Journal of Molecular Biology
Regular articleCrystal structure of the DNA polymerase processivity factor of T4 bacteriophage1
Introduction
Highly processive DNA polymerases in bacteria and eukaryotes achieve remarkable speed in DNA replication by tethering their catalytic subunits to ring-shaped proteins (e.g. see Huang et al 1981, Kuriyan and O’Donnell 1993, Stillman 1994, Stukenberg et al 1991). These proteins, known as sliding clamps, encircle DNA and allow the polymerases to move rapidly without detachment from the template. This principle was first established for the β-clamp of Escherichia coli DNA polymerase III complex, which allows the polymerase to move for thousands of nucleotides along the template without dissociation (reviewed by Kelman & O’Donnell, 1995).
The crystal structure of the β-clamp showed that two molecules of β form a ring-shaped dimer that can girdle duplex DNA without steric hindrance (Kong et al., 1992). Although the overall charge of the protein is negative, the distribution of charged residues within the protein is such that the space inside the ring is a region of positive electrostatic potential. The structure provided an immediate explanation for how the protein can move rapidly along DNA without dissociation, since it revealed the basis for a non-specific topological interaction with DNA.
The connection between structure and function that is so apparent in the β-clamp naturally raised the question as to whether the processivity factors for other DNA polymerases adopt a similar ring-shaped structure. The bacterial β-clamp is a dimer of ∼40 kDa subunits, whereas the eukaryotic DNA polymerase processivity factor PCNA and gp45 (protein encoded by gene 45) are trimers of ∼25 kDa subunits. The internal symmetry of the β-ring suggested that PCNA and gp45 could form similar rings if each monomer of these proteins contained two of the three domains of the β-clamp (Kong et al., 1992). While this proposal has an attractive simplicity to it, the amino acid sequences of these proteins are completely unrelated and the truth of this proposal can be established only by crystallographic analysis. The crystal structures of yeast and human PCNA have been determined and have confirmed that the principles established for the bacterial processivity factor are readily transferred to the eukaryotic system Gulbis et al 1996, Krishna et al 1994.
The bacteriophage T4 replication apparatus has served as an important model system for understanding the principles underlying high-speed DNA replication. Replication of T4 DNA is performed by four proteins. These are the DNA polymerase (gp43), the processivity clamp (gp45) and two proteins (gp44 and gp62) that make up an ATP-dependent clamp-loader assembly that loads the clamp onto DNA. The gene 45 protein of phage T4 is the original “sliding clamp”, identified by Alberts and colleagues as one of the proteins responsible for the remarkably processive replication of DNA by T4 DNA polymerase and for its ability to pass through DNA hairpins (Huang et al., 1981). gp45 acts also as a transcriptional activator for late genes, by interacting with RNA polymerase (Herendeen et al., 1989).
The gp45 protein has been shown to form trimers in the absence of DNA (Jarvis et al., 1989), and cryo-electron microscopic images suggest that it is a disk-shaped protein that encircles DNA (Gogol et al., 1992). Although we have speculated that its architecture would resemble that of E. coli β subunit and eukaryotic PCNA (Kong et al., 1992), the lack of any significant sequence similarity between these proteins requires that the crystal structure of gp45 be determined. This is particularly the case, since the sequence alignment on which we based the proposal that the β-clamp, PCNA and gp45 would resemble each other (Kong et al., 1992) was shown by our subsequent crystallographic analysis of PCNA to be almost completely in error. The structure of PCNA and the β-clamp do, however, resemble each other closely (Krishna et al., 1994).
Here, we present the crystal structure of the gene 45 protein of T4 bacteriophage, gp45, which forms a ring-shaped trimer in the crystal. A structure-based sequence alignment shows that the sequence identity between gp45 and yeast PCNA is only 10 %. Despite this lack of similarity in their sequences, the structures of the two proteins are striking similar and it seems probable that they interact with DNA in a similar manner. Here, we focus on a discussion of the architectural elements of gp45 and a comparison of this structure to that of PCNA. We have provided preliminary versions of the coordinates of gp45 to other researchers in advance of publication. Several other groups have commented on the implications of our structure for gp45 function in DNA replication and RNA transcription Alley et al 1999, Pietroni et al 1997, Soumillion et al 1998, Wong and Geiduschek 1998.
Section snippets
The overall structure of gp45 resembles that of PCNA
The structure of gp45 was determined by multiple isomorphous replacement (MIR), using two mercury derivatives that were obtained by mutating two residues in the protein separately to cysteine. The final model for the protein was refined against X-ray data to 2.4 Å (see Materials and Methods). The crystals contain a trimer of gp45 in the asymmetric unit, which forms a ring (Figure 1). The longest dimensions across the outer surface of the ring are ∼85 Å, and the ring has an internal diameter of
Conclusions
DNA polymerases involved in chromosomal replication are notable in being very poorly processive in the absence of their cognate processivity factors. The coupling of the polymerase to a separate DNA-bound protein in order to achieve high processivity allows for proper regulation of the polymerase, as well as facilitating the rapid cycling of the polymerase from one site of discontinuous DNA replication to another. The striking similarity between the structures of the processivity factors from
Materials and methods
Recombinant gp45 protein was expressed in E. coli (strain DH5-α) from plasmid pTL151-W (Rush et al., 1989). Cultures were grown at 32 °C to mid-logarithmic phase and protein production was induced by shifting the temperature to 42 °C for three hours. The protein was purified by anion-exchange chromatography followed by affinity chromatography on heparin-Sepharose. Single-site cysteine mutants were generated using mutagenic primers and the polymerase chain reaction (PCR). Amplified fragments
Acknowledgements
We thank Xiadong Wu, Olga Livanis and Ramakoti Suresh for help with crystallization, and Ramakoti Suresh and Lonny Berman for help with X-ray data collection. This work was supported, in part, by a grant from the NIH to J.K. (45547) and to M.O.D. (38839). The National Synchrotron Light Source is supported by the United States Department of Energy Offices of Health and Environmental Research and of Basic Energy Sciences under prime contract DE-AC02-98CH10886, by the National Science Foundation,
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2019, Biophysical JournalCitation Excerpt :The sliding clamp binds the polymerase and tethers it to DNA, allowing the polymerase to replicate DNA without dissociation from the template. Structural studies reveal that sliding clamps have a pseudo-sixfold symmetry and individual subunits are arranged in a head-to-tail conformation (1,4–8). Clamps from bacterial species (4) are homodimeric, with three domains in each monomer, whereas eukaryotic and archaeal species (5,9–11) are trimers with two domains in each monomer.
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Edited by I. A. Wilson