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ABSTRACT: REV1, REV3, and REV7 are pivotal proteins in translesion DNA synthesis, which allows DNA synthesis even in the presence of DNA damage. REV1 and REV3 are error-prone DNA polymerases and function as inserter and extender polymerases in this process, respectively. REV7 interacts with both REV1 and REV3, acting as an adaptor that functionally links the two, although the structural basis of this collaboration remains unclear. Here, we show the crystal structure of the ternary complex, composed of the C-terminal domain of human REV1, REV7, and a REV3 fragment. The REV1 C-terminal domain adopts a four-helix bundle that interacts with REV7. A linker region between helices 2 and 3, which is conserved among mammals, interacts with the β-sheet of REV7. Remarkably, the REV7-binding interface is distinct from the binding site of DNA polymerase η or κ. Thus, the REV1 C-terminal domain might facilitate polymerase switching by providing a scaffold for both inserter and extender polymerases to bind. Our structure reveals the basis of DNA polymerase ζ (a complex of REV3 and REV7) recruitment to the stalled replication fork and provides insight into the mechanism of polymerase switching.
Journal of Biological Chemistry 08/2012; 287(40):33847-52. · 4.77 Impact Factor
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ABSTRACT: REV1, REV3 and REV7 are pivotal proteins in translesion DNA synthesis that allows DNA synthesis to continue even in the presence of DNA damage. REV1 and REV3 are error-prone DNA polymerases, while REV7 acts as an adaptor protein that links them together. A ternary complex of the C-terminal domain of human REV1 in complex with REV7 bound to a REV3 fragment has been crystallized. The crystals belonged to space group P3(1)21, with unit-cell parameters a = b = 74.7, c = 124.5 Å.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 08/2012; 68(Pt 8):962-4. · 0.51 Impact Factor
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ABSTRACT: Nanos is an RNA-binding protein that is involved in the development and maintenance of germ cells. In combination with Pumilio, Nanos binds to the 3' untranslated region of a messenger RNA and represses its translation. Nanos has two conserved Cys-Cys-His-Cys zinc-finger motifs that are indispensable for its function. In this study, we have determined the crystal structure of the zinc-finger domain of zebrafish Nanos, for the first time revealing that Nanos adopts a novel zinc-finger structure. In addition, Nanos has a conserved basic surface that is directly involved in RNA binding. Our results provide the structural basis for further studies to clarify Nanos function.
EMBO Reports 11/2010; 11(11):848-53. · 7.36 Impact Factor
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ABSTRACT: DNA polymerase ζ (Polζ) is an error-prone DNA polymerase involved in translesion DNA synthesis. Polζ consists of two subunits:
the catalytic REV3, which belongs to B family DNA polymerase, and the noncatalytic REV7. REV7 also interacts with REV1 polymerase,
which is an error-prone Y family DNA polymerase and is also involved in translesion DNA synthesis. Cells deficient in one
of the three REV proteins and those deficient in all three proteins show similar phenotype, indicating the functional collaboration
of the three REV proteins. REV7 interacts with both REV3 and REV1 polymerases, but the structure of REV7 or REV3, as well
as the structural and functional basis of the REV1-REV7 and REV3-REV7 interactions, remains unknown. Here we show the first
crystal structure of human REV7 in complex with a fragment of human REV3 polymerase (residues 1847–1898) and reveal the mechanism
underlying REV7-REV3 interaction. The structure indicates that the interaction between REV7 and REV3 creates a structural
interface for REV1 binding. Furthermore, we show that the REV7-mediated interactions are responsible for DNA damage tolerance.
Our results highlight the function of REV7 as an adapter protein to recruit Polζ to a lesion site. REV7 is alternatively called
MAD2B or MAD2L2 and also involved in various cellular functions such as signal transduction and cell cycle regulation. Our
results will provide a general structural basis for understanding the REV7 interaction.
