Structural analysis of a eukaryotic sliding DNA clamp–clamp loader complex

Bowman, Gregory D.; O’Donnell, Michael; Kuriyan, John

doi:10.1038/nature02585

Cited by 418 publications

(688 citation statements)

References 49 publications

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“…Oligomerization of DnaB, the SF3 helicases [46], and MCM proteins [47] appears to derive from a second domain that is static and also forms tight and extensive interactions with adjacent subunits through apparently inflexible interfaces. These properties classify this region as a 'collar' similar to that described earlier in the case of clamp-loaders [48,49]. The collar provides a rigid scaffold to direct the movements of the appended ATPase domains [29 ,30].…”

Section: Intersubunit Interactionsmentioning

confidence: 92%

On helicases and other motor proteins

Enemark

Joshua‐Tor

2008

Current Opinion in Structural Biology

192

238

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Helicases are molecular machines that utilize energy derived from ATP hydrolysis to move along nucleic acids and to separate base-paired nucleotides. The movement of the helicase can also be described as a stationary helicase that pumps nucleic acid. Recent structural data for the hexameric E1 helicase of papillomavirus in complex with single-stranded DNA and MgADP has provided a detailed atomic and mechanistic picture of its ATP-driven DNA translocation. The structural and mechanistic features of this helicase are compared with the hexameric helicase prototypes T7gp4 and SV40 T-antigen. The ATP-binding site architectures of these proteins are structurally similar to the sites of other prototypical ATP-driven motors such as F1-ATPase, suggesting related roles for the individual site residues in the ATPase activity. IntroductionHelicases are essential enzymes that unwind duplex DNA, RNA, or DNA-RNA hybrids. This unwinding is driven by consumption of input energy that is harnessed to separate base-paired oligonucleotides and also to maintain a unidirectional advancement of the helicase upon the nucleic acid substrate. This translocation can alternatively be described as an immobile helicase pumping nucleic acid. The energy for these transformations is derived from the hydrolysis of nucleotide triphosphate (NTP). Helicases can be depicted as an internal combustion engine with each individual NTPase site serving as one cylinder. Each individual cylinder follows a defined series of events: injection (ATP binding), compression (optimally positioning the site for hydrolysis), combustion (ATP hydrolysis/work generation), and exhaust (ADP and phosphate release). In a helicase, the individual combustion cylinders coordinate these actions to carry out the repetitive mechanical operation of prying open base pairs and/or actively translocating with respect to the nucleic acid substrate. Many other molecular motors utilize similar engines to carry out multiple diverse functions such as translocation of peptides in the case of ClpX, movement along cellular structures in the case of dyenin, and rotation about an axle as in F 1 -ATPase.Based upon conserved sequence motifs, helicases have been classified into six superfamilies [1,2 ]. An extensive review of these superfamilies has been provided recently [3 ]. Superfamily 1 (SF1) and superfamily 2 (SF2) helicases are very prevalent, generally monomeric, and participate in several diverse DNA and RNA manipulations. The other helicase superfamilies form hexameric rings (reviewed in [4]), as demonstrated by biochemistry [5][6][7][8] and electron microscopy studies [9][10][11][12][13][14][15][16][17], and often participate at the replication fork. All of these helicases bind and hydrolyze NTP at the interface between two recA-like domains. The binding site consists of a Walker A (P-loop) and a Walker B motif from the first domain and other elements such as an arginine finger from the other domain. The SF1 and SF2 helicases contain two recAlike domains coupled by a short linker, and t...

