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Titolo:
Creating a dynamic picture of the sliding clamp during T4 DNA polymerase holoenzyme assembly by using fluorescence resonance energy transfer
Autore:
Trakselis, MA; 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:
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
fascicolo: 15, volume: 98, anno: 2001,
pagine: 8368 - 8375
SICI:
0027-8424(20010717)98:15<8368:CADPOT>2.0.ZU;2-G
Fonte:
ISI
Lingua:
ENG
Soggetto:
PROTEIN-PROTEIN INTERACTIONS; SINGLE-STRANDED-DNA; ESCHERICHIA-COLI; CRYSTAL-STRUCTURE; REPLICATION FORK; ATP HYDROLYSIS; 3-DIMENSIONAL STRUCTURE; PROCESSIVITY FACTOR; CARBOXYL-TERMINUS; HELICASE-PRIMASE;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
62
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:
M.A. Trakselis et al., "Creating a dynamic picture of the sliding clamp during T4 DNA polymerase holoenzyme assembly by using fluorescence resonance energy transfer", P NAS US, 98(15), 2001, pp. 8368-8375

Abstract

The coordinated assembly of the DNA polymerase (gp43), the sliding clamp (gp45), and the clamp loader (gp44/62) to form the bacteriophage T4 DNA polymerase holoenzyme is a multistep process. A partially opened toroid-shaped gp45 is loaded around DNA by gp44/62 in an ATP-dependent manner. Gp43 bindsto this complex to generate the holoenzyme in which gp45 acts to topologically link gp43 to DNA, effectively increasing the processivity of DNA replication. Stopped-flow fluorescence resonance energy transfer was used to investigate the opening and closing of the gp45 ring during holoenzyme assembly. By using two site-specific mutants of gp45 along with a previously characterized gp45 mutant, we tracked changes in distances across the gp45 subunit interface through seven conformational changes associated with holoenzyme assembly. Initially, gp45 is partially open within the plane of the ring at one of the three subunit interfaces. On addition of gp44/62 and ATP, this interface of gp45 opens further in-plane through the hydrolysis of ATP. Addition of DNA and hydrolysis of ATP close gp45 in an out-of-plane conformation. The final holoenzyme is formed by the addition of gp43, which causes gp45 to close further in plane, leaving the subunit interface open slightly. This open interface of gp45 in the final holoenzyme state is proposed to interact with the C-terminal tail of gp43, providing a point of contact between gp45 and gp43. This study further defines the dynamic process of bacteriophage T4 polymerase holoenzyme assembly.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 01/10/20 alle ore 03:50:07