Hisao Masai and Ken-ichi Arai.

Department of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo, 108, Japan

Received 01/17/96; Accepted 02/17/96; On-line 03/01/96

3.1 Primosomal proteins for assembly of the phiX174-type primosome (table 1)

In E. coli, a primosome was first discovered from the study of SS to RF replication of single-stranded DNA phage phiX174 (1). Assembly of this primosome is initiated by recognition of a specific hairpin structure (called n' site or n'-pas [primosome assembly site]) present on the phiX174 genome by protein n' (later redesignated as PriA) (2, 3, 4), which is followed by assembly of a preprepriming complex through association of proteins n (PriB), n" (PriC), and i (DnaT). A heterohexmeric DnaB-DnaC protein joins the complex to form a preprimosome, which moves along a DNA strand while periodically associating with primase to generate primer RNAs (1, 5, 6). The preprimosome can be physically isolated by gel filtration, which can support efficient primer RNA synthesis and DNA chain elongation upon addition of primase and DNA polymerase III holoenzyme (5, 6).

3.2 n'-pas isolated from varoius plasmids

n'-pas on the phiX174 DNA can adopt a stable secondary structure which is resistant to exonuclease VII digestion and can activate ATPase activity of PriA protein (6, 7). On ColE1 plasmid, sequences which could support efficient SS to RF conversion of an origin-defective M13 phage were discovered (8). The sequences were discovered on a related plasmid, pBR322, which could stimulate ATPase activity of PriA protein when converted to single-strand (9).

Figure 1: Nucleotide sequences and possible stem-loop structures of n'-pas isolated from various plasmid DNAs. Eight newly isolated n'-pas in addition to previously reported ones were classified into five groups on the basis of their primary structures. n'-pas of phiX174 phage, R100 plasmid and F•f2b belong to group I, n'-pas on the lagging strand of ColE1-type plasmids (ssiA) and n' sites of F•f5, F•f7 and ColE2 plasmid to group II, n'-pas of the F•f2a to group III, n'-pas on the leading strand of ColE1-type plasmids (ssiB) to group IV, and n'-pas from Rts1 plasmid to group V. Each group is represented by the sequences of the first listing described above. The sequences indicated by boxes show some sequence similarities, although none of them are conserved among all the groups. The group I and group II were described before (10).

Later, it was demonstrated that these sequences could support assembly of the phiX174-type primosome in vitro (11, 12). We have screened the genomes of various plasmids for the presence of n'-pas by utilizing a derivative of single-stranded M13 phage DNA which lacks the complementary strand origin (13). This defective phage generates only small, turbid plaques by itself. However, upon cloning of a sequence capable of directing efficient primer RNA synthesis, it can form big, clear plaques (14). Enzymatic analyses of primer RNA synthesis revealed that many of these so called ssi (single-strand initiation sequences) were functionally equivalent to n'-pas (10). Comparison of phage- and plasmid-derived n'-pas lead to classification of them into five groups on the basis of their primary structures (15) (Figure 1). n'-pas sequences in each group are capable of forming a stable secondary structure. The significance of these secondary structures was suggested by the conservation of the base paring in the stems in spite of divergence of primary sequences within the members of each group.