Formation of Okazaki Pieces at the Escherichia coli Replication Fork In Vitro

Okazaki's model of discontinuous DNA synthesis, supported by the discovery of nascent DNA fragments at the replication fork (Okazaki pieces), provided a solution to the problem of how antiparallel DNA strands could be simultaneously replicated (Okazaki et al. 1969). Since Okazaki's initial observations, many investigators have shown that in a wide variety of biological systems nascent DNA at the replication fork is found in the form of Okazaki pieces which must be sealed to give normal-length mature DNA. It has been implicitly assumed in many cases that these DNA fragments arise by discontinuous DNA synthesis. However, recent studies on uracil incorpora-tion and excision have led to ambiguity about the origin of Okazaki pieces isolated at the replication fork. Our own work in the last few years has been done on an in vitro system for DNA replication in which Okazaki fragments are generated which very probably do arise by discontinuous DNA synthesis. In this system, however, it is also possible to produce Okazaki pieces which arise by mechanisms other than discontinuous DNA synthesis. In this paper we discuss the formation and distribu-tion of two distinguishable types of Okazaki pieces: those which arise by uracil excision and those which we believe arise by discontinuous DNA synthesis, i.e., are initiated de novo. Since the term Okazaki pieces has become a generic term for all the small inter-mediates found at the replication fork under normal conditions, we will continue to denote any small intermediates found at the replication fork as Okazaki pieces. In this paper, for clarity, those small inter-mediates which we believe are initiated de novo will be called initiated pieces and those formed by uracil excision, uracil-excision pieces.