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Titolo:
Tracking sliding clamp opening and closing during bacteriophage T4 DNA polymerase holoenzyme assembly
Autore:
Alley, SC; Abel-Santos, E; Benkovic, SJ;
Indirizzi:
Penn State Univ, Dept Chem, Wartik Lab 414, University Pk, PA 16802 USA Penn State Univ University Pk PA USA 16802 4, University Pk, PA 16802 USA
Titolo Testata:
BIOCHEMISTRY
fascicolo: 11, volume: 39, anno: 2000,
pagine: 3076 - 3090
SICI:
0006-2960(20000321)39:11<3076:TSCOAC>2.0.ZU;2-S
Fonte:
ISI
Lingua:
ENG
Soggetto:
III HOLOENZYME; CRYSTAL-STRUCTURE; REPLICATION FORK; COMPLEX; PROTEIN; SUBUNIT; PURIFICATION; HYDROLYSIS; DYNAMICS; PCNA;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
36
Recensione:
Indirizzi per estratti:
Indirizzo: Benkovic, SJ Penn State Univ, Dept Chem, Wartik Lab 414, University Pk, PA16802 USA Penn State Univ University Pk PA USA 16802 Pk, PA 16802 USA
Citazione:
S.C. Alley et al., "Tracking sliding clamp opening and closing during bacteriophage T4 DNA polymerase holoenzyme assembly", BIOCHEM, 39(11), 2000, pp. 3076-3090

Abstract

The bacteriophage T4 DNA polymerase holoenzyme, consisting of the DNA polymerase (gp43), the sliding clamp (gp45), and the clamp loader (gp44/62), isloaded onto DNA in an ATP-dependent, multistep reaction. The trimeric, ring-shaped gp45 is loaded onto DNA such that the DNA passes through the center of the ring. gp43 binds to this complex, thereby forming a topological link with the DNA and increasing its processivity. Using stopped-flow fluorescence-resonance energy transfer, we have investigated opening and closing of the gp45 ring during the holoenzyme assembly process. Two amino acids that lie on opposite sides of the gp45 subunit interface, W91 and V162C labeled with coumarin, were used as the fluorescence donor and acceptor, respectively Free in solution, gp45 has two closed subunit interfaces with W91 to V162-coumarin distances of 19 Angstrom and one open subunit interface with aW91 to V162C-coumarin distance of 40 Angstrom. Making the assumption that the distance across the two closed subunit. interfaces is unchanged during the holoenzyme assembly process, we have found that the distance across theopen subunit interface is first increased to greater than 45 Angstrom and is then decreased to 30 Angstrom during a 10-step assembly mechanism. The gp45 ring is not completely closed in the holoenzyme complex, consistent with previous evidence suggesting that the C-terminus of gp43 is inserted intothe gp45 subunit interface. Unexpectedly, ATP-hydrolysis events are coupled to only a fraction of the total distance change, with conformational changes linked to binding DNA and gp43 coupled to the majority of the total distance change. Using the nonhydrolyzable ATP analogue ATP-gamma-S results information of a nonproductive gp45 gp44/62 complex; however, adding an excess of ATP to this nonproductive complex results in rapid ATP/ATP-gamma-S exchange to yield a productive gp45 gp44/62 complex within seconds.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 01/10/20 alle ore 04:22:15