scholarly journals Baculovirus F-Box Protein LEF-7 Modifies the Host DNA Damage Response To Enhance Virus Multiplication

2013 ◽  
Vol 87 (23) ◽  
pp. 12592-12599 ◽  
Author(s):  
Jonathan K. Mitchell ◽  
Nathaniel M. Byers ◽  
Paul D. Friesen
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Viral Gene Expression ◽  
Virus Multiplication ◽  
Site Directed Mutagenesis ◽  
Viral Gene ◽  
Content Type ◽  
Baculovirus Infection ◽  
Damage Response ◽  
F Box Protein

The DNA damage response (DDR) of a host organism represents an effective antiviral defense that is frequently manipulated and exploited by viruses to promote multiplication. We report here that the large DNA baculoviruses, which require host DDR activation for optimal replication, encode a conserved replication factor, LEF-7, that manipulates the DDR via a novel mechanism. LEF-7 suppresses DDR-induced accumulation of phosphorylated host histone variant H2AX (γ-H2AX), a critical regulator of the DDR. LEF-7 was necessary and sufficient to block γ-H2AX accumulation caused by baculovirus infection or DNA damage induced by means of pharmacological agents. Deletion of LEF-7 from the baculovirus genome allowed γ-H2AX accumulation during virus DNA synthesis and impaired both very late viral gene expression and production of infectious progeny. Thus, LEF-7 is essential for efficient baculovirus replication. We determined that LEF-7 is a nuclear F-box protein that interacts with host S-phase kinase-associated protein 1 (SKP1), suggesting that LEF-7 acts as a substrate recognition component of SKP1/Cullin/F-box (SCF) complexes for targeted protein polyubiquitination. Site-directed mutagenesis demonstrated that LEF-7's N-terminal F-box is necessary for γ-H2AX repression andAutographa californicamultiple nucleopolyhedrovirus (AcMNPV) replication events. We concluded that LEF-7 expedites virus replication most likely by selective manipulation of one or more host factors regulating the DDR, including γ-H2AX. Thus, our findings indicate that baculoviruses utilize a unique strategy among viruses for hijacking the host DDR by using a newly recognized F-box protein.

scholarly journals Nucleotide Excision Repair Protein Rad23 Regulates Cell Virulence Independent of Rad4 in Candida albicans

mSphere ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Jia Feng ◽  
Shuangyan Yao ◽  
Yansong Dong ◽  
Jing Hu ◽  
Malcolm Whiteway ◽  
...  
Keyword(s):  
Dna Damage ◽  
Candida Albicans ◽  
Nucleotide Excision Repair ◽  
Dna Damage Response ◽  
Excision Repair ◽  
Content Type ◽  
Virulence Regulation ◽  
Nucleotide Excision ◽  
Damage Response

ABSTRACT In the pathogenic yeast Candida albicans, the DNA damage response contributes to pathogenicity by regulating cell morphology transitions and maintaining survival in response to DNA damage induced by reactive oxygen species (ROS) in host cells. However, the function of nucleotide excision repair (NER) in C. albicans has not been extensively investigated. To better understand the DNA damage response and its role in virulence, we studied the function of the Rad23 nucleotide excision repair protein in detail. The RAD23 deletion strain and overexpression strain both exhibit UV sensitivity, confirming the critical role of RAD23 in the nucleotide excision repair pathway. Genetic interaction assays revealed that the role of RAD23 in the UV response relies on RAD4 but is independent of RAD53, MMS22, and RAD18. RAD4 and RAD23 have similar roles in regulating cell morphogenesis and biofilm formation; however, only RAD23, but not RAD4, plays a negative role in virulence regulation in a mouse model. We found that the RAD23 deletion strain showed decreased survival in a Candida-macrophage interaction assay. Transcriptome sequencing (RNA-seq) and quantitative real-time PCR (qRT-PCR) data further revealed that RAD23, but not RAD4, regulates the transcription of a virulence factor, SUN41, suggesting a unique role of RAD23 in virulence regulation. Taking these observations together, our work reveals that the RAD23-related nucleotide excision pathway plays a critical role in the UV response but may not play a direct role in virulence. The virulence-related role of RAD23 may rely on the regulation of several virulence factors, which may give us further understanding about the linkage between DNA damage repair and virulence regulation in C. albicans. IMPORTANCE Candida albicans remains a significant threat to the lives of immunocompromised people. An understanding of the virulence and infection ability of C. albicans cells in the mammalian host may help with clinical treatment and drug discovery. The DNA damage response pathway is closely related to morphology regulation and virulence, as well as the ability to survive in host cells. In this study, we checked the role of the nucleotide excision repair (NER) pathway, the key repair system that functions to remove a large variety of DNA lesions such as those caused by UV light, but whose function has not been well studied in C. albicans. We found that Rad23, but not Rad4, plays a role in virulence that appears independent of the function of the NER pathway. Our research revealed that the NER pathway represented by Rad4/Rad23 may not play a direct role in virulence but that Rad23 may play a unique role in regulating the transcription of virulence genes that may contribute to the virulence of C. albicans.