Journal of Biological Chemistry 04/2010; 285(16):12299-12307. · 4.77 Impact Factor
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ABSTRACT: DNA polymerase zeta (Polzeta) is an error-prone DNA polymerase involved in translesion DNA synthesis. Polzeta consists of two subunits: the catalytic REV3, which belongs to B family DNA polymerase, and the noncatalytic REV7. REV7 also interacts with REV1 polymerase, which is an error-prone Y family DNA polymerase and is also involved in translesion DNA synthesis. Cells deficient in one of the three REV proteins and those deficient in all three proteins show similar phenotype, indicating the functional collaboration of the three REV proteins. REV7 interacts with both REV3 and REV1 polymerases, but the structure of REV7 or REV3, as well as the structural and functional basis of the REV1-REV7 and REV3-REV7 interactions, remains unknown. Here we show the first crystal structure of human REV7 in complex with a fragment of human REV3 polymerase (residues 1847-1898) and reveal the mechanism underlying REV7-REV3 interaction. The structure indicates that the interaction between REV7 and REV3 creates a structural interface for REV1 binding. Furthermore, we show that the REV7-mediated interactions are responsible for DNA damage tolerance. Our results highlight the function of REV7 as an adapter protein to recruit Polzeta to a lesion site. REV7 is alternatively called MAD2B or MAD2L2 and also involved in various cellular functions such as signal transduction and cell cycle regulation. Our results will provide a general structural basis for understanding the REV7 interaction.
Journal of Biological Chemistry 02/2010; 285(16):12299-307. · 4.77 Impact Factor
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ABSTRACT: REV7 is involved in various cellular functions including DNA replication, signal transduction and cell-cycle regulation. In DNA replication, REV7 interacts with REV3 and forms DNA polymerase zeta, which plays a central role in error-prone DNA synthesis. REV3 is a catalytic subunit and its activity is stimulated by REV7. To clarify the structural basis of the interaction between REV7 and REV3, human REV7 was crystallized in complex with a REV3 fragment. Two crystal forms were obtained. Crystal forms I and II belonged to space groups P2(1), with unit-cell parameters a = 43.8, b = 50.0, c = 107.3 A, beta = 96.9 degrees , and P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 76.6, c = 118.4 A, respectively.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 12/2009; 65(Pt 12):1302-5. · 0.51 Impact Factor
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ABSTRACT: Nanos is a highly conserved RNA-binding protein in higher eukaryotes and acts as a key regulator protein involved in translational control utilizing the 3' untranslated region of mRNA. The C-terminal domain of Nanos has two conserved and novel CCHC-type zinc-finger motifs that are responsible for the function of Nanos. To clarify the structural basis of the function of Nanos, the C-terminal domain (residues 59-159) of zebrafish Nanos was overexpressed, purified and crystallized. The crystal belonged to space group P6(3), with unit-cell parameters a = b = 100.9, c = 71.5 A, gamma = 120 degrees. Structure determination by the MAD/SAD method is now in progress.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 10/2009; 65(Pt 9):959-61. · 0.51 Impact Factor
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ABSTRACT: The crystal structure of 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) from Escherichia coli complexed with Mg(2+), NADPH and fosmidomycin was solved at 2.2 A resolution. DXR is the key enzyme in the 2-C-methyl-D-erythritol 4-phosphate pathway and is an effective target of antimalarial drugs such as fosmidomycin. In the crystal structure, electron density for the flexible loop covering the active site was clearly observed, indicating the well ordered conformation of DXR upon substrate binding. On the other hand, no electron density was observed for the nicotinamide-ribose portion of NADPH and the position of Asp149 anchoring Mg(2+) was shifted by NADPH in the active site.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 07/2007; 63(Pt 6):466-70. · 0.51 Impact Factor
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ABSTRACT: We have obtained the single-crystal X-ray crystallographic structures of the bisphosphonates [(1-isoquinolinylamino)methylene]-1,1-bisphosphonate and [[(5-chloro-2-pyridinyl)amino]methylene]-1,1-bisphosphonate, bound to the enzyme 1-deoxyxylulose-5-phosphate reductoisomerase (DXR, EC 1.1.1.267, also known as 2-C-methyl-d-erythritol-4-phosphate synthase), an important target for the development of antimalarial drugs. Our results indicate that both bisphosphonates bind into the fosmidomycin binding site. The aromatic groups are in a shallow hydrophobic pocket, and the phosphonate groups are involved in electrostatic interactions with Mg2+ or a cluster of carboxylic acid groups and lysine while the fosmidomycin phosphonate-binding site is occupied by a sulfate ion (as also observed in the DXR/NADP+ structure). The availability of these two new crystal structures opens up the possibility of the further development of bisphosphonates and related systems as DXR inhibitors and, potentially, as antiinfective agents.
Journal of the American Chemical Society 10/2004; 126(35):10824-5. · 9.91 Impact Factor
