Primers and Primases

DNA polymerases cannot initiate the synthesis of new DNA chains, they can only elongate pre-existing primers. The opposite polarities of the two strands of the double helix coupled with the 5′r3′ polarity of the polymerase means that replication occurs relatively continuously on one (leading) strand and discontinuously on the other (lagging) strand. The continuous strand probably needs to be primed once, usually at an origin. Nature has found many different ways of doing this, including the use of RNA primers made by an RNA polymerase (e.g. during replication of the plasmid ColE1 and mitochondrial DNA), preformed tRNA (by retroviral reverse transcriptases), DNA primers generated by endonucleolytic cleavage (by gpA of øX174) and even a serine OH group (adenoviral 55 kDa terminal protein). The discontinuous strand must be primed repeatedly to generate short nascent chains (Okazaki fragments) and the multiple RNA primers needed are invariably synthesized by a special primase, which is an integral part of the replication machinery (see Kornberg and Baker, 1992, for a comprehensive review). This means that RNA synthesis, by RNA polymerases or primases, is inevitably involved in primer synthesis. This review will concentrate on the additional roles played by RNA polymerases in initiating DNA synthesis, especially in eukaryotes. Unfortunately no clear view as to their role emerges. The current model for the initiation of replication at origins unifies the findings from many different organisms (Bramhill and Kornberg, 1988). A trans-acting initiator protein first binds to, and transiently unwinds, a specific site in the origin, before a helicase extends the unwound region. After recruitment of a primase-polymerase complex, RNA-primed DNA synthesis initiates on the leading strand. Next a processive polymerase captures the leading-strand primer and the primase-polymerase complex becomes exclusively involved in the synthesis of Okazaki fragments on the lagging strand. A critical step in this process is the unwinding of the duplex.