Abstract
The E. coli γ clamp loader is a pentameric complex of δ, δ′ and three γ subunits that opens and loads β-clamp proteins onto DNA in an ATP-dependent process essential for efficient DNA replication. ATP binding to the γ subunits promotes conformational changes that enable the clamp loader to bind and open the ring-shaped β-clamp homodimer. Here we report the nearly complete backbone and side-chain 1H, 13C and 15N NMR resonance assignments of the 242-residue truncated γ subunit of the clamp loader complex, which includes the N-terminal mini (domain I) and lid (domain II) domains. This construct represents the nucleotide binding module in the clamp loader complex and provides a model system for studies of conformational rearrangements of the clamp loader induced by nucleotide binding.
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References
Ahmadian MR, Stege P, Scheffzek K, Wittinghofer A (1997) Confirmation of the arginine-finger hypothesis for the GAP-stimulated GTP-hydrolysis reaction of Ras. Nat Struct Biol 4:686–689
Berjanskii MV, Wishart DS (2008) Application of the random coil index to studying protein flexibility. J Biomol NMR 40:31–48
Blinkowa AL, Walker JR (1990) Programmed ribosomal frameshifting generates the Escherichia coli DNA polymerase III gamma subunit from within the tau subunit reading frame. Nucleic Acids Res 18:1725–1729
Bloom LB (2009) Loading clamps for DNA replication and repair. DNA Repair (Amst) 8:570–578
Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6:277–293
Flower AM, McHenry CS (1990) The gamma subunit of DNA polymerase III holoenzyme of Escherichia coli is produced by ribosomal frameshifting. Proc Natl Acad Sci USA 87:3713–3717
Hedglin M, Kumar R, Benkovic SJ (2013) Replication clamps and clamp loaders. Cold Spring Harb Perspect Biol 5:a010165
Hingorani MM, O’Donnell M (1998) ATP binding to the Escherichia coli clamp loader powers opening of the ring-shaped clamp of DNA polymerase III holoenzyme. J Biol Chem 273:24550–24563
Jeruzalmi D, O’Donnell M, Kuriyan J (2001) Crystal structure of the processivity clamp loader gamma (gamma) complex of E. coli DNA polymerase III. Cell 106:429–441
Kelch BA (2016) Review: the lord of the rings: structure and mechanism of the sliding clamp loader. Biopolymers 105:532–546
Kelch BA, Makino DL, O’Donnell M, Kuriyan J (2012) Clamp loader ATPases and the evolution of DNA replication machinery. BMC Biol 10:34
Kodaira M, Biswas SB, Kornberg A (1983) The dnaX gene encodes the DNA polymerase III holoenzyme tau subunit, precursor of the gamma subunit, the dnaZ gene product. Mol Gen Genet 192:80–86
Kong XP, Onrust R, O’Donnell M, Kuriyan J (1992) Three-dimensional structure of the beta subunit of E. coli DNA polymerase III holoenzyme: a sliding DNA clamp. Cell 69:425–437
Lee W, Tonelli M, Markley JL (2015) NMRFAM-SPARKY: enhanced software for biomolecular NMR spectroscopy. Bioinformatics 31:1325–1327
Maciejewski MW, Schuyler AD, Gryk MR, Moraru II, Romero PR, Ulrich EL, Eghbalnia HR, Livny M, Delaglio F, Hoch JC (2017) NMRbox: a resource for biomolecular NMR computation. Biophys J 112:1529–1534
Neuwald AF, Aravind L, Spouge JL, Koonin EV (1999) AAA+: a class of chaperone-like ATPases associated with the assembly, operation, and disassembly of protein complexes. Genom Res 9:27–43
O’Donnell M, Onrust R, Dean FB, Chen M, Hurwitz J (1993) Homology in accessory proteins of replicative polymerases—E. coli to humans. Nucleic Acids Res 21:1–3
Oakley AJ, Prosselkov P, Wijffels G, Beck JL, Wilce MC, Dixon NE (2003) Flexibility revealed by the 1.85 A crystal structure of the beta sliding-clamp subunit of Escherichia coli DNA polymerase III. Acta Crystallogr D Biol Crystallogr 59:1192–1199
Podobnik M, Weitze TF, O’Donnell M, Kuriyan J (2003) Nucleotide-induced conformational changes in an isolated Escherichia coli DNA polymerase III clamp loader subunit. Structure 11:253–263
Pritchard AE, Dallmann HG, Glover BP, McHenry CS (2000) A novel assembly mechanism for the DNA polymerase III holoenzyme DnaX complex: association of δδ’ with DnaX(4) forms DnaX(3)δδ’. EMBO J 19:6536–6545
Sattler M, Schleucher J, Griesinger C (1999) Heteronuclear multidimensional NMR experiments for the structure determination of proteins in solution employing pulsed field gradients. Prog Nucl Magn Reson Spectrosc 34:93–158
Shen Y, Bax A (2013) Protein backbone and sidechain torsion angles predicted from NMR chemical shifts using artificial neural networks. J Biomol NMR 56:227–241
Tsuchihashi Z, Kornberg A (1989) ATP interactions of the tau and gamma subunits of DNA polymerase III holoenzyme of Escherichia coli. J Biol Chem 264:17790–17795
Tsuchihashi Z, Kornberg A (1990) Translational frameshifting generates the gamma subunit of DNA polymerase III holoenzyme. Proc Natl Acad Sci U S A 87:2516–2520
Walker JE, Saraste M, Runswick MJ, Gay NJ (1982) Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J 1:945–951
Ying J, Delaglio F, Torchia DA, Bax A (2017) Sparse multidimensional iterative lineshape-enhanced (SMILE) reconstruction of both non-uniformly sampled and conventional NMR data. J Biomol NMR 68:101–118
Zambrello MA, Schuyler AD, Maciejewski MW, Delaglio F, Bezsonova I, Hoch JC (2018) Nonuniform sampling in multidimensional NMR for improving spectral sensitivity. Methods 138–139:62–68
Acknowledgements
This work was supported by the National Institutes of Health R01GM123239 grant to P.J.B. and D.M.K. The authors thank Mr. Thomas Bregnard for helpful discussion.
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Mahdi, S., Bezsonova, I., Beuning, P.J. et al. NMR resonance assignments for the nucleotide binding domains of the E. coli clamp loader complex γ subunit. Biomol NMR Assign 15, 281–285 (2021). https://doi.org/10.1007/s12104-021-10018-7
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DOI: https://doi.org/10.1007/s12104-021-10018-7