Okazaki fragment, synthesis

Polymerase II (pol II) is mostly involved in proofreading and DNA repair. Polymerase I (pol I) completes chain synthesis between Okazaki fragments on the lagging strand. Eukaryotic cells have counterparts for each of these enzymes plus some additional ones. A comparison is shown in Table 36—6. 

Figure 36-16. The discontinuous poiymerization of deoxyribonucleotides on the lagging strand formation of Okazaki fragments during iagging strand DNA synthesis is illustrated. Okazaki fragments are 100-250 nt iong in eukaryotes, 1000-2000 bp in prokaryotes.

Figure 4.26 (a) DNA replication at low resolution (for example as seen by electron microscopy). Only one replication fork is visible and it appears that both strands of the parental DNA replicate continuously in the same direction, which cannot be the case, since the two strands of parental DNA are anti-parallel, (b) The problem is solved by the priming of DNA synthesis with short RNA primers, whose 3 -hydroxyl can be used by DNA polymerase, producing Okazaki fragments, while on the other strand, DNA synthesis is continuous. (From Voet and Voet, 2004. Reproduced with permission from John Wiley Sons., Inc.)... 

The lagging strand is synthesized discontinuously as a series of small fragments (about 1,000 nucleotides long) known as Okazaki fragments. Each Okazaki fragment is initiated by the synthesis of an RNA primer by primase, and then Completed by the synthesis of DNA using DNA polymerase III. Each fr ment is made in the 5 - 3 direction. 

Synthesis of DNA Leading strand Lading strand (Okazaki fragments) DNA polymerase III DNA polymerase lU DNA polymerase 6 DNA polymerase a... 

Lagging strand synthesis, as we have noted, is accomplished in short Okazaki fragments. First, an RNA... 

The replisome promotes rapid DNA synthesis, adding -1,000 nucleotides/s to each strand (leading and lagging). Once an Okazaki fragment has been completed, its RNA primer is removed and replaced with DNA by DNA polymerase I, and the remaining nick is sealed by DNA ligase (Fig. 25-15). 

Like bacteria, eukaryotes have several types of DNA polymerases. Some have been linked to particular functions, such as the replication of mitochondrial DNA. The replication of nuclear chromosomes involves DNA polymerase a, in association with DNA polymerase S. DNA polymerase a is typically a multisubunit enzyme with similar structure and properties in all eukaryotic cells. One subunit has a primase activity, and the largest subunit (Afr -180,000) contains the polymerization activity. However, this polymerase has no proofreading 3 —>5 exonuclease activity, making it unsuitable for high-fidelity DNA replication. DNA polymerase a is believed to function only in the synthesis of short primers (containing either RNA or DNA) for Okazaki fragments on the lagging strand. These primers... 

DNA in eukaryotic chromosomes is complexed with histone proteins in complexes called nucleosomes. These DNA-protein complexes are disassembled directly in front of the replication fork. The nucleosome disassembly may be rate-limiting for the migration of the replication forks, as the rate of migration is slower in eukaryotes than prokaryotes. The length of Okazaki fragments is also similar to the size of the DNA between nucleosomes (about 200 bp). One model that would allow the synthesis of new eukaryotic DNA and nucleosome formation would be the disassembly of the histones in front of the replication fork and then the reassembly of the histones on the two duplex strands. Histone synthesis is closely coupled to DNA replication. 

Okazaki fragment. A short segment of single-stranded DNA that is an intermediate in DNA synthesis. In bacteria, Okazaki fragments are 1,000-2,000 bases in length in eukaryotes, 100-200 bases in length. 

DNA synthesis proceeds in a 5 — 3 direction on each strand of the parental DNA. On the strand with 3 —>5 orientation (the leading strand) the new DNA is synthesized continuously. On the strand that has 5 —>3 orientation (the lagging strand) the DNA is synthesized discontinuously as a series of short Okazaki fragments that are then joined together. 

Fig. 4. Details of DNA replication, (a) Primase binds to the DNA template strand (thin line) and (b) synthesizes a short RNA primer (dotted line) (c) DNA polymerase III now extends the RNA primer by synthesizing new DNA (thick line) (d) during synthesis of the lagging stand, adjacent Okazaki fragments are separated by the RNA primers (e) the RNA primers are now removed and the gaps filled with DNA by DNA polymerase I (f) generating adjacent DNA fragments that are then (g) joined by DNA ligase.

Fig. 16-11 Synthesis of DNA as nascent or Okazaki fragments in one arm of the replication fork.

DNA replication in E. coli starts at a unique origin (oriC) and proceeds sequentially in opposite directions. More than 20 proteins are required for replication. An ATP-driven helicase unwinds the oriC region to create a replication fork. At this fork, both strands of parental DNA serve as templates for the synthesis of new DNA. A short stretch of RNA formed by primase, an RNA polymerase, primes DNA synthesis. One strand of DNA (the leading strand) is synthesized continuously, whereas the other strand (the lagging strand) is synthesized discontinuously, in the form of 1-kb fragments (Okazaki fragments). Both new strands are formed simultaneously by the concerted actions of the highly processive... 

DNA polymerase then incorporates a dNMP onto the 3" end of the primer and initiates lagging strand synthesis. The polymerase extends the primer for about 1,000 nucleotides until it comes in contact with the 5 end of the preceding primer. These short segments of RNA/DNA are known as Okazaki fragments (Fig 11.24). 

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