Abstract
Eleven single strand initiation sequences (ssi) were isolated from various plasmid genomes using a plaque-morphology assay. Out of seven ssi that require dnaB and dnaC functions for replication in a crude in vitro system, six use a phi X174 type priming mechanism, and a phi X174 type primosome is assembled at these sequences from the purified proteins, n'(priA), n(priB), n"(priC), dnaT, dnaB, dnaC, and primase. The same ssi potentiate dATPase activity of n' protein, and thus represent new n' protein recognition sequences (n'-pas). Based on sequence homology, two structural groups are evident. Two sequences show a strong homology with the phi X174 site, whereas three share extensive homology with the previously characterized n'-pas of ColE1, ssiA(ColE1). All the n'-pas have a potential to form stem and loop structures, although sequence homology between the two classes is absent. In addition to the phi X174 type priming, three ssi do not require either dnaB or dnaC function for replication, and use a G4 type priming, requiring only SSB and primase. The 5' ends of primer RNA synthesized by primase are localized within the vicinity of one of the three blocks of highly conserved nucleotide sequences. Deletions of parts of these conserved sequences result in loss of priming activity, suggesting that they are important for priming on the G4 type ssi, which are termed G site. The general significance of these two types of priming in initiation of lagging or leading strand synthesis as well as various modes of initiation at origins of replication are proposed.
Highlights
The general significance of these two types of priming in initiation of lagging or leading strand synthesis as well as various modes of initiation at origins of replication are proposed. In both prokaryotic and eukaryotic replicons, DNA chain elongation is generally initiated upon primer RNAs synthesized by primase or by RNA polymerase [1]
Primer RNAs are synthesized at multiple sites located throughout the $X174 genome due to the mobile nature of this protein complex [7, 8]. (iv) Primase can catalyze primer RNA synthesis on uncoated SS DNA in the presence of dnaB protein, without apparent sequence specificity [9]
Synthesis of primer RNA is a critical step in initiation of chain elongation
Summary
The general significance of these two types of priming in initiation of lagging or leading strand synthesis as well as various modes of initiation at origins of replication are proposed. In both prokaryotic and eukaryotic replicons, DNA chain elongation is generally initiated upon primer RNAs synthesized by primase or by RNA polymerase [1]. (iii) Primer RNA synthesis on SSB-coated 4X174 DNA requires the action of a set of seven prepriming proteins for primase to recognize priming signals [5]. (iv) Primase can catalyze primer RNA synthesis on uncoated SS DNA in the presence of dnaB protein, without apparent sequence specificity (general priming) [9] Primer RNAs are synthesized at multiple sites located throughout the $X174 genome due to the mobile nature of this protein complex [7, 8]. (iv) Primase can catalyze primer RNA synthesis on uncoated SS DNA in the presence of dnaB protein, without apparent sequence specificity (general priming) [9]
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