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Section: Intersubunit Interactionsmentioning

confidence: 92%

On helicases and other motor proteins

Enemark

Joshua‐Tor

2008

Current Opinion in Structural Biology

192

238

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“…Conversely, the affinity of PCNA for p15 is higher than for a PIPbox fragment of the p66 subunit of the replicative polymerase d (Pold; K d ¼ 15.4 mM, measured by ITC at 30°C) 29 , and similar to the translesion synthesis polymerase Z (TLS PolZ; K d ¼ 0.4 mM, measured by surface plasmon resonance at 25°C) 33 . Pold is a four-subunit complex of 239 kDa, much larger than PCNA, which possibly occludes (by binding and steric hindrance) more than one PIP-box-binding groove, as observed in the complexes of large proteins with PCNA rings 34,35 . PolZ is a 78-kDa multidomain protein with an ubiquitin-binding domain that increases its affinity for PCNA when ubiquitylated at K164 after ultraviolet irradiation 36 .…”

Section: Discussionmentioning

confidence: 99%

Structure of p15PAF–PCNA complex and implications for clamp sliding during DNA replication and repair

et al. 2015

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The intrinsically disordered protein p15 PAF regulates DNA replication and repair by binding to the proliferating cell nuclear antigen (PCNA) sliding clamp. We present the structure of the human p15 PAF -PCNA complex. Crystallography and NMR show the central PCNA-interacting protein motif (PIP-box) of p15 PAF tightly bound to the front-face of PCNA. In contrast to other PCNA-interacting proteins, p15 PAF also contacts the inside of, and passes through, the PCNA ring. The disordered p15 PAF termini emerge at opposite faces of the ring, but remain protected from 20S proteasomal degradation. Both free and PCNA-bound p15 PAF binds DNA mainly through its histone-like N-terminal tail, while PCNA does not, and a model of the ternary complex with DNA inside the PCNA ring is consistent with electron micrographs. We propose that p15 PAF acts as a flexible drag that regulates PCNA sliding along the DNA and facilitates the switch from replicative to translesion synthesis polymerase binding.

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“…In the crystal structure of the yeast clamp loader complex replication factor C (RFC) bound to PCNA, PCNA is closed and has the same flat, disk-like organization that is observed in the crystal structures of isolated clamps (17). The right-handed spiral organization of the clamp loader subunits suggested a mechanism for DNA recognition, because the pitch of the RFC spiral is approximately congruent with the helical geometry of duplex DNA (17).…”

mentioning

confidence: 96%

Out-of-plane motions in open sliding clamps: Molecular dynamics simulations of eukaryotic and archaeal proliferating cell nuclear antigen

Kazmirski

Zhao

Bowman

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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Sliding clamps are ring-like multimeric proteins that encircle duplex DNA and serve as mobile DNA-bound platforms that are essential for efficient DNA replication and repair. Sliding clamps are placed on DNA by clamp loader complexes, in which the clamp-interacting elements are organized in a right-handed spiral assembly. To understand how the flat, ring-like clamps might interact with the spiral interaction surface of the clamp loader complex, we have performed molecular dynamics simulations of sliding clamps (proliferating cell nuclear antigen from the budding yeast, humans, and an archaeal species) in which we have removed one of the three subunits so as to release the constraint of ring closure. The simulations reveal significant structural fluctuations corresponding to lateral opening and out-of-plane distortions of the clamp, which result principally from bending and twisting of the ␤-sheets that span the intermolecular interfaces, with smaller but similar contributions from ␤-sheets that span the intramolecular interfaces within each subunit. With the integrity of these ␤-sheets intact, the predominant fluctuations seen in the simulations are oscillations between lateral openings and right-handed spirals. The tendency for clamps to adopt a right-handed spiral conformation implies that once opened, the conformation of the clamp can easily match the spiraling of clamp loader subunits, a feature that is intrinsic to the recognition of DNA and subsequent hydrolysis of ATP by the clamp-bound clamp loader complex.clamp loader ͉ DNA replication ͉ DNA sliding clamps ͉ ␤-sheet distortion ͉ conformational transitions

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Structural analysis of a eukaryotic sliding DNA clamp–clamp loader complex

Cited by 418 publications

References 49 publications

On helicases and other motor proteins

On helicases and other motor proteins

Structure of p15PAF–PCNA complex and implications for clamp sliding during DNA replication and repair

Out-of-plane motions in open sliding clamps: Molecular dynamics simulations of eukaryotic and archaeal proliferating cell nuclear antigen

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