scholarly journals MCMBP maintains genome integrity by protecting the MCM subunits from degradation

2019 ◽  
Author(s):  
Venny Santosa ◽  
Masato T. Kanemaki
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Transcriptional Activation ◽  
Damage Accumulation ◽  
Nuclear Protein ◽  
De Novo ◽  
Genome Integrity ◽  
De Novo Synthesis ◽  
Eukaryotic Dna ◽  
High Level

AbstractThe hetero-hexameric MCM2–7 helicase plays a central role in eukaryotic DNA replication. The expression of MCM2–7 is maintained at a high level for creating dormant origins, which are important for maintaining genome integrity. However, other than transcriptional activation for the de novo synthesis, little is known about how cells maintain a high level of MCM2–7. We show that human MCMBP is a short-lived nuclear protein associating mainly with MCM5, 6, and 7. Loss of MCMBP down-regulates MCM2–7, leading to replication stress and DNA-damage accumulation. Our work demonstrates MCMBP protects the MCM subunits from degradation and suggests its chaperone-like role to achieve a high level of functional MCM2–7 using the nascent and recycled subunits.

scholarly journals Human PrimPol Discrimination against Dideoxynucleotides during Primer Synthesis

Genes ◽  
2021 ◽  
Vol 12 (10) ◽  
pp. 1487
Author(s):  
Gustavo Carvalho ◽  
Alberto Díaz-Talavera ◽  
Patricia A. Calvo ◽  
Luis Blanco ◽  
María I. Martínez-Jiménez
Keyword(s):  
Dna Replication ◽  
De Novo ◽  
Manganese Ions ◽  
De Novo Synthesis ◽  
Replicative Stress ◽  
Mitochondrial Dna Replication ◽  
Low Efficiency ◽  
A Chain ◽  
Dna Primers ◽  
Fold Excess

PrimPol is required to re-prime DNA replication at both nucleus and mitochondria, thus facilitating fork progression during replicative stress. ddC is a chain-terminating nucleotide that has been widely used to block mitochondrial DNA replication because it is efficiently incorporated by the replicative polymerase Polγ. Here, we show that human PrimPol discriminates against dideoxynucleotides (ddNTP) when elongating a primer across 8oxoG lesions in the template, but also when starting de novo synthesis of DNA primers, and especially when selecting the 3′nucleotide of the initial dimer. PrimPol incorporates ddNTPs with a very low efficiency compared to dNTPs even in the presence of activating manganese ions, and only a 40-fold excess of ddNTP would significantly disturb PrimPol primase activity. This discrimination against ddNTPs prevents premature termination of the primers, warranting their use for elongation. The crystal structure of human PrimPol highlights Arg291 residue as responsible for the strong dNTP/ddNTP selectivity, since it interacts with the 3′-OH group of the incoming deoxynucleotide, absent in ddNTPs. Arg291, shown here to be critical for both primase and polymerase activities of human PrimPol, would contribute to the preferred binding of dNTPs versus ddNTPs at the 3′elongation site, thus avoiding synthesis of abortive primers.

scholarly journals Cdc6 degradation requires phosphodegron created by GSK-3 and Cdk1 for SCFCdc4 recognition in Saccharomyces cerevisiae

2015 ◽  
Vol 26 (14) ◽  
pp. 2609-2619 ◽  
Author(s):  
Amr Al-Zain ◽  
Lea Schroeder ◽  
Alina Sheglov ◽  
Amy E. Ikui
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Ubiquitin Ligase ◽  
Genome Integrity ◽  
Binding Motif ◽  
Dependent Manner ◽  
Cyclin Dependent Kinase ◽  
Origin Firing ◽  
C Terminus ◽  
Prereplicative Complex