scholarly journals Guanine Quadruplex DNA Regulates Gamma Radiation Response of Genome Functions in the Radioresistant Bacterium Deinococcus radiodurans

2019 ◽  
Vol 201 (17) ◽  
Author(s):  
Shruti Mishra ◽  
Reema Chaudhary ◽  
Sudhir Singh ◽  
Swathi Kota ◽  
Hari S. Misra
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Deinococcus Radiodurans ◽  
Dna Structure ◽  
Protein Translation ◽  
Content Type ◽  
Cellular Processes ◽  
G4 Dna ◽  
Guanine Quadruplex ◽  
Damage Response

ABSTRACT Guanine quadruplex (G4) DNA/RNA are secondary structures that regulate the various cellular processes in both eukaryotes and bacteria. Deinococcus radiodurans, a Gram-positive bacterium known for its extraordinary radioresistance, shows a genomewide occurrence of putative G4 DNA-forming motifs in its GC-rich genome. N-Methyl mesoporphyrin (NMM), a G4 DNA structure-stabilizing drug, did not affect bacterial growth under normal conditions but inhibited the postirradiation recovery of gamma-irradiated cells. Transcriptome sequencing analysis of cells treated with both radiation and NMM showed repression of gamma radiation-responsive gene expression, which was observed in the absence of NMM. Notably, this effect of NMM on the expression of housekeeping genes involved in other cellular processes was not observed. Stabilization of G4 DNA structures mapped at the upstream of recA and in the encoding region of DR_2199 had negatively affected promoter activity in vivo, DNA synthesis in vitro and protein translation in Escherichia coli host. These results suggested that G4 DNA plays an important role in DNA damage response and in the regulation of expression of the DNA repair proteins required for radioresistance in D. radiodurans. IMPORTANCE Deinococcus radiodurans can recover from extensive DNA damage caused by many genotoxic agents. It lacks LexA/RecA-mediated canonical SOS response. Therefore, the molecular mechanisms underlying the regulation of DNA damage response would be worth investigating in this bacterium. D. radiodurans genome is GC-rich and contains numerous islands of putative guanine quadruplex (G4) DNA structure-forming motifs. Here, we showed that in vivo stabilization of G4 DNA structures can impair DNA damage response processes in D. radiodurans. Essential cellular processes such as transcription, DNA synthesis, and protein translation, which are also an integral part of the double-strand DNA break repair pathway, are affected by the arrest of G4 DNA structure dynamics. Thus, the role of DNA secondary structures in DNA damage response and radioresistance is demonstrated.

scholarly journals Baculovirus Infection Induces a DNA Damage Response That Is Required for Efficient Viral Replication

2011 ◽  
Vol 85 (23) ◽  
pp. 12547-12556 ◽  
Author(s):  
N. Huang ◽  
W. Wu ◽  
K. Yang ◽  
A. L. Passarelli ◽  
G. F. Rohrmann ◽  
...  
Keyword(s):  
Dna Damage ◽  
Viral Replication ◽  
Dna Damage Response ◽  
Baculovirus Infection ◽  
Damage Response ◽  
Efficient Viral Replication

Phosphorylation of FANCA on S1449 Is Important for Fanconi Anemia (FA) Pathway Function.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 186-186
Author(s):  
Natalie B. Collins ◽  
Andrei Tomashevski ◽  
Gary M. Kupfer
Keyword(s):  
Dna Damage ◽  
Homologous Recombination ◽  
Dna Damage Response ◽  
Fanconi Anemia ◽  
Phosphorylation Site ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Damage Response ◽  
And Function ◽  
Mutant Cells

Abstract Previous work in our lab and others has shown that the Fanconi anemia proteins, FANCG and FANCA, are phosphoproteins. FANCG is phosphorylated at mitosis, and these phosphorylations are required for proper exit from chromatin at mitosis. FANCG is also phosphorylated after DNA damage, with the phosphorylation site required for wild-type sensitivity to DNA damaging agents. FANCA is also phosphorylated after DNA damage and localized to chromatin, but the site and significance of this phosphorylation were previously unknown. Mass spectrometry of FANCA revealed one phosphopeptide with phosphorylation on serine 1449. Site-directed mutagenesis of this residue to alanine (S1449A) abolished a slower mobility form of FANCA seen after MMC treatment. Furthermore, the S1449A mutant failed to completely correct the MMC hypersensitivity of FA-A mutant cells. S1449A mutant cells displayed lower than wild-type levels of FANCD2 monoubiquitination following DNA damage, and an increased number of gross chromosomal aberrations were seen in metaphase spreads from S1449A mutant cells when compared to wild type cells. Using a GFP reporter substrate to measure homologous recombination, cells expressing the S1449A FANCA failed to completely correct the homologous recombination defect seen in FA cells. Taken together, cells expressing FANCA S1449A display a variety of FA-associated phenotypes, suggesting that the phosphorylation of S1449 is a functionally significant event. The DNA damage response in human cells is, in large part, coordinated by phosphorylation events initiated by apical kinases ATM and ATR. S1449 is found in a consensus ATM site, therefore studies are underway to determine if ATM or ATR is the kinase responsible for FANCA phosphorylation at S1449. Phosphorylation is a crucial process in transducing the DNA damage response, and phosphorylation of FA proteins appears critical to both localization and function of the proteins. Understanding how phosphorylation marks are placed on FANCA will give insight into the role of FANCA in the DNA damage response.

