A zinc ribbon protein in DNA replication: primer synthesis and macromolecular interactions by the bacteriophage T4 primase.
The gene product 61 primase protein from bacteriophage T4 was expressed as an intein fusion and purified to homogeneity. The primase binds one zinc ion, which is coordinated by four cysteine residues to form a zinc ribbon motif. Factors that influence the rate of priming were investigated, and a physiologically relevant priming rate of approximately 1 primer per second per primosome was achieved. Primase binding to the single-stranded binding protein (1 primase:4 gp32 monomers; K(d) approximately 860 nM) and to the helicase protein in the presence of DNA and ATP-gamma-S (1 primase:1 helicase monomer; K(d) approximately 100 nM) was investigated by isothermal titration calorimetry (ITC). Because the helicase is hexameric, the inferred stoichiometry of primase binding as part of the primosome is helicase hexamer:primase in a ratio of 1:6, suggesting that the active primase, like the helicase, might have a ring-like structure. The primase is a monomer in solution but binds to single-stranded DNA (ssDNA) primarily as a trimer (K(d) approximately 50-100 nM) as demonstrated by ITC and chemical cross-linking. Magnesium is required for primase-ssDNA binding. The minimum length of ssDNA required for stable binding is 22-24 bases, although cross-linking reveals transient interactions on oligonucleotides as short as 8 bases. The association is endothermic at physiologically relevant temperatures, which suggests an overall gain in entropy upon binding. Some possible sources of this gain in entropy are discussed.
Valentine
A M
AM
Department of Chemistry, The Pennsylvania State University, 415 Wartik Laboratory, University Park, Pennsylvania 16802, USA.
Ishmael
F T
FT
Shier
V K
VK
Benkovic
S J
SJ
D017122:Bacteriophage T4 / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D004265:DNA Helicases / Q000378:metabolism; D019915:DNA Primase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D004261:DNA Replication / Q000235:genetics / Q000502:physiology*; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D013816:Thermodynamics
D003432:Cross-Linking Reagents; D017931:DNA Primers; D004277:DNA, Single-Stranded; D046911:Macromolecular Substances; D019915:DNA Primase; D004265:DNA Helicases