Three potential secondary structures, stem-loops I, II, and III, are contained in the phage G4 origin of complementary DNA strand synthesis, G4oric, and are believed to be involved in its recognition by dnaG-encoded primase and the synthesis of primer RNA. In a previous publication [Sakai et al., Gene 71 (1988) 323-330], we suggested that base pairing between the loops of stem-loops I and II, and/or II and III, might play a role in G4oric function. To test this hypothesis, site-directed mutagenesis was used to construct mutants which carried base substitutions in loops I, II and III that destroyed possible interloop base pairing. These mutations, however, did not seriously affect G4oric activity. This indicates that base pairing between the loops is not essential for G4oric functional activity, and also that base substitutions which do not affect the secondary structure of stem-loops I, II and III, do not affect G4oric activity. To complete an analysis of the effects of altering the structure of the G4oric stem-loops, insertions were made into stem-loop III. In contrast to stem-loops I and II, all insertions into stem-loop III destroyed in vivo G4oric activity.
Bibliographical noteFunding Information:
We thank Dr. N. Nomura for providing us with bacterial and phage strains. We also thank Dr. B. Goldschmidt for synthesizing the oligos used for mutagenesisa nd Ms. A. Siegel for typing the manuscript. This work was supported in part by Public Health Service Grant GM38292 to G.N.G.
Copyright 2014 Elsevier B.V., All rights reserved.
- DNA replication origin
- M13 phage vector
- loop-loop interaction
- recombinant DNA
- site-directed mutagenesis
- stem-loop structures