Since most archaea are extremophilic and difficult to cultivate, our current knowledge of their biology is confined largely to comparative genomics and biochemistry. Haloferax volcanii offers great promise as a model organism for archaeal genetics, but until now there has been a lack of a wide variety of selectable markers for this organism. We describe here isolation of H. volcanii leuB and trpA genes encoding 3-isopropylmalate dehydrogenase and tryptophan synthase, respectively, and development of these genes as a positive selection system. ⌬leuB and ⌬trpA mutants were constructed in a variety of genetic backgrounds and were shown to be auxotrophic for leucine and tryptophan, respectively. We constructed both integrative and replicative plasmids carrying the leuB or trpA gene under control of a constitutive promoter. The use of these selectable markers in deletion of the lhr gene of H. volcanii is described.Less than 25 years ago, the archaea were virtually unknown. We now recognize that these organisms represent one of the fundamental domains of life (32) and constitute a significant fraction of the total biomass (9). Acceptance of the distinct status of the archaea has been largely due to genome sequencing projects (6). The data from the sequenced genomes have revealed that in spite of their prokaryotic morphology, the archaea have numerous similarities with eukaryotes (5), particularly in enzymes involved in core processes such as transcription (2), translation (10), and DNA replication (23). As the archaeal transcription and replication systems are considerably less complex than those found in eukaryotes, they are more amenable to analysis. Notwithstanding this sequence similarity, the archaea have a unique identity (12), which is best exemplified by novel enzymes such as the Holliday junction resolvase Hjc (19).In order to realize the true potential of the archaea, both as stripped-down models to dissect more complex eukaryotic systems and as a source of novel enzymes, it is essential to harness the power of genetics to underpin advances in biochemistry and genomics. Haloferax volcanii is an obligate halophile (25) that is genetically stable and grows aerobically in both complex and minimal media (24). It has some of the best genetic tools among the archaea, including a transformation system (8), reporter genes (17), shuttle vectors with antibiotic resistance (14,16,22), auxotrophic markers (26), and a recently developed gene knockout system based on the pyrE2 gene (3) (Fig. 1A), which encodes orotate phosphoribosyl transferase and is involved in uracil biosynthesis.Since the number of selectable genetic markers in H. volcanii is still limited, we wished to supplement it with additional markers. We describe here the isolation of H. volcanii leuB and trpA genes encoding 3-isopropylmalate dehydrogenase and tryptophan synthase, respectively. Deletion of leuB, a homologue of the widely used Saccharomyces cerevisiae LEU2 gene (1), confers leucine auxotrophy in minimal medium. Deletion of the trpA gene (20) co...

Chromosome duplication normally initiates via the assembly of replication fork complexes at defined origins1,2. DNA synthesis by any one fork is thought to cease when it meets another travelling in the opposite direction, at which stage the replication machinery may simply dissociate before the nascent strands are finally ligated. But what actually happens is not clear. Here we present evidence consistent with the idea that every fork collision has the potential to threaten genomic integrity. In Escherichia coli this threat is kept at bay by RecG DNA translocase3 and by single-strand DNA exonucleases. Without RecG, replication initiates where forks meet via a replisome assembly mechanism normally associated with fork repair, replication restart and recombination4,5, establishing new forks with the potential to sustain cell growth and division without an active origin. This potential is realised when roadblocks to fork progression are reduced or eliminated. It relies on the chromosome being circular, reinforcing the idea that replication initiation is triggered repeatedly by fork collision. The results reported raise the question of whether replication fork collisions have pathogenic potential for organisms that exploit multiple origins to replicate each chromosome.

SummaryNucleoprotein complexes present challenges to genome stability by acting as potent blocks to replication. One attractive model of how such conflicts are resolved is direct targeting of blocked forks by helicases with the ability to displace the blocking protein-DNA complex. We show that Rep and UvrD each promote movement of E. coli replisomes blocked by nucleoprotein complexes in vitro, that such an activity is required to clear protein blocks (primarily transcription complexes) in vivo, and that a polarity of translocation opposite that of the replicative helicase is critical for this activity. However, these two helicases are not equivalent. Rep but not UvrD interacts physically and functionally with the replicative helicase. In contrast, UvrD likely provides a general means of protein-DNA complex turnover during replication, repair, and recombination. Rep and UvrD therefore provide two contrasting solutions as to how organisms may promote replication of protein-bound DNA.