Volume 13, Issue 1, Pages 143-153 (January 2005) Crystal Structure and Functional Implications of Pyrococcus furiosus Hef Helicase Domain Involved in Branched DNA Processing  Tatsuya Nishino, Kayoko Komori, Daisuke Tsuchiya, Yoshizumi Ishino, Kosuke Morikawa  Structure  Volume 13, Issue 1, Pages 143-153 (January 2005) DOI: 10.1016/j.str.2004.11.008 Copyright © 2005 Elsevier Ltd Terms and Conditions

Figure 1 Crystal Structure of Hef Helicase (A) Hef domain organization. Three structural domains of Hef are indicated by ovals. The N-terminal helicase domain forms Hef helicase, whereas the C-terminal nuclease and HhH domains form Hef nuclease. Structural domains within the helicase region are colored differently. (B) Overall structure of Hef helicase. Three structural domains are colored differently. Domain 1 is shown in red, domain 2 is shown in green, and domain 3 is shown in blue. Bound phosphate is shown as a ball-and-stick model. Disordered loop in domain 3 is shown as dotted line. (C) Close-up view of the helicase motifs and the bound phosphate. Motif I (magenta), Motif II (cyan), and Motif VI (yellow) are denoted by different colors. Side chains involved in phosphate recognition and domain-domain interactions are shown by a ball-and-stick model. (D) Structure-sequence alignment. Primary sequences of Hef (Pyrococcus furiosus), Mph1 (Saccharomyces cerevisiae), KIAA1596 (Homo sapiens), two Dicer proteins (Caenorhabditis elegans and Drosophila melanogaster), Helicard (Mus musculus), and RIG1 (Homo sapiens) are shown. The alignment was carried out by using ClustalW, with manual corrections. Identical residues are colored yellow, and conserved residues are colored green. Helicase motifs are boxed with Roman numeral numbering on top. Arrowheads on the top of the sequences indicate mutation sites of Hef. Helices are shown as cylinders, and β strands are shown as arrows. The coloring of the domains is the same as in (B). Structure 2005 13, 143-153DOI: (10.1016/j.str.2004.11.008) Copyright © 2005 Elsevier Ltd Terms and Conditions

Figure 2 Hef and Taq DNA Polymerase Structural Comparison Stereo view of Hef domain 2 (residues 213–337, green) and the taq DNA polymerase thumb domain (pdb code 3ktq, residues 454–599, blue), shown in a ribbon diagram. Structure 2005 13, 143-153DOI: (10.1016/j.str.2004.11.008) Copyright © 2005 Elsevier Ltd Terms and Conditions

Figure 3 Structural Comparisons between Hef, RecG, and RecQ Two orthogonal views of Hef helicase (left), RecG (middle, pdb code 1gm5), and RecQ (right, pdb code 1oyy) are shown in a ribbon diagram. The coloring scheme is the same as in Figure 1. N-terminal helicase fold, red; C-terminal helicase fold, blue; additional domains, green. Proteins were superimposed on Hef helicase, based on the similarity of their helicase core domains. Structure 2005 13, 143-153DOI: (10.1016/j.str.2004.11.008) Copyright © 2005 Elsevier Ltd Terms and Conditions

Figure 4 Mutational Analyses of Hef Helicase (A) Ribbon diagram of the Hef helicase domain colored as in Figure 1B. Side chains of the mutated residues are shown in yellow. (B and C) (B) Fork-structured DNA and (C) Holliday junction dissociation by Hef helicase. HefN546 and mutant proteins (5 or 50 nM) were each incubated with 2 nM 32P-labeled fork-structured DNA with a (B) gap (FG4) or (C) Holliday junction DNA (HSL) in helicase buffer at 37°C or 55°C, respectively, for 30 min. The products were analyzed by 12% PAGE, followed by autoradiography. The structures of the substrates and products are indicated by the schematic representations, and the asterisks in the schemes indicate 32P-labeled strands. Structure 2005 13, 143-153DOI: (10.1016/j.str.2004.11.008) Copyright © 2005 Elsevier Ltd Terms and Conditions

Figure 5 DNA-Dependent ATPase Activity by Hef Helicase (A–C) ATPase activities of (A) HefN546, (B) HefN546/R371E, and (C) HefN546/Δd2. Purified HefN546 and mutant proteins (each at 50 nM) were incubated with 1 mM (A) or 0.05 mM (B and C) ATP and [α-32P]ATP (25 pCi/μl) in cleavage buffer, in the presence or absence of duplex DNA (D, 400 nM), fork-structured DNA (FM, 400 nM), and Holliday junction DNA (HSL, 400 nM) at 60°C for 0, 5, 10, 15, 20, 30, 40, and 60 min. Aliquots of reactions were analyzed by thin layer chromatography. The amounts of ATP and ADP were quantified from the auto radiograms by using a laser-excited image analyzer (BAS5000, Fuji Film). The graph was plotted according to the amount of ADP formed by the hydrolysis of ATP, after subtraction of the background radioactivity from the reaction without protein at each point. Structure 2005 13, 143-153DOI: (10.1016/j.str.2004.11.008) Copyright © 2005 Elsevier Ltd Terms and Conditions

Figure 6 Models of Hef in Complex with Branched Structure DNA Hef is shown as the surface representation whose orientation is the same as in Figure 1B. The surface was colored according to its electrostatic surface potential at + or − 10 kB T/e for positive (blue) or negative (red) charge potential by using the program GRASP (Nicholls, 1993). DNA is shown as schematic double helices. (A) Model 1. Domain 2 interacts with the double-stranded region of the branched structure, and the fork-structured DNA is recognized by the coordinated action of all three domains. (B) Model 2. Domain 2 directly interacts with a moiety near the junction, and the helicase core domains support double- or single-stranded DNA binding in a mode similar to the case of RecG. Structure 2005 13, 143-153DOI: (10.1016/j.str.2004.11.008) Copyright © 2005 Elsevier Ltd Terms and Conditions