Atomic structures and functional implications of the archaeal RecQ-like helicase Hjm.
BACKGROUND
Pyrococcus furiosus Hjm (PfuHjm) is a structure-specific DNA helicase that was originally identified by in vitro screening for Holliday junction migration activity. It belongs to helicase superfamily 2, and shares homology with the human DNA polymerase Theta (PolTheta), HEL308, and Drosophila Mus308 proteins, which are involved in DNA repair. Previous biochemical and genetic analyses revealed that PfuHjm preferentially binds to fork-related Y-structured DNAs and unwinds their double-stranded regions, suggesting that this helicase is a functional counterpart of the bacterial RecQ helicase, which is essential for genome maintenance. Elucidation of the DNA unwinding and translocation mechanisms by PfuHjm will require its three-dimensional structure at atomic resolution.


RESULTS
We determined the crystal structures of PfuHjm, in two apo-states and two nucleotide bound forms, at resolutions of 2.0-2.7 A. The overall structures and the local conformations around the nucleotide binding sites are almost the same, including the side-chain conformations, irrespective of the nucleotide-binding states. The architecture of Hjm was similar to that of Archaeoglobus fulgidus Hel308 complexed with DNA. An Hjm-DNA complex model, constructed by fitting the five domains of Hjm onto the corresponding Hel308 domains, indicated that the interaction of Hjm with DNA is similar to that of Hel308. Notably, sulphate ions bound to Hjm lie on the putative DNA binding surfaces. Electron microscopic analysis of an Hjm-DNA complex revealed substantial flexibility of the double stranded region of DNA, presumably due to particularly weak protein-DNA interactions. Our present structures allowed reasonable homology model building of the helicase region of human PolTheta, indicating the strong conformational conservation between archaea and eukarya.


CONCLUSIONS
The detailed comparison between our DNA-free PfuHjm structure and the structure of Hel308 complexed with DNA suggests similar DNA unwinding and translocation mechanisms, which could be generalized to all of the members in the same family. Structural comparison also implied a minor rearrangement of the five domains during DNA unwinding reaction. The unexpected small contact between the DNA duplex region and the enzyme appears to be advantageous for processive helicase activity.
Oyama
Takuji
T
The Takara Bio Endowed Division, Institute for Protein Research, Osaka University, Open Laboratories of Advanced Bioscience and Biotechnology (OLABB), 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan.
Oka
Hayato
H
Mayanagi
Kouta
K
Shirai
Tsuyoshi
T
Matoba
Kyoko
K
Fujikane
Ryosuke
R
Ishino
Yoshizumi
Y
Morikawa
Kosuke
K
D019843:Archaeal Proteins / Q000737:chemistry* / Q000648:ultrastructure; D019611:Archaeoglobus fulgidus / Q000201:enzymology; D019641:DNA, Archaeal / Q000378:metabolism / Q000648:ultrastructure; D006801:Humans; D008854:Microscopy, Electron; D008958:Models, Molecular; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D020027:Pyrococcus furiosus / Q000201:enzymology*; D053484:RecQ Helicases / Q000737:chemistry* / Q000648:ultrastructure; D016415:Sequence Alignment; D040681:Structural Homology, Protein
D019843:Archaeal Proteins; D019641:DNA, Archaeal; D053484:RecQ Helicases