Structure of the Escherichia coli DNA polymerase III epsilon-HOT proofreading complex.

Thomas W Kirby,Robert E London,Roel M Schaaper,Lars C Pedersen,Scott Harvey,Anna K Chikova,Sergey Chalov,Eugene F DeRose,Fred W Perrino

doi:10.1074/jbc.m606917200

Abstract

The epsilon subunit of Escherichia coli DNA polymerase III possesses 3'-exonucleolytic proofreading activity. Within the Pol III core, epsilon is tightly bound between the alpha subunit (DNA polymerase) and subunit. Here, we present the crystal structure of epsilon in complex with HOT, the bacteriophage P1-encoded homolog of , at 2.1 A resolution. The epsilon-HOT interface is defined by two areas of contact: an interaction of the previously unstructured N terminus of HOT with an edge of the epsilon central beta-sheet as well as interactions between HOT and the catalytically important helix alpha1-loop-helix alpha2 motif of epsilon. This structure provides insight into how HOT and, by implication, may stabilize the epsilon subunit, thus promoting efficient proofreading during chromosomal replication.

Highlights

The precise mechanisms by which cells are able to duplicate their DNA with both high accuracy (Ͻ1 error/1010 bases replicated) and speed are of major interest
Chromosomal replication is performed by multisubunit replicases that conduct the simultaneous, coordinated replication of the leading and lagging strands
Among the model systems currently being investigated, the best understood is that of the bacterium Escherichia coli [1, 2]

Summary

EXPERIMENTAL PROCEDURES

HOT and ⑀186 were expressed and purified as previously described [7, 17]. The complex of ⑀186 and HOT was prepared by mixing 2 volumes of a 3.8 mg/ml solution of ⑀186 with 1 volume of 3 M Tris1⁄7HCl, pH 7.0, and adding 0.028 volumes of 62 mg/ml HOT, yielding a ratio of HOT:⑀186 of 1.03:1. Crystals of the ⑀186-HOT complex were obtained at 4 °C by mixing, in a sitting drop tray, 1 ␮l of a mg/ml complex in mM Tris, pH 7.5, 100 mM NaCl, 5 mM MnSO4, and 5 mM TMP with 1 ␮l of the reservoir solution consisting of 0.1 M Tris1⁄7HCl, pH 8.0, and 22% polyethylene glycol 6000. Crystals were harvested and transferred to a stabilization solution consisting of 0.1 M Tris, pH 7.5, 23% polyethylene glycol 6000, 100 mM NaCl, 5 mM MnSO4, and 5 mM TMP. Crystals were transferred in four steps from the stabilization solution to the cryosolution consisting of 0.1 M Tris, pH 7.5, 25% polyethylene glycol 6000, 15% ethylene glycol, 200 mM NaCl, 5 mM MnSO4, and 5 mM TMP. The two main helices of HOT from the Protein Data Bank coordinates 1SE7 were placed separately into the electron density manually using the pro-

Ramachandran statisticsd

RESULTS AND DISCUSSION

Residue Atom Secondary Residue Atom Secondary