scholarly journals Phosphorylation-Dependent Regulation of the DNA Damage Response of Adaptor Protein KIBRA in Cancer Cells

2016 ◽  
Vol 36 (9) ◽  
pp. 1354-1365 ◽  
Author(s):  
Jayadev Mavuluri ◽  
Swarnalatha Beesetti ◽  
Rohan Surabhi ◽  
Joachim Kremerskothen ◽  
Ganesh Venkatraman ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Cancer Cells ◽  
Dna Damage Response ◽  
Adaptor Protein ◽  
Adaptor Proteins ◽  
Site Directed Mutagenesis ◽  
Scaffolding Protein ◽  
Response Factors ◽  
Damage Response

Multifunctional adaptor proteins encompassing various protein-protein interaction domains play a central role in the DNA damage response pathway. In this report, we show that KIBRA is a physiologically interacting reversible substrate of ataxia telangiectasia mutated (ATM) kinase. We identified the site of phosphorylation in KIBRA as threonine 1006, which is embedded within the serine/threonine (S/T) Q consensus motif, by site-directed mutagenesis, and we further confirmed the same with a phospho-(S/T) Q motif-specific antibody. Results from DNA repair functional assays such as the γ-H2AX assay, pulsed-field gel electrophoresis (PFGE), Comet assay, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, and clonogenic cell survival assay using stable overexpression clones of wild-type (wt.) KIBRA and active (T1006E) and inactive (T1006A) KIBRA phosphorylation mutants showed that T1006 phosphorylation on KIBRA is essential for optimal DNA double-strand break repair in cancer cells. Further, results from stable retroviral short hairpin RNA-mediated knockdown (KD) clones of KIBRA and KIBRA knockout (KO) model cells generated by a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system showed that depleting KIBRA levels compromised the DNA repair functions in cancer cells upon inducing DNA damage. All these phenotypic events were reversed upon reconstitution of KIBRA into cells lacking KIBRA knock-in (KI) model cells. All these results point to the fact that phosphorylated KIBRA might be functioning as a scaffolding protein/adaptor protein facilitating the platform for further recruitment of other DNA damage response factors. In summary, these data demonstrate the imperative functional role of KIBRAper se(KIBRA phosphorylation at T1006 site as a molecular switch that regulates the DNA damage response, possibly via the nonhomologous end joining [NHEJ] pathway), suggesting that KIBRA could be a potential therapeutic target for modulating chemoresistance in cancer cells.

scholarly journals Rad53- and Chk1-Dependent DNA Damage Response Pathways Cooperatively Promote Fungal Pathogenesis and Modulate Antifungal Drug Susceptibility

mBio ◽  
2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Kwang-Woo Jung ◽  
Yeonseon Lee ◽  
Eun Young Huh ◽  
Soo Chan Lee ◽  
Sangyong Lim ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Drug Susceptibility ◽  
Antifungal Drugs ◽  
Antifungal Drug ◽  
Content Type ◽  
Evolutionarily Conserved ◽  
Damage Response ◽  
Fungal Dna ◽  
Living Organisms

