Faculty Opinions recommendation of Defective telomere lagging strand synthesis in cells lacking WRN helicase activity.

Enteroviruses belong to the genus Enterovirus of the family Picornaviridae and include four human enterovirus groups (EV A-D), and the epidemic of enteroviruses such as human enterovirus A71 (EV-A71) and Coxsackievirus-A16 (CVA16) is a threat to global public health. Enteroviral 2C is the most conserved nonstructural protein among all enteroviruses and possesses the RNA helicase activity that plays pivotal roles during enteroviral life cycles, which makes 2C an attractive target for developing the anti-enteroviral drugs. In this study, we designed a peptide, named 2CL, based on the structure of EV-A71 2C. This peptide effectively impaired the oligomerization of EV-A71 2C protein, and inhibited the RNA helicase activities of 2C proteins encoded by EV-A71 and CVA16, and both of which belong to EV-A, and showed potent antiviral efficacy against EV-A71 and CVA16 in cells. Moreover, the 2CL treatment elicited a strong in vivo protective efficacy against lethal EV-A71 challenge. Besides, the antiviral strategy of targeting the 2C helicase activity can be applied to inhibit the replication of EV-B. Either 2CL or B-2CL, the peptide redesigned based on the 2CL-corresponding sequence of EV-Bs, exerted effective antiviral activity against two important EV-Bs, Coxsackievirus B3 and Echovirus 11. Together, our findings demonstrated that targeting the helicase activity of 2C by rationally designed peptide is an efficient antiviral strategy against enteroviruses, and the 2CL and B-2CL showed promising clinical potentials to be further developed as broad-spectrum anti-enteroviral drugs. Importance Enteroviruses are a large group of positive-sense single-stranded RNA viruses, and include numerous human pathogens, such as enterovirus A71 (EV-A71), coxsackieviruses, and echoviruses. However, no approved antiviral drug is available. Enteroviral 2C is the most conserved nonstructural protein among all enteroviruses and contains the RNA helicase activity critical for the viral life cycle. Herein, according to the structure of EV-A71 2C, we designed a peptide that effectively inhibited the RNA helicase activities of EV-A71-and coxsackievirus A16 (CVA16)-encoded 2C proteins. Moreover, this peptide exerted potent antiviral effects against EV-A71 and CVA16 in cells and elicited therapeutic efficacy against lethal EV-A71 challenge in vivo. Furthermore, we demonstrated that the strategy of targeting the 2C helicase activity can be used to other relevant enteroviruses, including coxsackievirus B3 and echovirus 11. In summary, our findings provide compelling evidence that the designed peptides targeting the helicase activity of 2C could be broad-spectrum antiviral for enteroviruses.

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Dna2 Mutants Reveal Interactions with Dna Polymerase α and Ctf4, a Pol α Accessory Factor, and Show That Full Dna2 Helicase Activity Is Not Essential for Growth

Genetics ◽

10.1093/genetics/151.4.1459 ◽

1999 ◽

Vol 151 (4) ◽

pp. 1459-1470 ◽

Cited By ~ 8

Author(s):

Tim Formosa ◽

Thalia Nittis

Keyword(s):

Dna Polymerase ◽

Synthetic Lethality ◽

Helicase Domain ◽

Helicase Activity ◽

Yeast Saccharomyces Cerevisiae ◽

Α Subunit ◽

Dna Polymerase Α ◽

Accessory Factor ◽

Related Proteins ◽

Lagging Strand

Abstract Mutations in the gene for the conserved, essential nuclease-helicase Dna2 from the yeast Saccharomyces cerevisiae were found to interact genetically with POL1 and CTF4, which encode a DNA Polymerase α subunit and an associated protein, suggesting that Dna2 acts in a process that involves Pol α. DNA2 alleles were isolated that cause either temperature sensitivity, sensitivity to alkylation damage, or both. The alkylation-sensitive alleles clustered in the helicase domain, including changes in residues required for helicase activity in related proteins. Additional mutations known or expected to destroy the ATPase and helicase activities of Dna2 were constructed and found to support growth on some media but to cause alkylation sensitivity. Only damage-sensitive alleles were lethal in combination with a ctf4 deletion. Full activity of the Dna2 helicase function is therefore not needed for viability, but is required for repairing damage and for tolerating loss of Ctf4. Arrest of dna2 mutants was RAD9 dependent, but deleting this checkpoint resulted in either no effect or suppression of defects, including the synthetic lethality with ctf4. Dna2 therefore appears to act in repair or lagging strand synthesis together with Pol α and Ctf4, in a role that is optimal with, but does not require, full helicase activity.

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BCR/ABL Stimulates WRN Helicase Activity To Facilitate DNA Double-Strand Breaks Repair.

Blood ◽

10.1182/blood.v110.11.1024.1024 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1024-1024

Author(s):

Artur Slupianek ◽

Stanislaw Jozwiakowski ◽

Dariusz Pytel ◽

Tomasz Poplawski ◽

Michal Nowicki ◽

...

