Molecular Genetic Methods to Study DNA Replication Protein Function in Haloferax volcanii, A Model Archaeal Organism.

Abstract

Successful high-fidelity chromosomal DNA replication is fundamental to all forms of cellular life and requires the complex interplay of a variety of essential and nonessential protein factors in a spatially and temporally coordinated manner. Much of what is known about the enzymes and mechanisms of chromosome replication has come from analysis of simple microbial model systems, such as yeast and archaea. Archaea possess a highly simplified eukaryotic-like replication apparatus, making them an excellent model for gaining novel insights into conserved aspects of protein function at the heart of the replisome. Amongst the thermophilic archaea, a number of species have proved useful for biochemical analysis of protein function, but few of these organisms are suited to genetic analysis. One archaeal organism that is genetically tractable is the mesophilic euryarchaeon Haloferax volcanii, a halophile that grows aerobically in high salt medium at an optimum temperature of 40-45 °C and with a doubling time of 2-3 h. The Hfx. volcanii genome has been sequenced and a range of methods have been developed to allow reverse genetic analysis of protein function in vivo, including techniques for gene replacement and gene deletion, transcriptional regulation, point mutation and gene tagging. Here we briefly summarize current knowledge of the chromosomal DNA replication machinery in the haloarchaea before describing in detail the molecular methods available to probe protein structure and function within the Hfx. volcanii replication apparatus.