![]() ![]() Thus, null mutants of the genes encoding DNA polymerase I (polA) or DNA ligase (lig) cannot be isolated. If Okazaki fragments are not connected efficiently, a daughter DNA molecule synthesized on the lagging strand will suffer from double-strand DNA breaks upon the next round of DNA replication. coli mutant cells deficient in DNA polymerase I or DNA ligase accumulate large amounts of Okazaki fragments. This process is very important for cells to maintain a high fidelity of DNA replication, since the primase has no proofreading capacity and the mismatch repair system is not effective on errors in the primer synthesis.Į. coli DNA ligase cannot connect RNA to DNA. DNA polymerase I, assisted by RNaseH, removes the primers and then fills the resulting gaps, to enable the short fragments to be joined by DNA ligase (6). SSB is required for the initiation of Okazaki fragment synthesis, as well as for chain elongation by DNA polymerase III holoenzyme. Thus, the interaction between primase and DnaB at the replication fork is the primary regulator of the cycle of Okazaki fragment synthesis. In vitro experiments suggest that the primase is recruited anew from solution for each cycle of Okazaki fragment synthesis and that association of primase with the replication fork occurs via a protein-protein interaction with the helicase, DnaB (5). Primase synthesizes RNA primer in a manner dependent on DnaB protein, which acts as a major replicative DNA helicase at the replication fork (4). Those are primase, single-strand DNA binding protein (SSB), DNA polymerase III, DNA polymerase I, ribonuclease H (RNaseH), and DNA Ligase (see Discontinuous DNA Replication). The distinction between prokaryotic and eukaryotic replication may be due to a difference in the replication machinery or in the structure of the chromosomes.Īt least six proteins are involved in the initiation, elongation, and completion of Okazaki fragments in E. In eukaryotic cells, the rate of fork movement is slow, 10 to 100 residues per second, but the time required for completion of one Okazaki fragment is the same as in the prokaryotic cells. coli, the replication fork proceeds at 1000 residues per second, and one Okazaki fragment is synthesized every 1 to 2 sec. The velocity of the replication fork movement, which represents the rate of leading-strand DNA synthesis, has been estimated from the size of a replicon and the length of its replication period. This difference seems to reflect a difference between prokaryotes and eukaryotes in the rate of chain elongation on the leading-strand template. In eukaryotic cells, however, the Okazaki fragments are between 100 and 200 nucleotides (3). In a wide variety of bacteriophages and prokaryote cells, the size of the nascent DNA is between 10 nucleotides. RNA linked to Okazaki fragments can also be identified, although the RNA primers are removed quickly in vivo (2). This means that the Okazaki fragments have a very short life-time and are connected to a long stretch of DNA in cells. After the chase, with subsequent exposure to high concentrations of unlabeled precursors, the radioactive precursor was found exclusively in high-molecular-weight DNA. To make such an analysis possible, Okazaki and colleagues developed a pulse-chase labeling technique, by which much of the nucleotide precursor label could be captured as the nascent DNA. Those molecules sediment with sedimentation coefficients of about 8 S to 10 S in alkaline sucrose gradients, corresponding to chain lengths of 1000 to 2000 residues. Okazaki fragments were first identified during a course of study on the most recently synthesized, or nascent, DNA molecules in the replication of bacteriophage T4 DNA (1). In other words, the Okazaki fragment is a unit of discontinuous DNA replication on the lagging strand. The short stretches of DNA attached to RNA primers on the lagging strand during DNA replication are called Okazaki fragments, after their discoverer (see Primer and Leading and Lagging Strands). ![]()
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