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19 pages, 3172 KiB

Open AccessArticle

CSB Regulates Pathway Choice in Response to DNA Replication Stress Induced by Camptothecin

by Nicole L. Batenburg, John R. Walker and Xu-Dong Zhu

Int. J. Mol. Sci. 2023, 24(15), 12419; https://doi.org/10.3390/ijms241512419 - 4 Aug 2023

Cited by 1 | Viewed by 2441

Topoisomerase inhibitor camptothecin (CPT) induces fork stalling and is highly toxic to proliferating cells. However, how cells respond to CPT-induced fork stalling has not been fully characterized. Here, we report that Cockayne syndrome group B (CSB) protein inhibits PRIMPOL-dependent fork repriming in response [...] Read more.

Topoisomerase inhibitor camptothecin (CPT) induces fork stalling and is highly toxic to proliferating cells. However, how cells respond to CPT-induced fork stalling has not been fully characterized. Here, we report that Cockayne syndrome group B (CSB) protein inhibits PRIMPOL-dependent fork repriming in response to a low dose of CPT. At a high concentration of CPT, CSB is required to promote the restart of DNA replication through MUS81–RAD52–POLD3-dependent break-induced replication (BIR). In the absence of CSB, resumption of DNA synthesis at a high concentration of CPT can occur through POLQ–LIG3-, LIG4-, or PRIMPOL-dependent pathways, which are inhibited, respectively, by RAD51, BRCA1, and BRCA2 proteins. POLQ and LIG3 are core components of alternative end joining (Alt-EJ), whereas LIG4 is a core component of nonhomologous end joining (NHEJ). These results suggest that CSB regulates fork restart pathway choice following high-dosage CPT-induced fork stalling, promoting BIR but inhibiting Alt-EJ, NHEJ, and fork repriming. We find that loss of CSB and BRCA2 is a toxic combination to genomic stability and cell survival at a high concentration of CPT, which is likely due to accumulation of ssDNA gaps, underscoring an important role of CSB in regulating the therapy response in cancers lacking functional BRCA2. Full article

(This article belongs to the Special Issue Latest Progress in DNA Damage and DNA Repair)

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29 pages, 2462 KiB

Open AccessReview

The Adaptive Mechanisms and Checkpoint Responses to a Stressed DNA Replication Fork

by Joanne Saldanha, Julie Rageul, Jinal A. Patel and Hyungjin Kim

Int. J. Mol. Sci. 2023, 24(13), 10488; https://doi.org/10.3390/ijms241310488 - 22 Jun 2023

Cited by 7 | Viewed by 4761

Abstract

DNA replication is a tightly controlled process that ensures the faithful duplication of the genome. However, DNA damage arising from both endogenous and exogenous assaults gives rise to DNA replication stress associated with replication fork slowing or stalling. Therefore, protecting the stressed fork [...] Read more.

DNA replication is a tightly controlled process that ensures the faithful duplication of the genome. However, DNA damage arising from both endogenous and exogenous assaults gives rise to DNA replication stress associated with replication fork slowing or stalling. Therefore, protecting the stressed fork while prompting its recovery to complete DNA replication is critical for safeguarding genomic integrity and cell survival. Specifically, the plasticity of the replication fork in engaging distinct DNA damage tolerance mechanisms, including fork reversal, repriming, and translesion DNA synthesis, enables cells to overcome a variety of replication obstacles. Furthermore, stretches of single-stranded DNA generated upon fork stalling trigger the activation of the ATR kinase, which coordinates the cellular responses to replication stress by stabilizing the replication fork, promoting DNA repair, and controlling cell cycle and replication origin firing. Deregulation of the ATR checkpoint and aberrant levels of chronic replication stress is a common characteristic of cancer and a point of vulnerability being exploited in cancer therapy. Here, we discuss the various adaptive responses of a replication fork to replication stress and the roles of ATR signaling that bring fork stabilization mechanisms together. We also review how this knowledge is being harnessed for the development of checkpoint inhibitors to trigger the replication catastrophe of cancer cells. Full article

(This article belongs to the Special Issue Endogenous DNA Damage and Repair)

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28 pages, 2136 KiB

Open AccessReview

Regulation of Cardiac Cav1.2 Channels by Calmodulin

by Masaki Kameyama, Etsuko Minobe, Dongxue Shao, Jianjun Xu, Qinghua Gao and Liying Hao

Int. J. Mol. Sci. 2023, 24(7), 6409; https://doi.org/10.3390/ijms24076409 - 29 Mar 2023

Cited by 7 | Viewed by 3998

Abstract

Cav1.2 Ca²⁺ channels, a type of voltage-gated L-type Ca²⁺ channel, are ubiquitously expressed, and the predominant Ca²⁺ channel type, in working cardiac myocytes. Cav1.2 channels are regulated by the direct interactions with calmodulin (CaM), a Ca²⁺-binding protein that [...] Read more.

