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

How does PriA protein initiate DNA replication from D-loops or R-loops? The cloned oriM sequences do not have n'-pas (44, 45), and there may not be any functional n'-pas on the E. coli chromosome, since extensive screening using an origin-defective M13 phage vector failed to detect any defined sequences with n'-pas activity (N. Nomura et al., unpublished results). Therefore, it is unlikely that n'-pas on D-loops or on R-loops are recognized by PriA protein. We consider a possibility that PriA protein recognizes a part of the structural features of D-loops and R-loops. Absolute requirement of PriA protein for replication of pBR322 lacking n'-pas in the wild-type and in rnhA mutants supports this idea, since replication of this plasmid presents a model for D-loop or R-loop dependent replication.

Interestingly, we noticed that the nucleotide binding domain of PriA protein possesses a low but significant homology to that of RecG protein, which is an RNA helicase specific for R-loops (data not shown; 46). Our preliminary data suggests that PriA protein interacts with an R-loop structure, although ATPase is not activated by this interaction (H. Masai et al., unpublished result). The presence of PriA homologues in two other distantly related bacteria suggests that PriA-dependent replication from D-loops and R-loops may be conserved throughout the eubacteria species. It will be essential to develop an in vitro replication system in which replication is specifically initiated from a D-loop or from an R-loop.

Multiple modes of primosome assembly, which we have demonstrated in E. coli, may operate in replication of the eukaryotic chromosomes as well. Chromosomal replication in eukaryotic cells is strictly regulated so that it is coordinated with various cell cycle events. At the same time, choice of replication origins and sequence specificity in origin recognition can vary depending on developmental stages (47) or state of transcription in the vicinity of the origin sequences (48, 49). In early cleaving embryo, the S phases are completed in a matter of several minutes. This extraordinary rate of DNA replication is achieved by increasing the numbers of replication origins fired during the short S phase. The replication origin in the intergenic space of b-globin region is fired early in S phase in blood cells where the gene is actively transcribed, whereas it is replicated late in the cells which do not express globins (50, 51). Flexibility in origin selection was also indicated by genetic studies of yeast Saccharomyces cerevisiae, in which it was shown that deletion of an actively firing origin from a chromosome can be generally torelated, and that in some cases normally inactive replication origins can be activated to compensate for the loss of active origins (52). It is not known whether these variations in origin usage reflect the switch of replication modes or changes in the chromatin structures in the origin region. We also know very little about the origin selection and mode of initiation during premeiotic DNA synthesis, which could be different from those of mitotic DNA replication. It is of interest whether a recombination-dependent replication pathway similar to the one discovered in E. coli exists in eukaryotic cells. If PriA protein is an "initiator" for DNA replication from D-loops and R-loops, which are commonly found in eukaryotic cells as well, it may well be conserved in eukaryotes.