Fluorescence monitoring of T4 polymerase holoenzyme accessory protein interactions during loading of the sliding clamp onto the template-primer junction

Journal of Molecular Biology
G J LathamP H von Hippel

Abstract

Assembly of the T4 polymerase holoenzyme requires coordinated interactions among the core polymerase and the clamp loader (gp44/62) and sliding clamp (gp45) accessory proteins. Here we describe the creation of a mutant of gp45 that can be uniquely modified by fluorescent probes within each protein monomer at a site-specific cysteine residue. The fluorescently labeled gp45 was shown to have the same biological activity as the wild-type protein. These strike "labeled" gp45 adducts were then used in steady-state and fluorescence polarization studies to monitor the interaction of gp45 with the gp44/62 clamp-loading complex and template-primer DNA in the presence and absence of ATP, and of the non-hydrolyzable analog, adenosine 5'-O-(3-thiotriphosphate). We find that a complex of ATP-activated gp44/62 with appropriately labeled gp45 shows significant fluorescent enhancement (and an increase in fluorescent anisotropy), that can be partially reversed by interaction with template-primer DNA. Fluorescence-monitored binding curves between gp45 and ATP-activated gp44/62 reveal that the two protein complexes bind with a 1:1 stoichiometry. Analysis shows that these methods can be used to follow the ATP-driven loading of gp45 onto the templa...Continue Reading

Citations

Feb 2, 1999·The EMBO Journal·J TurnerM O'Donnell
Mar 11, 2003·Microbiology and Molecular Biology Reviews : MMBR·Eric S MillerWolfgang Rüger
Jun 8, 2001·Annual Review of Biochemistry·S J BenkovicF Salinas
Jul 19, 2001·Proceedings of the National Academy of Sciences of the United States of America·M A TrakselisS J Benkovic
Oct 27, 1999·Proceedings of the National Academy of Sciences of the United States of America·G J LathamP H von Hippel
Apr 12, 2014·Biochemistry·Shuangluo Xia, William H Konigsberg
Sep 7, 2006·Nature Reviews. Molecular Cell Biology·Chiara Indiani, Mike O'Donnell
May 10, 2020·The Analyst·Xuelin JinKyubong Jo
Jul 17, 1998·Current Opinion in Chemical Biology·D J SextonS J Benkovic
Sep 12, 2001·Trends in Biochemical Sciences·M A TrakselisS J Benkovic
Sep 24, 2004·Biochemical and Biophysical Research Communications·Nicole SommerWolfgang Rüger
Jun 20, 1998·Biochemistry·D J SextonS J Benkovic

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