Cav1.2 Ca²⁺ channels, a type of voltage-gated L-type Ca²⁺ channel, are ubiquitously expressed, and the predominant Ca²⁺ channel type, in working cardiac myocytes. Cav1.2 channels are regulated by the direct interactions with calmodulin (CaM), a Ca²⁺-binding protein that causes Ca²⁺-dependent facilitation (CDF) and inactivation (CDI). Ca²⁺-free CaM (apoCaM) also contributes to the regulation of Cav1.2 channels. Furthermore, CaM indirectly affects channel activity by activating CaM-dependent enzymes, such as CaM-dependent protein kinase II and calcineurin (a CaM-dependent protein phosphatase). In this article, we review the recent progress in identifying the role of apoCaM in the channel ‘rundown’ phenomena and related repriming of channels, and CDF, as well as the role of Ca²⁺/CaM in CDI. In addition, the role of CaM in channel clustering is reviewed. Full article

(This article belongs to the Special Issue Calcium Channels and Calcium-Binding Proteins)

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26 pages, 6801 KiB

Open AccessFeature PaperEditor’s ChoiceReview

PrimPol: A Breakthrough among DNA Replication Enzymes and a Potential New Target for Cancer Therapy

by Alberto Díaz-Talavera, Cristina Montero-Conde, Luis Javier Leandro-García and Mercedes Robledo

Biomolecules 2022, 12(2), 248; https://doi.org/10.3390/biom12020248 - 3 Feb 2022

Cited by 10 | Viewed by 6172 | Correction

Abstract

DNA replication can encounter blocking obstacles, leading to replication stress and genome instability. There are several mechanisms for evading this blockade. One mechanism consists of repriming ahead of the obstacles, creating a new starting point; in humans, PrimPol is responsible for carrying out [...] Read more.

DNA replication can encounter blocking obstacles, leading to replication stress and genome instability. There are several mechanisms for evading this blockade. One mechanism consists of repriming ahead of the obstacles, creating a new starting point; in humans, PrimPol is responsible for carrying out this task. PrimPol is a primase that operates in both the nucleus and mitochondria. In contrast with conventional primases, PrimPol is a DNA primase able to initiate DNA synthesis de novo using deoxynucleotides, discriminating against ribonucleotides. In vitro, PrimPol can act as a DNA primase, elongating primers that PrimPol itself sythesizes, or as translesion synthesis (TLS) DNA polymerase, elongating pre-existing primers across lesions. However, the lack of evidence for PrimPol polymerase activity in vivo suggests that PrimPol only acts as a DNA primase. Here, we provide a comprehensive review of human PrimPol covering its biochemical properties and structure, in vivo function and regulation, and the processes that take place to fill the gap-containing lesion that PrimPol leaves behind. Finally, we explore the available data on human PrimPol expression in different tissues in physiological conditions and its role in cancer. Full article

(This article belongs to the Collection Feature Papers in Molecular Genetics)

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17 pages, 1859 KiB

Open AccessArticle

Human PrimPol Discrimination against Dideoxynucleotides during Primer Synthesis

by Gustavo Carvalho, Alberto Díaz-Talavera, Patricia A. Calvo, Luis Blanco and María I. Martínez-Jiménez

Genes 2021, 12(10), 1487; https://doi.org/10.3390/genes12101487 - 24 Sep 2021

Cited by 6 | Viewed by 2568

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue Mechanisms of Replication of Damaged DNA)

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13 pages, 2059 KiB

Open AccessArticle

Interleukin-15 after Near-Infrared Photoimmunotherapy (NIR-PIT) Enhances T Cell Response against Syngeneic Mouse Tumors

by Yasuhiro Maruoka, Aki Furusawa, Ryuhei Okada, Fuyuki Inagaki, Hiroaki Wakiyama, Takuya Kato, Tadanobu Nagaya, Peter L. Choyke and Hisataka Kobayashi

Cancers 2020, 12(9), 2575; https://doi.org/10.3390/cancers12092575 - 10 Sep 2020

Cited by 28 | Viewed by 3604

Abstract

Near infrared photoimmunotherapy (NIR-PIT) is a newly developed and highly selective cancer treatment that employs a monoclonal antibody (mAb) conjugated to a photo-absorber dye, IRDye700DX, which is activated by 690 nm light. Cancer cell-targeted NIR-PIT induces rapid necrotic/immunogenic cell death (ICD) that induces [...] Read more.

