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

Abstract

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.

Keywords: Archaea; DNA repair; DNA replication; Haloferax volcanii; homologous recombination.

Figure 1.

Structural components of the replisome. The CMG replicative helicase complex (RecJ:MCM:GINS in H. volcanii) unwinds DNA to expose single-stranded DNA (ssDNA). It remains unknown which of the four RecJ proteins in H. volcanii forms part of the CMG complex. The ssDNA is protected from damage by binding protein RPA and is used as a template for the synthesis of RNA primers by the primase activities of PriS and PriL. Replicative DNA polymerases (PolB1 and PolD) extend the RNA primer to initiate DNA replication. Clamp loader RFC removes primases from the replication fork and the open DNA structure is held in place by the sliding clamp PCNA. H. volcanii gene loci (HVO_#) for each component of the replisome are indicated.

Figure 2.

Base excision repair. The damaged base (red) is recognized and removed by DNA glycosylases, which cleave the N-glycosyl bond between the damaged base and the sugar to generate an apurinic or apyrimidinic (AP) site. AP endonucleases cleave 5′ of the abasic site or ß lyase cleaves 3′ of the site, and the backbone is removed by phosphodiesterases. Short-patch BER uses DNA polymerases (PolB1; HVO_0858 in H. volcanii) to insert the missing nucleotide (purple) with DNA ligases (LigA; HVO_1565 or LigN; HVO_3000) linking the newly synthesized nucleotide to the sugar backbone. In long-patch BER, DNA polymerases insert 2–6 nucleotides at the gap to generate a flap structure. Flap endonuclease Fen1 (HVO_2873) cleaves the displaced strand and DNA ligases seal the DNA backbone.

Figure 3.

Nucleotide excision repair. In H. volcanii, bulky DNA adducts (red) are recognized by UvrA (HVO_0393). UvrB (HVO_0029) initiates DNA unwinding around the damage site through its helicase activity. Incisions 5′ and 3′ to the damaged bases are carried out by the endonuclease UvrC (HVO_3006). Unwinding of the damaged strand is carried out by a helicase such as UvrD (HVO_0415). The remaining gap is filled (purple) by replicative DNA polymerase PolB1 (HVO_0858) and the newly synthesized DNA is attached to the backbone by the activity of DNA ligases (LigA; HVO_1565 or LigN; HVO_3000).

Figure 4.

Double-strand break repair. Double-strand break repair in H. volcanii occurs by either microhomology-mediated end joining (MMEJ) or homologous recombination (HR). MMEJ begins with end resection at the break site which is carried out by Rad50 (HVO_0854) and Mre11 (HVO_0853). Homologous DNA strands around the break (red) is annealed and any displaced DNA is trimmed by Fen1 (HVO_2873). Gaps in both strands are filled (purple) by replicative DNA polymerase PolB1 (HVO_0858) and ligated by DNA ligases; in H. volcanii either LigA (HVO_1565) or LigN (HVO_3000). HR also involves initial end resection at the break site by the combined activities of Rad50 and Mre11. The single-stranded DNA ends are bound by the recombinase RadA (HVO_0104), aided by the recombination mediator RadB (HVO_2383), which then promotes the search for homologous DNA duplex (red). Strand exchange with the homologous duplex generates a D-loop (displacement loop) with a 3′ invading end, from which DNA is synthesized (purple) by the action of replicative polymerase PolB1 (HVO_0858). At this stage, either a non-crossover event occurs due to displacement of the invading strand by helicases such as Hel308 (HVO_0014) or Hef (HVO_3010); the free strand anneals with and is ligated to the other end of the DNA break using either LigA (HVO_1565) or LigN (HVO_3000). Alternatively, a Holliday junction is formed that is processed by resolvases Hjc (HVO_0170) or Hef (HVO_3010) to cut the four-way DNA junction, leading to either a crossover or non-crossover event between recombining chromosomes. The remaining nicks in DNA are resolved by either LigA or LigN.