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Structure and Mechanism of the DNA Polymerase Processivity Clamp Loader D

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Structure and Mechanism of the DNA Polymerase Processivity Clamp Loader D Structure and Mechanism of the DNA Polymerase Processivity Clamp Loader D. Jeruzalmi, M. O’Donnell, and J. Kuriyan The Rockefeller University, New York, NY ABSTRACT: plex with a clamp subunit (β:δ). These structures were The replication of a genome is a fundamental biologi- solved using data measured at the National Synchro- cal process that every organism must perform in order tron Light Source (X-25) at the Brookhaven National to transmit its genetic makeup to its progeny. We have Laboratory, at the Structural Biology Center at the Ad- been studying the molecular machines that are used vanced Photon Source (ID-19), the Stanford Synchro- to rapidly and accurately replicate the DNA genome of tron Radiation Laboratory (9.2), and the Advanced Light bacteria. In addition to the polymerase (the actual copy- Source (5.02) at the Lawrence Berkeley Laboratory. ing machine), organisms have a set of assemblies that The crystal structure of the β:δ assembly reveals enable the polymerase to perform its jobs more rap- that the δ subunit binds to the C-terminal side of the idly. One of these assemblies is the processivity clamp, processivity clamp at a location near to, but not at the which is a ring shaped protein that encircles and slides dimer interface (Figure 1) (3). We used a mutated β β along DNA and physically tethers the polymerase to (I272A, L273A) form of ( 1), which exists as a stable the DNA that is being replicated. The main problem monomer in solution and which binds to the wrench that we have been studying is how does a ring-shaped subunit (δ) with a 50 fold higher affinity than wild type sliding clamp become assembled onto DNA, which is (9). The δ subunit, which adopts the same fold as the a long polymer. Organisms have solved this problem other clamp loader subunits, places its N-terminal do- by using a clamp loader machine that physically opens main (β-interacting element) into a binding site com- β the ring and loads it onto DNA. We have determined posed of two domains (2 & 3) on 1. Two highly con- the structure of an intact and functional clamp loader served hydrophobic δ residues (F73, L74) are wedged machine. This structure, along with a second structure into a hydrophobic pocket on the clamp. Binding of the of the clamp, caught in the act of being opened, has δ subunit requires a conformational change in β that allowed us to construct a model of the clamp loading renders the clamp interface incapable of closing. With reaction. respect to its structure in the dimeric clamp, β from the β:δ complex adopts a conformation of reduced curva- Chromosomal replicases from prokaryotes to eu- ture. This observation along with molecular dynamics karyotes are organized around three functional com- simulations suggest a spring-loaded mechanism in ponents, a DNA polymerase, a ring-shaped processivity which the β ring opens spontaneously once a dimer clamp, and a clamp-loading machine. DNA polymerases interface is perturbed by the β wrench. catalyze the addition of nucleotides to primed tem- plate DNA and, in isolation, are poorly processive enzymes (4,10,13). Presence of the processivity clamp, dramatically increase processivity. Processivity clamps are ring shaped proteins com- posed of 2 (eubacteria) or 3 (phage, archae, eu- karyote) subunits (5). The clamps confer processivity by encircling DNA and physically teth- ering the polymerase subunit to the DNA as it tracks along the template (11). Assembly of slid- ing clamps on DNA is performed by the clamp loader assembly in a reaction fueled by ATP (12). In bacteria, the clamp loader is composed of 7 γ, δ, δ, χ, ψ β unique subunits ( ) and the 2 dimer is the processivity clamp. The clamp loader is an AAA+ ATPase and is the bacterial homologue of the eukaryotic replication factor C (RFC) (7, 8). We have determined the structure of the bacterial γ δδ δ β:δ clamp loader 3 ’ and that of the wrench in com- Figure 1. Structure of the complex. 