation prior to S phase entry in synchronized cells was similarly shown to not impact replication timing patterns. These results suggest that cohesin-mediated genome architecture is not required for the execution of replication timing patterns in S phase, nor for the establishment of replication timing domains in G1.

Bacterial DNA repair genes and their eukaryotic homologues: 4. The role of nucleotide excision DNA repair (NER) system in mammalian cells.

2007 ◽  
Vol 54 (3) ◽  
pp. 469-482 ◽  
Author(s):  
Leena Maddukuri ◽  
Dominika Dudzińska ◽  
Barbara Tudek
Keyword(s):  
Dna Repair ◽  
Mammalian Cells ◽  
Excision Repair ◽  
Wide Spectrum ◽  
Genetic Disorders ◽  
Dna Lesions ◽  
Entire Genome ◽  
Sequence Of Events ◽  
Nucleotide Excision

The eukaryotic cell encounters more than one million various kinds of DNA lesions per day. The nucleotide excision repair (NER) pathway is one of the most important repair mechanisms that removes a wide spectrum of different DNA lesions. NER operates through two sub pathways: global genome repair (GGR) and transcription-coupled repair (TCR). GGR repairs the DNA damage throughout the entire genome and is initiated by the HR23B/XPC complex, while the CSB protein-governed TCR process removes DNA lesions from the actively transcribed strand. The sequence of events and the role of particular NER proteins are currently being extensively discussed. NER proteins also participate in other cellular processes like replication, transcription, chromatin maintenance and protein turnover. Defects in NER underlay severe genetic disorders: xeroderma pigmentosum (XP), Cockayne syndrome (CS) and trichothiodystrophy (TTD).

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