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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


, : Interaction between the T4 helicase-loading protein (gp59) and the DNA polymerase (gp43): a locking mechanism to delay replication during replisome assembly. Biochemistry 44(7): 2305-2318

The T4 helicase-loading protein (gp59) has been proposed to coordinate leading- and laggingstrand 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' fwdarw 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 Cys 169 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 laggingstrand holoenzyrne can occur without leading-strand DNA replication.

US$19.90

PMID: 15709743

DOI: 10.1021/bi0479508


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