Sliding Clamp of the Bacteriophage T4 Polymerase Has Open and Closed Subunit Interfaces in Solution

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• 作者：Stephen C. Alley ; Vincent K. Shier ; Ernesto Abel-Santos ; Daniel J. Sexton ; Patrice Soumillion ; and Stephen J. Benkovic
• 刊名：Biochemistry
• 出版年：1999
• 出版时间：June 15, 1999
• 年：1999
• 卷：38
• 期：24
• 页码：7696 - 7709
• 全文大小：283K
• 年卷期：v.38,no.24(June 15, 1999)
• ISSN：1520-4995

文摘

The sliding clamps of bacteriophage T4 (gp45), Escherichia coli (mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle"> clamp), and yeast (PCNA)are required for processive DNA synthesis by their cognate DNA polymerases. The X-ray crystal structuresof all three of these clamps have been shown to be closed, circular complexes. This paper reportsinvestigations of the solution structure of bacteriophage T4 gp45 by analytical ultracentrifugation,fluorescence, and hydrodynamic modeling. Mutants of gp45 with inter- and intrasubunit disulfide bondswere created to alter the solution structure of gp45, with additional mutagenesis used to investigate theimportance of the proline-rich loop region found between the two domains of each gp45 monomer. Thewild-type gp45 trimer assembles from monomers cooperatively with a dissociation constant of 0.21 mages/entities/mgr.gif">M²and values between 0.088 and 0.32 mages/entities/mgr.gif">M² for the mutants. Velocity ultracentrifugation experiments showedthat wild-type gp45 possesses a sedimentation coefficient strongly dependent on concentration, typical ofasymmetric or elongated molecules, that when extrapolated to zero concentration yields a sedimentationcoefficient of 4.0 S. The loop and the disulfide mutants exhibited sedimentation coefficients with littleconcentration dependence, typical of symmetric or spherical molecules, that when extrapolated to zeroconcentration yielded sedimentation coefficients of 4.4-4.8 S. The lower sedimentation coefficient inthe former case is consistent with wild-type gp45 being more asymmetric or elongated than the mutantforms. Fluorescence-resonance energy-transfer experiments were used to measure the distance betweentwo amino acids (W91 and V162C-coumarin) on opposite sides of the gp45 subunit interface. For anintrasubunit disulfide mutant, the distance between these two amino acids was determined to be 19 Å (14Å in the X-ray crystal structure), consistent with a closed complex. For the mutants without intrasubunitdisulfides, the efficiency of fluorescence-resonance energy transfer was in accord with a model of gp45being an open complex composed of two closed subunit interfaces and a third open interface separatedby a distance of 35-38 Å. The collective data supplemented with hydrodynamic modeling were consistentwith gp45 subunit separation achieved within the plane of the gp45 ring.