ABSTRACT
Cells depend on robust DNA damage recognition and repair systems to maintain genomic integrity for survival in a mutagenic environment. In the pathogenic yeast Candida albicans, a subset of genes involved in the response to DNA damage-induced genome instability and morphological changes has been found to regulate virulence. To better understand the virulence-linked DNA repair network, we screened for methyl methane sulfonate (MMS) sensitivity within the GRACE conditional expression collection and identified 56 hits. One of these potential DNA damage repair-associated genes, a HOF1 conditional mutant, unexpectedly had a previously characterized function in cytokinesis. Deletion of HOF1 resulted in MMS sensitivity and genome instability, suggesting Hof1 acts in the DNA damage response. By probing genetic interactions with distinct DNA repair pathways, we found that Hof1 is genetically linked to the Rad53 pathway. Furthermore, Hof1 is down-regulated in a Rad53-dependent manner and its importance in the MMS response is reduced when Rad53 is overexpressed or when RAD4 or RAD23 is deleted. Together, this work expands our understanding of the C. albicans DNA repair network and uncovers interplay between the cytokinesis regulator Hof1 and the Rad53-mediated checkpoint.
Subject(s)
Candida albicans/cytology , Candida albicans/metabolism , Cell Cycle Checkpoints , DNA Damage , Fungal Proteins/metabolism , Methyl Methanesulfonate/toxicity , Candida albicans/drug effects , Cell Cycle Checkpoints/drug effects , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Epistasis, Genetic/drug effects , Fungal Proteins/chemistry , Genomic Instability/drug effects , Models, Biological , Mutation/genetics , Protein DomainsABSTRACT
Among actinomycetes, chromosome organization and segregation studies have been limited to Streptomyces coelicolor, Corynebacterium glutamicum, and Mycobacterium spp. There are differences with respect to ploidy and chromosome organization pattern in these bacteria. Here, we report on chromosome replication, organization, and segregation in Rhodococcus erythropolis PR4, which has a circular genome of 6.5 Mbp. The origin of replication of R. erythropolis PR4 was identified, and the DNA content in the cell under different growth conditions was determined. Our results suggest that the number of origins increases as the growth medium becomes rich, suggesting an overlapping replication cell cycle in this bacterium. Subcellular localization of the origin region revealed polar positioning in minimal and rich media. The terminus, which is the last region to be replicated and segregated, was found to be localized at the cell center in large cells. The middle markers corresponding to the 1.5-Mb and 4.7-Mb loci did not overlap, suggesting discontinuity in the segregation of the two arms of the chromosome. Chromosome segregation was not affected by inhibiting cell division. Deletion of parA or parB affected chromosome segregation. Unlike in C. glutamicum and Streptomyces spp., diploidy or polyploidy was not observed in R. erythropolis PR4. Our results suggest that R. erythropolis is different from other members of Actinobacteria; it is monoploid and has a unique chromosome segregation pattern. This is the first report on chromosome organization, replication, and segregation in R. erythropolis PR4.IMPORTANCE Rhodococci are highly versatile Gram-positive bacteria with high bioremediation potential. Some rhodococci are pathogenic and have been suggested as emerging threats. No studies on the replication, segregation, and cell cycle of these bacteria have been reported. Here, we demonstrate that the genus Rhodococcus is different from other actinomycetes, such as members of the genera Corynebacterium, Mycobacterium, and Streptomyces, with respect to ploidy and chromosome organization and segregation. Such studies will be useful not only in designing better therapeutics pathogenic strains in the future but also for studying genome maintenance in strains used for bioremediation.
Subject(s)
Chromosome Segregation , Chromosomes, Bacterial/genetics , Ploidies , Rhodococcus/genetics , Bacterial Proteins/physiology , Cell Cycle , DNA Replication , Replication OriginABSTRACT
Mitochondrial DNA (mtDNA) lacks the protection provided by the nucleosomes in the nuclear DNA and does not have a DNA repair mechanism, making it highly susceptible to damage, which can lead to mtDNA depletion. mtDNA depletion compromises the efficient function of cells and tissues and thus impacts negatively on health. Here, we describe a brief and easy protocol to quantify mtDNA copy number by determining the mtDNA/nDNA ratio. The procedure has been validated using a cohort of young and aged mice. © 2017 by John Wiley & Sons, Inc.
