Roles of the G site and φX174-type primosome assembly site in priming of leading-strand synthesis: initiation by a mobile primosome and replication-fork arrest by RepA protein bound to oriR

Abstract

Bacterial replicons often contain single-strand initiation sequences ( ssi) such as a G site (a sequence recognized by a dnaG-encoded primase for the synthesis of primer RNA) and a primosome assembly site ( pas) near the DNA replication origin ( ori). The R1 plasmid contains a G site downstream from oriR, which serves for the priming of the leading-strand synthesis of this plasmid. On the other hand, the F, R6K and Rts1 plasmids carry pas at similar locations relative to the respective ori. In order to assess the functional significance of these pas, R1 plasmid derivatives carrying an n'- pas (φX 174-type pas) in place of the G site were constructed and their replication properties were examined in vitro. Deletion of the G site in the R1 plasmid resulted in a nearly 80% reduction of total DNA synthesis in vitro, which was recovered to the wild-type (wt) level by inserting the G4 complementary ori. Furthermore, insertion of an n'- pas on the leading-strand template restored the in vitro replicative activity to a level 70% of wt. This recovery was dependent on the assembly of the φX174-type primosome, which efficiently primed leading-strand synthesis and moved toward the oriR. However, the R1 plasmid derivative containing the n'- pas replicated unidirectionally in vitro, probably due to the anti-helicase activity of the RepA protein bound to oriR, which was shown by helicase assays using partial heteroduplexes as substrates. These results indicate that the pas present near the ori of various replicons serves for the assembly of a mobile primosome, which can prime leading-strand synthesis, although the movement of this primosome may be blocked by Rep protein bound to the ori.