2 - 33 Science Highlights Binding of the β subunit requires a substantial con- minal domains of the subunits form a circular collar that formational change in the β clamp that renders it inca- supports an asymmetric arrangement of the N-termi- pable of forming a closed ring. Clamp opening by the δ nal ATP binding domains of the γ motor and the struc- wrench exploits a peculiar structural feature at the β turally related domains of the δ’ stator and the δ wrench. dimer interface. One element of the subunit interface The nucleotide free clamp loader crystallizes with the of the dimeric clamp consists of the interfacial helix β-interacting element of the δ wrench accessible to sol- (α1”), whose last three residues (A271, I272 and L273) vent and not buried within the assembly. This observa- are distorted from ideal geometry. This distortion al- tion was unexpected since the clamp loader has no lows the side chains of residues I272 and L273 to fit detectible interaction with the clamp in the absence of into hydrophobic pockets on the surface of Domain I of nucleotide. The 3 ATP binding sites on the γ motor sub- the second β monomer. In the β:δ assembly, helix α1” units are located near clamp loader subunit interfaces. is essentially undistorted, leaving it in a conformation The structure crystallizes with only 2 of 3 sites avail- that is incapable of forming a well-packed dimer inter- able for ATP binding; the third site is blocked by the face with the second member of the dimer. Distortion presence of structural elements from a neighboring of helix α1” is directly coupled to the binding of δ to β subunit. We hypothesize that the structure in the crys- through a 5 residue loop in β (residues 274 to 278) that tal represents an intermediate in the clamp loading re- immediately follows it and which interacts closely with action. Nucleotide hydrolysis likely involves a conserved δ β δ β in the : complex. The structure of this loop in 2 is arginine residue from a neighboring subunit in a man- well defined due to the presence of a β turn between ner reminiscent to mechanisms proposed for other residues 275 and 278, but its conformation would pre- AAA+ ATPases (e.g. p97, NSF). The structure suggests vent the binding of δ. a mechanism by which the g complex switches between β β β δ β δ In comparison to 2, 1 of the : complex is sys- a closed state, in which the -interacting element of β δ tematically distorted. Superposition of the 1 onto do- is hidden by , and an open form similar to the crystal β δ β main 2 of 2 reveals a strikingly relaxed structure. Do- structure, in which is free to bind to . main 1 is rotated by 12° and domain 3 by 5° away from Crystal structures of assemblies that are members the central cavity. We have chosen to explain the ob- of the (AAA+, ATPases associated with various cellu- servation that the largest deviation from the dimeric lar activities) family have emphasized the importance clamp occurs distant from the β binding site by propos- of subunit interfaces in the coupling of ATP binding/ ing that the closed β dimer is under spring tension. The hydrolysis to biological function and inter-subunit com- wrench subunit might therefore open the clamp by dis- munication. For example, in the NSF (6) (14) or P97 rupting one of the dimer interfaces; clamp opening (15)assembly, the AAA+ domains are arranged as a would occur spontaneously through release of the hexamer with nucleotides bound near subunit inter- spring tension. A series of molecular dynamics calculations recapitulate the structure of the relaxed β monomer starting from the strained dimeric clamp. The conformational change seen in the β subunit allows a plausible model of the opened clamp to be con- structed. Superposition of the relaxed β 1 onto domain 1 and the paired do- main 3 at the interface that remains closed results in the appearance of a ≈15Å gap at the disrupted interface. This gap is large enough to accommo- date single stranded DNA and we have proposed that inter-domain flexibility in addition to interaction with other sub- units of the clamp loader might permit duplex DNA to enter/exit the clamp. The 2.7/3.0 Å crystal structure of the clamp loader reveals a pentameric arrangement of subunits, with a stoichi- δ γ δ γ ometry : 3: (Figure 2) (2). The C-ter- Figure 2. Structure of the complex. NSLS Activity Report 2001 2 - 34 faces. The majority of protein contacts to nucleotide Occupancy of all the nucleotide binding sites would then come from the recA-like fold of one subunit, while in- expose the beta interaction element for contacts with ter-subunit communication and nucleotide hydrolysis the sliding clamp as seen in the structure of the β:δ are mediated, at least in part, by completion of the bind- assembly. Presence of primed-template, which has ing site through donation of a lys (NSF) or arg (p97) been properly positioned by the clamp loader within residue from an adjacent subunit. While the AAA+ pro- the opened clamp, would trigger hydrolysis of ATP. Hy- teins NSF and P97 crystallized as symmetric oligomers, drolysis of ATP would return the clamp loader to its the processivity clamp loader has been captured with ‘closed’ state and lead to release of clamp. The released the AAA+ modules deployed asymmetrically. Though clamp would close on DNA at the primer template junc- a result of packing in the crystal lattice, this asymmetry tion.
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