Atomic structures and functional implications of the archaeal RecQ-like helicase Hjm - PubMed (original) (raw)

Atomic structures and functional implications of the archaeal RecQ-like helicase Hjm

Takuji Oyama et al. BMC Struct Biol. 2009.

Abstract

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.

Conclusion: 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.

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Figures

Figure 1

Figure 1

Structure of Pyrococcus furiosus Hjm. (A) The Form 2 apo structure is shown as a ribbon representation with a color spectrum from blue (N-terminus) to red (C-terminus). (B) and (C) Omit |_F_o-_F_c| maps (2.5 σ) for ADP in Form 1 (B) and AMPPCP in Form 2 (C) are shown with the final models. (D) Comparison of the nucleotide binding sites between Hjm and Hel308. Hjm is represented in blue, and the key residues interacting with the ATP analog are highlighted. Segments of A. fulgidus Hel308 (pink) should undergo structural changes to bind the nucleotide, while S. solfataricus Hel308 (green) could bind the nucleotide with slight rearrangements in the pocket. A. fulgidus Hel308 residues, which should sterically clash with the nucleotide, are indicated by magenta arrows.

Figure 2

Figure 2

Structural comparison of _Pfu_Hjm with the A. fulgidus Hel308-DNA complex. (A) Hjm (colored as in Figure 1) was fitted to Hel308 (protein and DNA are colored grey and blue, respectively), by superimposing each domain individually. First, Domain 2 of Hjm was fitted, and then the other domains were separately moved to the best-fit positions. Shift values on the second fitting are indicated. (B) Sulfate ion binding sites in the Hjm Form 1 crystals. The protein is represented as a surface colored by electrostatic potential, calculated by the program GRASP [46]. The DNA structure is that in the Hel308-DNA complex. The boxed region is shown by a transparent surface to show the sulfate ions located inside of the protein.

Figure 3

Figure 3

Electron microscopy of the _Pfu_Hjm-DNA complex. (A) Representative electron microscopic images of the complex. (B) The sequence and structure of the 3' overhang DNA. (C) Class averages of the Hjm-DNA complex, obtained from 3599 particles. The box size is 204 Å. (D) Single particle 3D reconstruction of the complex. Surface representations of the complex are shown from two orthogonal orientations. The atomic structure of the Hjm protein, shown as a cyan ribbon, is fitted into the density. The double-stranded DNA molecule, colored magenta, is fitted to the protruded region. (E) Comparison with the crystal structure of the A. fulgidus Hel308-DNA complex. The A. fulgidus binary complex, shown as a green and yellow ribbon, was fitted to the map using the Hel308 protein, and the location of the protruded double-stranded DNA is shown for comparison.

Figure 4

Figure 4

Schematic view of the structural alignment. (A) Domain structure conservation of _Pfu_Hjm with relevant DNA binding proteins. SIZ, ORP, RMS, and IDN are the number of residues aligned, percent superposed residues (% overlap), RMSD (Å), and sequence identity (%) of each domain to Hjm, respectively. PDB and CHN are the PDB code and chain ID of the solutions, respectively. Abbreviations, Hjm; _Pfu_Hjm, RecQ; E. coli RecQ, ArgR; E. coli Arginine repressor, TFIIF; Transcription initiation factor IIF, SRP; single recognition particle protein, ERCC-1; Human DNA endonuclease ERCC-1, RuvA; E. coli Holliday junction binding protein RuvA. (B) Structure comparison of Hjm with other relevant proteins. Structures are aligned using the two well-conserved helicase domains (cyan and green), to highlight the variation of the other domains and those orientation. (C) Superposition of _Pfu_Hjm (blue) and the homology model of the human DNA polymerase Θ (orange) helicase domain, shown in stereo figure. The major-insertions (with more than two residues) of the DNA polymerase Θ were colored green. The insertions were localized to the peripherals of the molecule, and the central crafts of the proteins are mostly intact. The seventeen cysteine residues of the DNA polymerase Θ are shown in stick models, and colored according to their possible characteristics (buried, grey; exposed, yellow; disulfide bond, red)

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