title: Interaction between the T4 helicase-loading protein (gp59) and the DNA polymerase (gp43): a locking mechanism to delay replication during replisome assembly.
abstract: 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.
authors.affiliation: Department of Chemistry, Pennsylvania State University, 104 Chemistry Building, University Park, Pennsylvania 16802, USA.
authors.forename: Jun
authors.initials: J
authors.lastname: Xi
authors.forename: Zhihao
authors.initials: Z
authors.lastname: Zhuang
authors.forename: Zhiquan
authors.initials: Z
authors.lastname: Zhang
authors.forename: Tzvia
authors.initials: T
authors.lastname: Selzer
authors.forename: Michelle M
authors.initials: MM
authors.lastname: Spiering
authors.forename: Gordon G
authors.initials: GG
authors.lastname: Hammes
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Benkovic
mesh_terms: D017122:Bacteriophage T4 / Q000201:enzymology* / Q000235:genetics; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000378:metabolism; D004265:DNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry / Q000378:metabolism; D004261:DNA Replication / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D005092:Exonucleases / Q000037:antagonists & inhibitors / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry / Q000378:metabolism; D019384:Nucleic Acid Synthesis Inhibitors; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011499:Protein Processing, Post-Translational / Q000235:genetics; D012093:Replicon / Q000235:genetics; D013379:Substrate Specificity; D013698:Templates, Genetic; D014764:Viral Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D003432:Cross-Linking Reagents; D017931:DNA Primers; D004268:DNA-Binding Proteins; D009692:Nucleic Acid Heteroduplexes; D019384:Nucleic Acid Synthesis Inhibitors; D014764:Viral Proteins; C069983:gene 43 protein, Enterobacteria phage T4; C091225:gene 59 protein, Enterobacteria phage T4; D004259:DNA-Directed DNA Polymerase; D005092:Exonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; C040158:GTAC-specific type II deoxyribonucleases; D004265:DNA Helicases