Differences in homologous recombination and maintenance of heteropolyploidy between Haloferax volcanii and Haloferax mediterranei.

Polyploidy, the phenomenon of having more than one copy of the genome in an organism, is common among haloarchaea. While providing short-term benefits for DNA repair, polyploidy is generally regarded as an "evolutionary trap" that by the notion of the Muller's ratchet will inevitably conclude in the species' decline or even extinction due to a gradual reduction in fitness. In most reported cases of polyploidy in archaea, the genetic state of the organism is considered as homoploidy i.e. all copies of the genome are identical. Here we demonstrate that while this is indeed the prevalent genetic status in the halophilic archaeon Haloferax volcanii, its close relative H. mediterranei maintains a prolonged heteroploidy state in a nonselective environment once a second allele is introduced. Moreover, a strong genetic linkage was observed between two distant loci in H. mediterranei indicating a low rate of homologous recombination while almost no such linkage was shown in H. volcanii indicating a high rate of recombination in the latter species. We suggest that H. volcanii escapes Muller's ratchet by means of an effective chromosome-equalizing gene-conversion mechanism facilitated by highly active homologous recombination, whereas H. mediterranei must elude the ratchet via a different, yet to be elucidated mechanism.

Genetic Manipulation of Haloferax Species.

In this chapter, we describe the reverse genetics methodology behind generating a targeted gene deletion or replacement in archaeal species of the genus Haloferax, which are renowned for their ease of manipulation. Individual steps in the method include the design of a gene-targeting vector, its use in transforming Haloferax to yield "pop-in" and "pop-out" clones, and techniques for validating the genetically manipulated strain. The vector carries DNA fragments of 500-1000 bp that flank the gene of interest (or a mutant allele), in addition to the pyrE2 gene for uracil biosynthesis (Bitan-Banin et al. J Bacteriol 185:772-778, 2003). The latter is used as a selectable marker for the transformation of Haloferax, wherein the vector integrates by homologous recombination at the genomic locus to generate the "pop-in" strain; this is also known as allele-coupled exchange. Culturing of these transformants in nonselective broth and subsequent plating on 5-fluoroorotic acid (5-FOA)-containing media selects for excision of the vector, yielding either wild type or mutant "pop-out" clones. These 5-FOA-resistant clones are screened to confirm the desired mutation, using a combination of phenotypic assays, colony hybridization and Southern blotting. The pop-in/pop-out method allows for the recycling of the pyrE2 marker to enable multiple gene deletions to be carried out in a single strain, thereby providing insights into the function of multiple proteins and how they interact in their respective cellular pathways.

Unraveling the antitrypanosomal mechanism of benznidazole and related 2-nitroimidazoles: From prodrug activation to DNA damage.

Nitroheterocycles represent an important class of compound used to treat trypanosomiasis. They often function as prodrugs and can undergo type I nitroreductase (NTR1)-mediated activation before promoting their antiparasitic activities although the nature of these downstream effects has yet to be determined. Here, we show that in an NTR1-dependent process, benznidazole promotes DNA damage in the nuclear genome of Trypanosoma brucei, providing the first direct link between activation of this prodrug and a downstream trypanocidal mechanism. Phenotypic and protein expression studies revealed that components of the trypanosome's homologous recombination (HR) repair pathway (TbMRE11, γH2A, TbRAD51) cooperate to resolve the benznidazole-induced damage, indicating that the prodrug-induced lesions are most likely double stand DNA breaks, while the sequence/recruitment kinetics of these factors parallels that in other eukaryotes HR systems. When extended to other NTR1-activated 2-nitroimidazoles, some were shown to promote DNA damage. Intriguingly, the lesions induced by these required TbMRE11 and TbCSB activities to fix leading us to postulate that TbCSB may operate in systems other than the transcription-coupled nucleotide excision repair pathway. Understanding how existing trypanosomal drugs work will aid future drug design and help unlock novel reactions/pathways that could be exploited as targets for therapeutic intervention.

Haloferax volcanii-a model archaeon for studying DNA replication and repair.

The tree of life shows the relationship between all organisms based on their common ancestry. Until 1977, it comprised two major branches: prokaryotes and eukaryotes. Work by Carl Woese and other microbiologists led to the recategorization of prokaryotes and the proposal of three primary domains: Eukarya, Bacteria and Archaea. Microbiological, genetic and biochemical techniques were then needed to study the third domain of life. Haloferax volcanii, a halophilic species belonging to the phylum Euryarchaeota, has provided many useful tools to study Archaea, including easy culturing methods, genetic manipulation and phenotypic screening. This review will focus on DNA replication and DNA repair pathways in H. volcanii, how this work has advanced our knowledge of archaeal cellular biology, and how it may deepen our understanding of bacterial and eukaryotic processes.

Deciphering the interstrand crosslink DNA repair network expressed by Trypanosoma brucei.

Interstrand crosslinks (ICLs) represent a highly toxic form of DNA damage that can block essential biological processes including DNA replication and transcription. To combat their deleterious effects all eukaryotes have developed cell cycle-dependent repair strategies that co-opt various factors from 'classical' DNA repair pathways to resolve such lesions. Here, we report the first systematic dissection of how ICL repair might operate in the Trypanosoma brucei, the causative agent of African trypanosomiasis, and demonstrated that this diverged eukaryote expresses systems that show some intriguing differences to those mechanisms present in other organisms. Following the identification of trypanosomal homologues encoding for CSB, EXO1, SNM1, MRE11, RAD51 and BRCA2, gene deletion coupled with phenotypic studies demonstrated that all the above factors contribute to this pathogen's ICL REPAIRtoire with their activities split across two epistatic groups. We postulate that one network, which encompasses TbCSB, TbEXO1 and TbSNM1, may operate throughout the cell cycle to repair ICLs encountered by transcriptional detection mechanisms while the other relies on homologous recombination enzymes (MRE11, RAD51 and BRCA2) that together help resolve lesions responsible for the stalling of DNA replication forks. This study not only sheds light on the conservation and divergence of ICL repair in one of only a handful of protists that can be studied genetically, but offers the promise of developing or exploiting ICL-causing agents as new anti-parasite therapies.