To ensure genome integrity, DNA replication takes place only once per cell cycle and is tightly controlled by cyclin-dependent kinase (Cdk1). Cdc6p is part of the prereplicative complex, which is essential for DNA replication. Cdc6 is phosphorylated by cyclin-Cdk1 to promote its degradation after origin firing to prevent DNA rereplication. We previously showed that a yeast GSK-3 homologue, Mck1 kinase, promotes Cdc6 degradation in a SCFCdc4-dependent manner, therefore preventing rereplication. Here we present evidence that Mck1 directly phosphorylates a GSK-3 consensus site in the C-terminus of Cdc6. The Mck1-dependent Cdc6 phosphorylation required priming by cyclin/Cdk1 at an adjacent CDK consensus site. The sequential phosphorylation by Mck1 and Clb2/Cdk1 generated a Cdc4 E3 ubiquitin ligase–binding motif to promote Cdc6 degradation during mitosis. We further revealed that Cdc6 degradation triggered by Mck1 kinase was enhanced upon DNA damage caused by the alkylating agent methyl methanesulfonate and that the resulting degradation was mediated through Cdc4. Thus, Mck1 kinase ensures proper DNA replication, prevents DNA damage, and maintains genome integrity by inhibiting Cdc6.

Regulatory Factors Associated with Synthesis of the Osmolyte Glycine Betaine in the Halophilic MethanoarchaeonMethanohalophilus portucalensis

1999 ◽  
Vol 65 (2) ◽  
pp. 828-833 ◽  
Author(s):  
Mei-Chin Lai ◽  
Daw-Renn Yang ◽  
Ming-Jen Chuang
Keyword(s):  
Glycine Betaine ◽  
De Novo ◽  
Compatible Solutes ◽  
In Vitro Assays ◽  
Intracellular Accumulation ◽  
De Novo Synthesis ◽  
Factors Associated ◽  
High Level

ABSTRACT The halophilic methanoarchaeon Methanohalophilus portucalensis can synthesize de novo and accumulate β-glutamine, N ɛ-acetyl-β-lysine, and glycine betaine (betaine) as compatible solutes (osmolytes) when grown at elevated salt concentrations. Both in vivo and in vitro betaine formation assays in this study confirmed previous nuclear magnetic resonance 13C-labelling studies showing that the de novo synthesis of betaine proceeded from glycine, sarcosine, and dimethylglycine to form betaine through threefold methylation. Exogenous sarcosine (1 mM) effectively suppressed the intracellular accumulation of betaine, and a higher level of sarcosine accumulation was accompanied by a lower level of betaine synthesis. Exogenous dimethylglycine has an effect similar to that of betaine addition, which increased the intracellular pool of betaine and suppressed the levels of N ɛ-acetyl-β-lysine and β-glutamine. Both in vivo and in vitro betaine formation assays with glycine as the substrate showed only sarcosine and betaine, but no dimethylglycine. Dimethylglycine was detected only when it was added as a substrate in in vitro assays. A high level of potassium (400 mM and above) was necessary for betaine formation in vitro. Interestingly, no methylamines were detected without the addition of KCl. Also, high levels of NaCl and LiCl (800 mM) favored sarcosine accumulation, while a lower level (400 mM) favored betaine synthesis. The above observations indicate that a high sarcosine level suppressed multiple methylation while dimethylglycine was rapidly converted to betaine. Also, high levels of potassium led to greater amounts of betaine, while lower levels of potassium led to greater amounts of sarcosine. This finding suggests that the intracellular levels of both sarcosine and potassium are associated with the regulation of betaine synthesis inM. portucalensis.

scholarly journals C. elegans THSC/TREX-2 deficiency causes replication stress and genome instability

2021 ◽  
Author(s):  
Angelina Zheleva ◽  
Lola P Camino ◽  
Nuria Fernández-Fernández ◽  
María García-Rubio ◽  
Peter Askjaer ◽  
...  
Keyword(s):  
Dna Damage ◽  
Damage Accumulation ◽  
Genome Instability ◽  
Underlying Mechanism ◽  
Rnase H ◽  
Genome Integrity ◽  
Dna Damage Checkpoint ◽  
C Elegans ◽  
Mrnp Biogenesis

