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Interaction between the T4 helicase-loading protein (gp59) and the DNA polymerase (gp43): a locking mechanism to delay replication during replisome assembly
Journal article   Peer reviewed

Interaction between the T4 helicase-loading protein (gp59) and the DNA polymerase (gp43): a locking mechanism to delay replication during replisome assembly

Jun Xi, Zhihao Zhuang, Zhiquan Zhang, Tzvia Selzer, Michelle M Spiering, Gordon G Hammes and Stephen J Benkovic
Biochemistry (Easton), v 44(7), pp 2305-2318
22 Feb 2005
DOI: https://doi.org/10.1021/bi0479508
PMID: 15709743
Featured in Collection :   UN Sustainable Development Goals @ Drexel

Abstract

Protein Processing, Post-Translational - genetics Substrate Specificity Templates, Genetic DNA-Directed DNA Polymerase - chemistry DNA Primers - chemistry Nucleic Acid Heteroduplexes - metabolism Viral Proteins - metabolism DNA-Binding Proteins - metabolism Nucleic Acid Synthesis Inhibitors Bacteriophage T4 - enzymology DNA Primers - metabolism Exonucleases - metabolism Nucleic Acid Conformation DNA Helicases - genetics DNA Helicases - chemistry Cross-Linking Reagents - chemistry Viral Proteins - chemistry Models, Molecular Viral Proteins - genetics Bacteriophage T4 - genetics Viral Proteins - antagonists & inhibitors DNA-Binding Proteins - genetics DNA-Binding Proteins - chemistry Nucleic Acid Heteroduplexes - chemistry Protein Interaction Mapping Cross-Linking Reagents - metabolism DNA Helicases - metabolism Deoxyribonucleases, Type II Site-Specific - metabolism Exonucleases - antagonists & inhibitors DNA Replication - genetics Protein Binding Fluorescence Resonance Energy Transfer - methods DNA-Directed DNA Polymerase - metabolism Replicon - genetics
The T4 helicase-loading protein (gp59) has been proposed to coordinate leading- and lagging-strand DNA synthesis by blocking leading-strand synthesis during the primosome assembly. In this work, we unambiguously demonstrate through a series of biochemical and biophysical experiments, including single-molecule fluorescence microscopy, that the inhibition of leading-strand holoenzyme progression by gp59 is the result of a complex formed between gp59 and leading-strand polymerase (gp43) on DNA that is instrumental in preventing premature replication during the assembly of the T4 replisome. We find that both the polymerization and 3' --> 5' exonuclease activities of gp43 are totally inhibited within this complex. Chemical cross-linking of the complex followed by tryptic digestion and peptide identification through matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry identified Cys169 of gp43 and Cys215 of gp59 as residues in a region of a protein-protein contact. With the available crystal structures for both gp43 and gp59, a model of the complex was constructed based on shape complementarity, revealing that parts of the C-terminal domain from gp59 insert into the interface created by the thumb and exonuclease domains of gp43. This insertion effectively locks the polymerase into a conformation where switching between the pol and editing modes is prevented. Thus, continued assembly of the replisome through addition of the primosome components and elements of the lagging-strand holoenzyme can occur without leading-strand DNA replication.

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34 citations in Web of Science
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Collaboration types
Domestic collaboration
Web of Science research areas
Biochemistry & Molecular Biology
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