Keyword(s):

Dna Damage ◽

Werner Syndrome ◽

Genotoxic Stress ◽

Double Strand Breaks ◽

Leukemia Cells ◽

Helicase Activity ◽

Strand Breaks ◽

Increased Sensitivity ◽

Repair Pathways ◽

Wrn Helicase

Abstract DNA damage and defects in DNA repair pathways may severely predispose to genomic instability which is one of the major factors associated with the formation and progression of chronic myelogenous leukemia (CML). However, leukemia cells transfected by BCR/ABL seem to be better equipped to survive DNA damage generated by reactive oxygen species (ROS) and external factors through activation of double-strand breaks (DSBs) repair by homologous recombination (HR) and non-homologous end-joining (NHEJ). A genome-wide screen was performed to identify genes regulated by BCR/ABL kinase and involved in DSBs repair. Werner syndrome protein (WRN), which exhibits helicase and exonuclease activity, was upregulated in CML cells. WRN is capable of unwinding various DNA structures associated with progressing replication forks as well as promoting Holliday junctions formed as intermediates in DNA recombination. Moreover, the helicase can directly interact with a variety of proteins involved in DSBs repair including Ku complex (NHEJ), and RAD51 (HR). Lack of WRN protein in Werner syndrome is characterized by accumulation of DSBs, genomic instability and a high incidence of cancer. Here we present evidence that BCR/ABL induced the expression of WRN mRNA and protein by activation of c-MYC transcription and inhibition of caspase-dependent cleavage, respectively. Immunoprecipitation and pull-down studies indicated that WRN is phosphorylated by BCR/ABL, and that BCR/ABL SH2 domain interacts directly with phospho-Y1346 of WRN. Drug sensitivity assays performed after downregulation of WRN expression by shWRN in BCR/ABL-positive cells have demonstrated an increased sensitivity to genotoxic stress induced by cisplatin and oxidative stress caused by H2O2. Experiments using TUR90010 lymphoblast cell line established from a Werner syndrome patient (3724C>T) and transfected with BCR/ABL confirmed that WRN plays an important role in response to DNA damage in CML cells. Further studies revealed that BCR/ABL-positive leukemia cells exert an enhanced WRN-mediated helicase activity. Bone marrow cells derived from transgenic mice expressing the helicase-defective WRN mutant (K577M) and transfected with BCR/ABL display increased sensitivity to cisplatin compared to those obtained from the wild-type littermates. The role of WRN in BCR/ABL-induced DSBs repair pathways, HR and NHEJ, was examined. NHEJ activity was measured in nuclear cell lysates of BCR/ABL-positive leukemia cells using linearized double-stranded plasmid as a substrate. Removal of WRN by immunoprecipitation did not affect the efficacy of NHEJ reaction. HR was assessed using cells containing one copy of the modified gene for GFP containing a unique I-SceI restriction site with two stop codons as a recombination reporter and a truncated fragment of the GFP gene as a template for homologous repair. A HR event restores functional GFP expression. Downregulation of WRN protein by shRNA abrogated HR activity induced by BCR/ABL. Therefore BCR/ABL-dependent overexpression of WRN helicase seemed to be important for HR, but not NHEJ. Finally, an enhanced interaction between WRN and RAD51 upon DNA damage in BCR/ABL-positive cells supported that conclusion. In summary, BCR/ABL-mediated overexpression and enhanced activation of WRN helicase played an essential role in response of CML cells to elevated numbers of DBSs induced by oxidative and genotoxic stress.

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Mcm10 promotes rapid isomerization of CMG-DNA for replisome bypass of lagging strand DNA blocks

eLife ◽

10.7554/elife.29118 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 41

Author(s):

Lance D Langston ◽

Ryan Mayle ◽

Grant D Schauer ◽

Olga Yurieva ◽

Daniel Zhang ◽

...

Keyword(s):

Cell Types ◽

The Other ◽

Replication Protein ◽

Helicase Activity ◽

Central Channel ◽

Steric Exclusion ◽

Dna Strand ◽

Dna Complex ◽

Lagging Strand

Replicative helicases in all cell types are hexameric rings that unwind DNA by steric exclusion in which the helicase encircles the tracking strand only and excludes the other strand from the ring. This mode of translocation allows helicases to bypass blocks on the strand that is excluded from the central channel. Unlike other replicative helicases, eukaryotic CMG helicase partially encircles duplex DNA at a forked junction and is stopped by a block on the non-tracking (lagging) strand. This report demonstrates that Mcm10, an essential replication protein unique to eukaryotes, binds CMG and greatly stimulates its helicase activity in vitro. Most significantly, Mcm10 enables CMG and the replisome to bypass blocks on the non-tracking DNA strand. We demonstrate that bypass occurs without displacement of the blocks and therefore Mcm10 must isomerize the CMG-DNA complex to achieve the bypass function.

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Cytoplasmic Tubules in Cells Infected with Laryngotracheitis Virus

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100063792 ◽

1969 ◽

Vol 27 ◽

pp. 376-377

Author(s):

A. M. Watrach

Keyword(s):

Tissue Culture ◽

Small Proportion ◽

Chicken Embryo ◽

Culture Cells ◽

Tissue Culture Cells ◽

Morphologic Characteristics ◽

Infectious Laryngotracheitis ◽

Laryngotracheitis Virus ◽

In Cells

During a study of the development of infectious laryngotracheitis (LT) virus in tissue culture cells, unusual tubular formations were found in the cytoplasm of a small proportion of the affected cells. It is the purpose of this report to describe the morphologic characteristics of the tubules and to discuss their possible association with the development of virus.The source and maintenance of the strain of LT virus have been described. Prior to this study, the virus was passed several times in chicken embryo kidney (CEK) tissue culture cells.

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