The oligomeric T4 primase is the functional form during replication.
Replisome DNA primases are responsible for the synthesis of short RNA primers required for the initiation of repetitive Okazaki fragment synthesis on the lagging strand during DNA replication. In bacteriophage T4, the primase (gp61) interacts with the helicase (gp41) to form the primosome complex, an interaction that greatly stimulates the priming activity of gp61. Because gp41 is hexameric, a question arises as to whether gp61 also forms a hexameric structure during replication. Several results from this study support such a structure. Titration of the primase/single-stranded DNA binding followed by fluorescence anisotropy implicated a 6:1 stoichiometry. The observed rate constant, k(cat), for priming was found to increase with the primase concentration, implicating an oligomeric form of the primase as the major functional species. The generation of hetero-oligomeric populations of the hexameric primase by controlled mixing of wild type and an inactive mutant primase confirmed the oligomeric nature of the most active primase form. Mutant primases defective in either the N- or C-terminal domains and catalytically inactive could be mixed to create oligomeric primases with restored catalytic activity suggesting an active site shared between subunits. Collectively, these results provide strong evidence for the functional oligomerization of gp61. The potential roles of gp61 oligomerization during lagging strand synthesis are discussed.
Yang
Jingsong
J
Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Xi
Jun
J
Zhuang
Zhihao
Z
Benkovic
Stephen J
SJ
D017122:Bacteriophage T4 / Q000201:enzymology* / Q000235:genetics*; D004247:DNA / Q000096:biosynthesis; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019915:DNA Primase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004261:DNA Replication / Q000502:physiology*; D005454:Fluorescence Polarization; D016296:Mutagenesis; D017434:Protein Structure, Tertiary; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
C022262:Okazaki fragments; D014764:Viral Proteins; C063983:gene 41 protein, Enterobacteria phage T4; D004247:DNA; D019915:DNA Primase; D004265:DNA Helicases