Transcription is an essential process of DNA metabolism, yet it makes DNA more susceptible to DNA damage. THSC/TREX-2 is a conserved eukaryotic protein complex with a key role in mRNP biogenesis and maturation that prevents genome instability. One source of such instability is linked to transcription as shown in yeast and human cells, but the underlying mechanism and whether is universal is still unclear. To get further insight in the putative role of THSC/TREX-2 in genome integrity we have used Caenorhabditis elegans mutants of the THP-1 and DSS-1 members of THSC/TREX-2. These mutants show similar defective meiosis, DNA damage accumulation and activation of the DNA damage checkpoint. However, they differ regarding replication defects as determined by dUTP incorporation in the germline. Interestingly, this specific thp-1 phenotype can be partially rescued by overexpression of RNase H. Furthermore, both mutants show a mild increase in the H3S10P mark previously shown to be linked to DNA-RNA hybrid-mediated genome instability. These data support the view that both THSC/TREX-2 factors prevent transcription-associated DNA damage derived from DNA-RNA hybrid accumulation by separate means.

scholarly journals Topoisomerase 1 dependent R-loop deficiency as a mechanism underlying oncogene-induced replication stress and genomic instability

2020 ◽  
Author(s):  
Dan Sarni ◽  
Alon Shtrikman ◽  
Yifat S. Oren ◽  
Batsheva Kerem
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Genomic Instability ◽  
Cancer Progression ◽  
Replication Stress ◽  
Genome Integrity ◽  
Replication Rate ◽  
Topoisomerase 1 ◽  
Replicative Stress ◽  
Complex Process

AbstractDNA replication is a complex process that is tightly regulated to ensure faithful genome duplication, and its perturbation leads to DNA damage and genomic instability. Oncogene expression triggers replicative stress that can lead to genetic instability, driving cancer progression. Thus, revealing the molecular basis for oncogene-induced replication stress is important for understanding of oncogenesis. Here we show that the activation of mutated HRAS leads to a non-canonical replication stress characterized by accelerated replication rate, inducing DNA damage. Mutated HRAS increases topoisomerase 1 (TOP1) expression, which leads to reduced levels of RNA-DNA hybrids (R-loops), driving fork acceleration and damage formation. Restoration of the perturbed replication either by restoration of TOP1 levels or directly by mild replication inhibition results in a dramatic reduction in DNA damage. The findings highlight the importance of TOP1 equilibrium in the regulation of R-loop homeostasis to ensure faithful DNA replication and genome integrity that when dysregulated can be a mechanism of oncogene-induced DNA damage.

scholarly journals Role of Histone Methylation in Maintenance of Genome Integrity

2021 ◽  
Author(s):  
Arjamand Mushtaq ◽  
Ulfat Syed Mir ◽  
Clayton R Hunt ◽  
Shruti Pandita ◽  
Wajahat W Tantray ◽  
...  
Keyword(s):  
Dna Damage ◽  
Histone Methylation ◽  
Genomic Stability ◽  
Genome Integrity ◽  
Post Translational Modifications ◽  
Damage Repair ◽  
Repair Protein ◽  
Eukaryotic Dna ◽  
Cellular Pathways

Packaging of the eukaryotic DNA genome with histone and other proteins forms a chromatin structure that regulates the outcome of all DNA mediated processes. The cellular pathways that ensure genomic stability detect and repair DNA damage through mechanisms which are critically dependent upon chromatin structures established by histones and, particularly, transient histone post-translational modifications . Though subject to a range of modifications, histone methylation is especially crucial for DNA damage repair as the methylated histones often form platforms for subsequent repair protein binding at damaged sites. In this review, we highlight and discuss how histone methylation impacts the maintenance of genome integrity through effects related to DNA repair and repair pathway choice.

