So far, the extremely halophilic archaeon Haloferax volcanii has the best genetic tools among the archaea. However, the lack of an efficient gene knockout system for this organism has hampered further genetic studies. In this paper we describe the development of pyrE-based positive selection and counterselection systems to generate an efficient gene knockout system. The H. volacanii pyrE1 and pyrE2 genes were isolated, and the pyrE2 gene was shown to code for the physiological enzyme orotate phosphoribosyl transferase. A ⌬pyrE2 strain was constructed and used to isolate deletion mutants by the following two steps: (i) integration of a nonreplicative plasmid carrying both the pyrE2 wild-type gene, as a selectable marker, and a cloned chromosomal DNA fragment containing a deletion in the desired gene; and (ii) excision of the integrated plasmid after selection with 5-fluoroorotic acid. Application of this gene knockout system is described.

Recombination plays a central role in the repair of broken chromosomes in all eukaryotes. We carried out a systematic study of mitotic recombination. Using several assays, we established the chronological sequence of events necessary to repair a single double-strand break. Once a chromosome is broken, yeast cells become immediately committed to recombinational repair. Recombination is completed within an hour and exhibits two kinetic gaps. By using this kinetic framework we also characterized the role played by several proteins in the recombinational process. In the absence of Rad52, the broken chromosome ends, both 5 and 3, are rapidly degraded. This is not due to the inability to recombine, since the 3 single-stranded DNA ends are stable in a strain lacking donor sequences. Rad57 is required for two consecutive strand exchange reactions. Surprisingly, we found that the Srs2 helicase also plays an early positive role in the recombination process.The process of recombination plays an essential role during meiosis and in DNA repair during vegetative growth. Doublestrand breaks (DSBs) arise frequently as a consequence of exposure to external insults or as a direct result of natural cell metabolism. Recombinational repair of DSBs is important in solving collapsed replication forks during DNA replication (20). If left unrepaired, DSBs result in broken chromosomes, genetic alterations, or cell death. Repair of DSBs (DSBR) takes place in eukaryotes by two competing processes: nonhomologous end joining and homologous recombination. In yeast cells, homologous recombination is the prevalent mechanism used (reviewed in reference 36).In a classic model for DSB-initiated recombination (53) (Fig. 1A), single-stranded degradation of the broken DNA molecule generates protruding 3Ј-OH ends that can invade homologous regions, creating a D-loop. The invasion process yields regions of heteroduplex DNA (hDNA) that may contain mismatches. The invading 3Ј end is then used to prime DNA synthesis. Eventually, the displaced donor strand pairs with single-stranded DNA (ssDNA) from the opposing end of the DSB, also serving as a template for DNA synthesis. Ligation results in the formation of a structure containing two Holliday junctions, which can be resolved to yield either crossover or noncrossover products. Mismatch repair of the hDNA may result in gene conversion events (Fig. 1A, left).An alternative model for gene conversion, termed the synthesis-dependent strand-annealing (SDSA) model (32), proposes that after DSB formation and resection, a single 3Ј single-stranded end invades the intact homologous template. DNA synthesis is followed by reannealing of the newly synthesized DNA with the opposite broken arm. In the basic version of this model (Fig. 1A, center), only gene conversion, and not crossover, can be obtained, although variations allowing crossing-over have been also proposed (reviewed in reference 36).Homologous recombination is catalyzed by a number of proteins encoded mostly by genes of the RAD52 epistasis group (36, 51). ...

A search of the complete genome sequence of the halophilic archaeon Halobacterium salinarum failed to identify a gene homologous to the thymidylate synthase (thyA) gene present in the closely related Haloferax volcanii. To understand the source of thymidine synthesis in Hbt. salinarum, a genomic library of Hbt. salinarum was constructed and used to complement a Hfx. volcanii thyA deletion mutation. The Hbt. salinarum ORF that complemented the thyA mutation shares sequence homology with ORFs found in numerous microorganisms that lack a thyA gene, including the recently discovered thyX of Helicobacter pylori. We also show that a homolog of the Hbt. salinarum ORF is present in Campylobacter jejuni and is able to complement an Escherichia coli thyA mutant under oxygen-limiting conditions.