Mutational analysis of primosome assembly sites. II. Role of secondary structure in the formation of active sites.
Based on their activity as effectors for the ATPase activity of Escherichia coli replication factor Y and as templates for primosome-directed DNA synthesis, single-point mutations in the L- and H-strand primosome assembly sites from pBR322 DNA have been grouped into four classes (Abarzúa, P., Soeller, W., and Marians, K. (1984) J. Biol. Chem. 259, 14286-14292). In this report, the effect of various ligands on the characteristic activities of primosome assembly site class II mutants has been examined. Both Mn2+ and spermidine can, at low levels, substitute for Mg2+ in the activation of wild-type sites as effectors for factor Y-catalyzed hydrolysis of ATP. Class II mutant sites characteristically require higher levels of these ligands for activation, suggesting that the specific higher order structure of an active primosome assembly site is maintained through base pairing within the single-stranded DNA sequence. This conclusion is supported by the following. 1) Excess levels of the E. coli single-stranded DNA-binding protein can inactivate wild-type sites at 1 mM Mg2+. Either the addition of NaCl to 80 mM or an increase in the Mg2+ concentration to 5 mM protects against this inactivation. Class II mutant sites, however, cannot be stabilized by 80 mM NaCl at 1 mM Mg2+, and only some class II mutants can be stabilized at 5 mM Mg2+. 2) Active second-site revertants, isolated in vivo and in vitro, of inactive primosome assembly sites containing multiple-base substitutions have mutated to restore lost base pairs in the proposed stem and loop structure of the sites.
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D004265:DNA Helicases; D004269:DNA, Bacterial / Q000032:analysis; D004268:DNA-Binding Proteins / Q000378:metabolism; D006868:Hydrolysis; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D009154:Mutation; D013095:Spermidine / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D008345:Manganese; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; D008274:Magnesium; D013095:Spermidine