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

4.1 oriC primosome (table 1)

The 4700 kb E. coli chromosome is replicated from oriC, located at 83 min on the chromosome, and initiation at oriC strictly depends on DnaA protein (16). ATP-bound DnaA protein binds to the oriC, and changes the conformation of the origin sequences (17, 18). Localized melting of the three repeats of 13mer sequences permits loading of DnaB/DnaC helicase. The oriC preprimosome, generated at the oriC sequence with DnaA, DnaB and DnaC proteins, can be isolated by sucrose gradient and addition of primase and DNA polymerase holoenzyme III together with SSB and gyrase to the isolated preprimosome can sustain DNA synthesis of the entire plasmid (19).

4.2 ABC primosome (table 1)

A ssi cloned from R6K plasmid did not support assembly of the phiX174-type primosome in vitro, nor did it support primer RNA synthesis with any known priming enzymes. We discovered that SS to RF replication of a single-stranded phage containing this ssi was dependent on DnaA protein. We were able to reconstitute the replication with purified proteins including DnaA, DnaB, DnaC, primase, SSB and DNA polymerase III holoenzyme (10). The ssi is capable of forming a secondary structure, and its stem contains a dnaA box sequence. DnaA protein specifically recognizes the stem and forms a complex which is isolatable by gel filtration. Hence, we have named this ssi A site and the complex ABC primosome.

In contrast to oriC plasmid replication in vitro, which requires ATP-form of DnaA protein, the ABC primosome can be assembled on A site with ADP-form of DnaA protein and the DnaA-A site complex can be isolated in the absence of ATP. However, formation of an isolatable preprimosome complex requires the presence of ATP or ATPgS, which stabilizes heterohexamers formed with DnaB and DnaC proteins. DnaB protein is delivered to the DnaA-A site complex by virtue of its association with DnaC, which may interact with DnaA protein. The preprimosome can translocate on SSB-coated single-stranded DNA with energy supplied by hydrolysis of ATP, dATP or dCTP (20). The hydrolysis of the nucleotide is required also to facilitate the release of DnaC protein from DnaB-DnaC complex (21).

DnaB protein hydrolyzes ATP, CTP and GTP but not deoexynucleotides (22, 23). In contrast, the isolated ABC preprimosome hydrolyzes dATP, dCTP and to some extent dGTP, in consistent with its ability to utilize these nucleotides for helicase activity (20). Helicase activity of the ABC preprimosome is maximally activated by 100 to 200 µM ATP, whereas more than 1 mM of ATP is required for maximum activation of the helicase activity by DnaB protein alone (23). The preprimosome contains DnaA and DnaB proteins but most likely not DnaC protein. Among the proteins required for the primosome assembly, only DnaB protein is capable of hydrolyzing nucleotides. These results indicate that assembly of the ABC preprimosome somehow leads to functional and/or structural alteration of DnaB helicase, which enables it to utilize low concentration of ATP as well as deoxyribonucleotides. dATP can not only support helicase activity but also support priming and replication by the ABC primosome, since the A site-dependent replication, which has been suppressed by the presence of ATPgS, can be reactivated by dATP in the absence of any ribonucleotides (20).

4.3 ABC primosome can support progression of replication forks

pBR322 plasmid contains an n'-pas on the lagging strand template, and efficient lagging strand synthesis depends on the assembly of the phiX174-type primosome at this n'-pas, in the absence of which replication intermediates containing only the nascent leading strand are accumulated in vitro (24, 25). Furthermore, lagging strand synthesis in vitro is completely suppressed in the presence of anti-DnaT protein antibody, which inhibits assembly of the phiX174-type primosome. Replacement of the n'-pas with A site on pBR322 restored the activity to synthesize lagging strand in the absence of DnaT protein (15). The same plasmid is capable of replication in the priA1::kan strain, in which the wild-type pBR322 cannot be replicated due to lack of the phiX174-type primosome assembly (26). These results demonstrate that ABC primosome can replace the phiX174-type primosome in replication of pBR322 for lagging strand synthesis and duplex unwinding at the replication fork.