Diverse functions of BRCA1 in the DNA damage response

2001 ◽  
Vol 3 (15) ◽  
pp. 1-11 ◽  
Author(s):  
Jong-Soo Lee ◽  
Jay H. Chung
Keyword(s):  
Breast Cancer ◽  
Dna Damage ◽  
Dna Damage Response ◽  
Nuclear Protein ◽  
Genome Integrity ◽  
Breast Cancers ◽  
Common Causes ◽  
Damage Response ◽  
Associated Proteins ◽  
Related Mortality

Breast cancer is one of the most common causes of cancer-related mortality in women. It affects more than one in nine women over their lifetime. Although most breast cancers are sporadic, the genetics and molecular biology of the heritable forms of breast cancer have provided valuable insights into not only breast cancer but also cancers in general. Among the mutations linked to heritable breast cancers, the mutations in the breast cancer 1 (BRCA1) gene are the best characterised. The BRCA1 gene encodes a nuclear protein that is important for maintaining genome integrity. However, a growing list of BRCA1-associated proteins suggests that BRCA1 has diverse and unexpected functions.

Characterization of Monocyte-Associated Factor V

1993 ◽  
Vol 70 (02) ◽  
pp. 273-280 ◽  
Author(s):  
Janos Kappelmayer ◽  
Satya P Kunapuli ◽  
Edward G Wyshock ◽  
Robert W Colman
Keyword(s):  
Monoclonal Antibody ◽  
Light Chain ◽  
Peripheral Blood ◽  
De Novo ◽  
Factor V ◽  
Blood Monocytes ◽  
De Novo Synthesis ◽  
Hep G2 ◽  
Hep G2 Cells ◽  
Peripheral Blood Monocytes

SummaryWe demonstrate that in addition to possessing binding sites for intact factor V (FV), unstimulated peripheral blood monocytes also express activated factor V (FVa) on their surfaces. FVa was identified on the monocyte surface by monoclonal antibody B38 recognizing FVa light chain and by human oligoclonal antibodies H1 (to FVa light chain) and H2 (to FVa heavy chain) using immunofluorescence microscopy and flow cytometry. On Western blots, partially cleaved FV could be identified as a 220 kDa band in lysates of monocytes. In addition to surface expression of FVa, monocytes also contain intracellular FV as detected only after permeabilization by Triton X-100 by monoclonal antibody B10 directed specifically to the Cl domain not present in FVa. We sought to determine whether the presence of FV in peripheral blood monocytes is a result of de novo synthesis.Using in situ hybridization, no FV mRNA could be detected in monocytes, while in parallel control studies, factor V mRNA was detectable in Hep G2 cells and CD18 mRNA in monocytes. In addition, using reverse transcriptase and the polymerase chain reaction, no FV mRNA was detected in mononuclear cells or in U937 cells, but mRNA for factor V was present in Hep G2 cells using the same techniques. These data suggest that FV is present in human monocytes, presumably acquired by binding of plasma FV, and that the presence of this critical coagulation factor is not due to de novo synthesis.

Platelets Stimulate Thromboplastin Synthesis in Human Endothelial Cells

1983 ◽  
Vol 49 (02) ◽  
pp. 069-072 ◽  
Author(s):  
U L H Johnsen ◽  
T Lyberg ◽  
K S Galdal ◽  
H Prydz
Keyword(s):  
Endothelial Cells ◽  
De Novo ◽  
Umbilical Vein ◽  
Time Dependent ◽  
De Novo Synthesis ◽  
Human Endothelial Cells ◽  
Tumor Promotor ◽  
Coagulation Assay ◽  
Platelet Aggregates ◽  
Umbilical Vein Endothelial Cells

SummaryHuman umbilical vein endothelial cells in culture synthesize thromboplastin upon stimulation with phytohaemagglutinin (PHA) or the tumor promotor 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The thromboplastin activity is further strongly enhanced in a time dependent reaction by the presence of gel-filtered platelets or platelet aggregates. This effect was demonstrable at platelet concentrations lower than those normally found in plasma, it may thus be of pathophysiological relevance. The thromboplastin activity increased with increasing number of platelets added. Cycloheximide inhibited the increase, suggesting that de novo synthesis of the protein component of thromboplastin, apoprotein III, is necessary.When care was taken to remove monocytes no thromboplastin activity and no apoprotein HI antigen could be demonstrated in suspensions of gel-filtered platelets, platelets aggregated with thrombin or homogenized platelets when studied with a coagulation assay and an antibody neutralization technique.

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