ABSTRACT Living organisms are constantly exposed to DNA damage stress caused by endogenous and exogenous events. Eukaryotic cells have evolutionarily conserved DNA damage checkpoint surveillance systems. We previously reported that a unique transcription factor, Bdr1, whose expression is strongly induced by the protein kinase Rad53 governs DNA damage responses by controlling the expression of DNA repair genes in the basidiomycetous fungus Cryptococcus neoformans. However, the regulatory mechanism of the Rad53-dependent DNA damage signal cascade and its function in pathogenicity remain unclear. Here, we demonstrate that Rad53 is required for DNA damage response and is phosphorylated by two phosphatidylinositol 3-kinase (PI3K)-like kinases, Tel1 and Mec1, in response to DNA damage stress. Transcriptome analysis revealed that Rad53 regulates the expression of several DNA repair genes in response to gamma radiation. We found that expression of CHK1, another effector kinase involved in the DNA damage response, is regulated by Rad53 and that CHK1 deletion rendered cells highly susceptible to DNA damage stress. Nevertheless, BDR1 expression is regulated by Rad53, but not Chk1, indicating that DNA damage signal cascades mediated by Rad53 and Chk1 exhibit redundant and distinct functions. We found that perturbation of both RAD53 and CHK1 attenuated the virulence of C. neoformans, perhaps by promoting phagosome maturation within macrophage, reducing melanin production, and increasing susceptibility to oxidative stresses. Furthermore, deletion of both RAD53 and CHK1 increased susceptibility to certain antifungal drugs such as amphotericin B. This report provides insight into the regulatory mechanism of fungal DNA damage repair systems and their functional relationship with fungal virulence and antifungal drug susceptibility. IMPORTANCE Genome instability is detrimental for living things because it induces genetic disorder diseases and transfers incorrect genome information to descendants. Therefore, living organisms have evolutionarily conserved signaling networks to sense and repair DNA damage. However, how the DNA damage response pathway is regulated for maintaining the genome integrity of fungal pathogens and how this contributes to their pathogenicity remain elusive. In this study, we investigated the DNA damage response pathway in the basidiomycete pathogen Cryptococcus neoformans, which causes life-threatening meningoencephalitis in immunocompromised individuals, with an average of 223,100 infections leading to 181,100 deaths reported annually. Here, we found that perturbation of Rad53- and Chk1-dependent DNA damage response pathways attenuated the virulence of C. neoformans and increased its susceptibility to certain antifungal drugs, such as amphotericin B and flucytosine. Therefore, our work paves the way to understanding the important role of human fungal DNA damage networks in pathogenesis and antifungal drug susceptibility.

scholarly journals A Noncanonical DNA Damage Checkpoint Response in a Major Fungal Pathogen

mBio ◽  
2020 ◽  
Vol 11 (6) ◽  
pp. e03044-20
Author(s):  
Erika Shor ◽  
Rocio Garcia-Rubio ◽  
Lucius DeGregorio ◽  
David S. Perlin
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Fungal Pathogen ◽  
Candida Glabrata ◽  
Genome Stability ◽  
S Phase ◽  
Eukaryotic Cells ◽  
Genome Integrity ◽  
Content Type ◽  
Damage Response

ABSTRACTDNA damage checkpoints are key guardians of genome integrity. Eukaryotic cells respond to DNA damage by triggering extensive phosphorylation of Rad53/CHK2 effector kinase, whereupon activated Rad53/CHK2 mediates further aspects of checkpoint activation, including cell cycle arrest and transcriptional changes. Budding yeast Candida glabrata, closely related to model eukaryote Saccharomyces cerevisiae, is an opportunistic pathogen characterized by high genetic diversity and rapid emergence of drug-resistant mutants. However, the mechanisms underlying this genetic variability are unclear. We used Western blotting and mass spectrometry to show that, unlike S. cerevisiae, C. glabrata cells exposed to DNA damage did not induce C. glabrata Rad53 (CgRad53) phosphorylation. Furthermore, flow cytometry analysis showed that, unlike S. cerevisiae, C. glabrata cells did not accumulate in S phase upon DNA damage. Consistent with these observations, time-lapse microscopy showed C. glabrata cells continuing to divide in the presence of DNA damage, resulting in mitotic errors and cell death. Finally, transcriptome sequencing (RNAseq) analysis revealed transcriptional rewiring of the DNA damage response in C. glabrata and identified several key protectors of genome stability upregulated by DNA damage in S. cerevisiae but downregulated in C. glabrata, including proliferating cell nuclear antigen (PCNA). Together, our results reveal a noncanonical fungal DNA damage response in C. glabrata, which may contribute to rapidly generating genetic change and drug resistance.IMPORTANCE In order to preserve genome integrity, all cells must mount appropriate responses to DNA damage, including slowing down or arresting the cell cycle to give the cells time to repair the damage and changing gene expression, for example to induce genes involved in DNA repair. The Rad53 protein kinase is a conserved central mediator of these responses in eukaryotic cells, and its extensive phosphorylation upon DNA damage is necessary for its activation and subsequent activity. Interestingly, here we show that in the opportunistic fungal pathogen Candida glabrata, Rad53 phosphorylation is not induced by DNA damage, nor do these cells arrest in S phase under these conditions, in contrast to the closely related yeast Saccharomyces cerevisiae. Instead, C. glabrata cells continue to divide in the presence of DNA damage, resulting in significant cell lethality. Finally, we show that a number of genes involved in DNA repair are strongly induced by DNA damage in S. cerevisiae but repressed in C. glabrata. Together, these findings shed new light on mechanisms regulating genome stability in fungal pathogens.

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