Near infrared photoimmunotherapy (NIR-PIT) is a newly developed and highly selective cancer treatment that employs a monoclonal antibody (mAb) conjugated to a photo-absorber dye, IRDye700DX, which is activated by 690 nm light. Cancer cell-targeted NIR-PIT induces rapid necrotic/immunogenic cell death (ICD) that induces antitumor host immunity including re-priming and proliferation of T cells. Interleukin-15 (IL-15) is a cytokine that activates natural killer (NK)-, B- and T-cells while having minimal effect on regulatory T cells (Tregs) that lack the IL-15 receptor. Here, we hypothesized that IL-15 administration with cancer cell-targeted NIR-PIT could further inhibit tumor growth by increasing antitumor host immunity. Three syngeneic mouse tumor models, MC38-luc, LL/2, and MOC1, underwent combined CD44-targeted NIR-PIT and short-term IL-15 administration with appropriate controls. Comparing with the single-agent therapy, the combination therapy of IL-15 after NIR-PIT inhibited tumor growth, prolonged survival, and increased tumor infiltrating CD8+ T cells more efficiently in tumor-bearing mice. IL-15 appears to enhance the therapeutic effect of cancer-targeted NIR-PIT. Full article

(This article belongs to the Special Issue Challenges and Opportunities for Effective Cancer Immunotherapies)

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12 pages, 2774 KiB

Open AccessArticle

Two Novel Peptide Toxins from the Spider Cyriopagopus longipes Inhibit Tetrodotoxin-Sensitive Sodium Channels

by Qingfeng Zhang, Yuxin Si, Li Yang, Li Wang, Shuijiao Peng, Yiming Chen, Minzhi Chen, Xi Zhou and Zhonghua Liu

Toxins 2020, 12(9), 529; https://doi.org/10.3390/toxins12090529 - 19 Aug 2020

Cited by 7 | Viewed by 4159

Abstract

Sodium channels play a critical role in the generation and propagation of action potentials in excitable tissues, such as nerves, cardiac muscle, and skeletal muscle, and are the primary targets of toxins found in animal venoms. Here, two novel peptide toxins (Cl6a and [...] Read more.

Sodium channels play a critical role in the generation and propagation of action potentials in excitable tissues, such as nerves, cardiac muscle, and skeletal muscle, and are the primary targets of toxins found in animal venoms. Here, two novel peptide toxins (Cl6a and Cl6b) were isolated from the venom of the spider Cyriopagopus longipes and characterized. Cl6a and Cl6b were shown to be inhibitors of tetrodotoxin-sensitive (TTX-S), but not TTX-resistant, sodium channels. Among the TTX-S channels investigated, Cl6a and Cl6b showed the highest degree of inhibition against NaV1.7 (half-maximal inhibitory concentration (IC₅₀) of 11.0 ± 2.5 nM and 18.8 ± 2.4 nM, respectively) in an irreversible manner that does not alter channel activation, inactivation, or repriming kinetics. Moreover, analysis of NaV1.7/NaV1.8 chimeric channels revealed that Cl6b is a site 4 neurotoxin. Site-directed mutagenesis analysis indicated that D816, V817, and E818 observably affected the efficacy of the Cl6b-NaV1.7 interaction, suggesting that these residues might directly affect the interaction of NaV1.7 with Cl6b. Taken together, these two novel peptide toxins act as potent and sustained NaV1.7 blockers and may have potential in the pharmacological study of sodium channels. Full article

(This article belongs to the Special Issue Animal Venoms and Their Components: Molecular Mechanisms of Action)

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15 pages, 1617 KiB

Open AccessReview

Mechanisms of DNA Replication and Repair: Insights from the Study of G-Quadruplexes

by Tracy M. Bryan

Molecules 2019, 24(19), 3439; https://doi.org/10.3390/molecules24193439 - 22 Sep 2019

Cited by 67 | Viewed by 8672

Abstract

G-quadruplexes are four-stranded guanine-rich structures that have been demonstrated to occur across the genome in humans and other organisms. They provide regulatory functions during transcription, translation and immunoglobulin gene rearrangement, but there is also a large amount of evidence that they can present [...] Read more.

