DNA replication in the archaea

Abstract

The archaeal DNA replication machinery bears striking similarity to that of eukaryotes and is clearly distinct from the bacterial apparatus. In recent years, considerable advances have been made in understanding the biochemistry of the archaeal replication proteins. Furthermore, a number of structures have now been obtained for individual components and higher-order assemblies of archaeal replication factors, yielding important insights into the mechanisms of DNA replication in both archaea and eukaryotes.

FIG. 1.

Components of the archaeal DNA replication machinery and chromatin proteins. Structure figures were prepared using Pymol (www.pymol.org), using the following PDB coordinates: Pyrobaculum Cdc6, 1FNN; Sulfolobus SSB, 1O71; Pyrococcus RFC small subunit, 1IQP; Sulfolobus Pol B1, 1S5J; Pyrococcus PCNA, 1ISQ; Archaeoglobus Fen1, 1RXW; Sulfolobus Alba, 1H0Y; Sulfolobus Sul7d, 1WTP; and Methanothermus histone HmfB, 1BFM. The image of the ligase structure was supplied by Y. Ishino (Fukuoka, Japan), and we obtained the image of the primase complex in collaboration with L. Pellegrini (Cambridge, United Kingdom).

FIG. 2.

Domain organization of MCM proteins. The N-terminal region, consisting of three domains, A, B, and C, is poorly conserved between different MCM proteins and is thought to be involved in regulation. Eukaryotic MCM proteins often have an additional N-terminal extension. The catalytic AAA+ domain is shown in blue-green. The helix-turn-helix (HTH) domain at the C terminus is not involved in DNA binding but may play a role in regulation of the complex.

FIG. 3.

Model of the architecture of the archaeal DNA replication fork. Parental DNA is indicated by black lines, and newly synthesized DNA is shown in red. RNA primers, synthesized by primase, are shown in blue. MCM is shown as a yellow hexameric assembly surrounding the leading-strand template. We propose that the MCM helicase translocates along this strand, unwinding the parental duplex ahead of the replication fork. Single-stranded DNA is bound by SSB (Sulfolobus nomenclature), shown as pink circles. MCM interacts with the archaeal GINS complex (brown), and GINS, in turn, is additionally capable of binding primase (light blue). We propose that GINS acts to couple MCM translocation on the leading-strand template with deposition of primase on the lagging-strand template. DNA polymerase (in salmon pink) acts to extend the RNA primers, and we indicate that two polymerases are coupled, although there is currently no evidence for this in archaeal systems. Each DNA Pol interacts with a trimer of PCNA (brown). PCNA can act as a platform for additional assembly of the flap endonuclease FEN1 (green) and DNA ligase 1 (Lig1 [blue]), as cartooned on the lagging strand-associated PCNA only.

FIG. 4.

Cartoon of the clamp loading process. A pentameric RFC (gray) containing one large subunit and four identical small subunits binds ATP and interacts with a ring of PCNA (blue) (step 1). RFC opens PCNA 34 Å in the plane of the ring, and DNA enters the ring (steps 2 and 3). The PCNA ring then closes around the DNA but leaves an out-of-plane gap of approximately 5 Å (step 4) before sealing shut (step 5). ATP is then hydrolyzed by RFC, and RFC leaves PCNA bound at the primer-template junction.