Abstract
Angomonas deanei coevolves in a mutualistic relationship with a symbiotic bacterium that divides in synchronicity with other host cell structures. Trypanosomatid mitochondrial DNA is contained in the kinetoplast and is composed of thousands of interlocked DNA circles (kDNA). The arrangement of kDNA is related to the presence of histone-like proteins, known as KAPs (kinetoplast-associated proteins), that neutralize the negatively charged kDNA, thereby affecting the activity of mitochondrial enzymes involved in replication, transcription and repair. In this study, CRISPR-Cas9 was used to delete both alleles of the A. deanei KAP4 gene. Gene-deficient mutants exhibited high compaction of the kDNA network and displayed atypical phenotypes, such as the appearance of a filamentous symbionts, cells containing two nuclei and one kinetoplast, and division blocks. Treatment with cisplatin and UV showed that Δkap4 null mutants were not more sensitive to DNA damage and repair than wild-type cells. Notably, lesions caused by these genotoxic agents in the mitochondrial DNA could be repaired, suggesting that the kDNA in the kinetoplast of trypanosomatids has unique repair mechanisms. Taken together, our data indicate that although KAP4 is not an essential protein, it plays important roles in kDNA arrangement and replication, as well as in the maintenance of symbiosis.
Highlights
The kinetoplast contains the mitochondrial DNA of trypanosomatids, which is arranged in a network of several thousand minicircles categorized into different classes and several dozen maxicircles that are virtually identical
The first model used to study the roles played by Kinetoplast-associated proteins (KAPs) was the monoxenic Crithidia fasciculata, where the disruption of the KAP1 gene generated viable cells with a phenotype of highly condensed kinetoplast contains the mitochondrial DNA (kDNA) fibers, which was similar to that observed when trypanosomatids were treated with nalidixic acid, an inhibitor of prokaryote topoisomerase II15,18
To delete KAP4, A. deanei was cotransfected with a repair template containing the neomycin resistance gene and 30 nt homologous to flanking KAP4 UTRs′, and 2 sgRNA templates were expressed in vivo by T7 RNA polymerase to insert double strand break (DSB) at the 5′ and 3′ ends of the gene
Summary
The kinetoplast contains the mitochondrial DNA (kDNA) of trypanosomatids, which is arranged in a network of several thousand minicircles categorized into different classes and several dozen maxicircles that are virtually identical. The minicircles duplicate as theta structures, by UMSBP, Pol 1B, and other proteins and subsequently migrate to the antipodal sites At this kinetoplast region, a primase enables the synthesis initiation of new DNA fragments following kDNA replication that involves more than 100 enzymes, such as universal minicircle sequence-binding protein (UMSBP) and polymerases. The duplicated network is separated by the basal body distance, since the kDNA is connected to it via the TAC structure This minicircle replication model was primarily based on findings obtained with Trypanosoma brucei and Crithidia fasciculata[4]. Downregulation of KAP6 promoted cell growth arrest and inhibition of covalently closed minicircle release, resulting in loss, shrinkage and disorganization of k DNA20
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