G-quadruplexes are four-stranded guanine-rich structures that have been demonstrated to occur across the genome in humans and other organisms. They provide regulatory functions during transcription, translation and immunoglobulin gene rearrangement, but there is also a large amount of evidence that they can present a potent barrier to the DNA replication machinery. This mini-review will summarize recent advances in understanding the many strategies nature has evolved to overcome G-quadruplex-mediated replication blockage, including removal of the structure by helicases or nucleases, or circumventing the deleterious effects on the genome through homologous recombination, alternative end-joining or synthesis re-priming. Paradoxically, G-quadruplexes have also recently been demonstrated to provide a positive role in stimulating the initiation of DNA replication. These recent studies have not only illuminated the many roles and consequences of G-quadruplexes, but have also provided fundamental insights into the general mechanisms of DNA replication and its links with genetic and epigenetic stability. Full article

(This article belongs to the Special Issue Recent Advances in Non-Canonical Nucleic Acid Structures)

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14 pages, 2686 KiB

Open AccessEditor’s ChoiceArticle

Dehydrocrenatidine Inhibits Voltage-Gated Sodium Channels and Ameliorates Mechanic Allodia in a Rat Model of Neuropathic Pain

by Fang Zhao, Qinglian Tang, Jian Xu, Shuangyan Wang, Shaoheng Li, Xiaohan Zou and Zhengyu Cao

Toxins 2019, 11(4), 229; https://doi.org/10.3390/toxins11040229 - 18 Apr 2019

Cited by 16 | Viewed by 4363

Abstract

Picrasma quassioides (D. Don) Benn, a medical plant, is used in clinic to treat inflammation, pain, sore throat, and eczema. The alkaloids are the main active components in P. quassioides. In this study, we examined the analgesic effect of dehydrocrenatidine (DHCT), a [...] Read more.

Picrasma quassioides (D. Don) Benn, a medical plant, is used in clinic to treat inflammation, pain, sore throat, and eczema. The alkaloids are the main active components in P. quassioides. In this study, we examined the analgesic effect of dehydrocrenatidine (DHCT), a β-carboline alkaloid abundantly found in P. quassioides in a neuropathic pain rat model of a sciatic nerve chronic constriction injury. DHCT dose-dependently attenuated the mechanic allodynia. In acutely isolated dorsal root ganglion, DHCT completely suppressed the action potential firing. Further electrophysiological characterization demonstrated that DHCT suppressed both tetrodotoxin-resistant (TTX-R) and sensitive (TTX-S) voltage-gated sodium channel (VGSC) currents with IC₅₀ values of 12.36 μM and 4.87 µM, respectively. DHCT shifted half-maximal voltage (V_1/2) of inactivation to hyperpolarizing direction by ~16.7 mV in TTX-S VGSCs. In TTX-R VGSCs, DHCT shifted V_1/2 of inactivation voltage to hyperpolarizing direction and V_1/2 of activation voltage to more depolarizing potential by ~23.9 mV and ~12.2 mV, respectively. DHCT preferred to interact with an inactivated state of VGSCs and prolonged the repriming time in both TTX-S and TTX-R VGSCs, transiting the channels into a slow inactivated state from a fast inactivated state. Considered together, these data demonstrated that the analgesic effect of DHCT was likely though the inhibition of neuronal excitability. Full article

(This article belongs to the Special Issue Biological Activities of Alkaloids: From Toxicology to Pharmacology)

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25 pages, 2860 KiB

Open AccessReview

PrimPol—Prime Time to Reprime

by Thomas A. Guilliam and Aidan J. Doherty

Genes 2017, 8(1), 20; https://doi.org/10.3390/genes8010020 - 6 Jan 2017

Cited by 49 | Viewed by 14836

Abstract

The complex molecular machines responsible for genome replication encounter many obstacles during their progression along DNA. Tolerance of these obstructions is critical for efficient and timely genome duplication. In recent years, primase-polymerase (PrimPol) has emerged as a new player involved in maintaining eukaryotic [...] Read more.

The complex molecular machines responsible for genome replication encounter many obstacles during their progression along DNA. Tolerance of these obstructions is critical for efficient and timely genome duplication. In recent years, primase-polymerase (PrimPol) has emerged as a new player involved in maintaining eukaryotic replication fork progression. This versatile replicative enzyme, a member of the archaeo-eukaryotic primase (AEP) superfamily, has the capacity to perform a range of template-dependent and independent synthesis activities. Here, we discuss the emerging roles of PrimPol as a leading strand repriming enzyme and describe the mechanisms responsible for recruiting and regulating the enzyme during this process. This review provides an overview and update of the current PrimPol literature, as well as highlighting unanswered questions and potential future avenues of investigation. Full article

(This article belongs to the Special Issue DNA Replication Controls)

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