A Noncanonical DNA Damage Checkpoint Response in a Major Fungal Pathogen

ABSTRACT DNA 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 trans...

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Autores principales:	Erika Shor, Rocio Garcia-Rubio, Lucius DeGregorio, David S. Perlin
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	Candida glabrata DNA damage checkpoints DNA damage response Rad53 cell division Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/113116fc5724411bb8d05db9c7e920c6
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spelling	oai:doaj.org-article:113116fc5724411bb8d05db9c7e920c62021-11-15T15:55:44ZA Noncanonical DNA Damage Checkpoint Response in a Major Fungal Pathogen10.1128/mBio.03044-202150-7511https://doaj.org/article/113116fc5724411bb8d05db9c7e920c62020-12-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.03044-20https://doaj.org/toc/2150-7511ABSTRACT DNA 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.Erika ShorRocio Garcia-RubioLucius DeGregorioDavid S. PerlinAmerican Society for MicrobiologyarticleCandida glabrataDNA damage checkpointsDNA damage responseRad53cell divisionMicrobiologyQR1-502ENmBio, Vol 11, Iss 6 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	Candida glabrata DNA damage checkpoints DNA damage response Rad53 cell division Microbiology QR1-502
spellingShingle	Candida glabrata DNA damage checkpoints DNA damage response Rad53 cell division Microbiology QR1-502 Erika Shor Rocio Garcia-Rubio Lucius DeGregorio David S. Perlin A Noncanonical DNA Damage Checkpoint Response in a Major Fungal Pathogen
description	ABSTRACT DNA 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.
format	article
author	Erika Shor Rocio Garcia-Rubio Lucius DeGregorio David S. Perlin
author_facet	Erika Shor Rocio Garcia-Rubio Lucius DeGregorio David S. Perlin
author_sort	Erika Shor
title	A Noncanonical DNA Damage Checkpoint Response in a Major Fungal Pathogen
title_short	A Noncanonical DNA Damage Checkpoint Response in a Major Fungal Pathogen
title_full	A Noncanonical DNA Damage Checkpoint Response in a Major Fungal Pathogen
title_fullStr	A Noncanonical DNA Damage Checkpoint Response in a Major Fungal Pathogen
title_full_unstemmed	A Noncanonical DNA Damage Checkpoint Response in a Major Fungal Pathogen
title_sort	noncanonical dna damage checkpoint response in a major fungal pathogen
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/113116fc5724411bb8d05db9c7e920c6
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A Noncanonical DNA Damage Checkpoint Response in a Major Fungal Pathogen

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