Replication Okazaki-fragments

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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.)... 

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... 

Yet another polymerase, DNA polymerase e, replaces DNA polymerase S in some situations, such as in DNA repair. DNA polymerase e may also function at the replication fork, perhaps playing a role analogous to that of the bacterial DNA polymerase I, removing the primers of Okazaki fragments on the lagging strand. 

DNA is synthesized in the 5 —>3 direction by DNA polymerases. At the replication fork, the leading strand is synthesized continuously in the same direction as replication fork movement the lagging strand is synthesized discontinuously as Okazaki fragments, which are subsequently ligated. 

Terms in bold are defined template 950 semiconservative replication 950 replication fork 951 origin 952 Okazaki fragments 952 leading strand 952 lagging strand 952 nucleases 952 exonuclease 952 endonuclease 952 DNA polymerase I 952 primer 954 primer terminus 954... 

Correct answer = C. Fluoroquinolones, such as ciprofloxacin, inhibit bacterial DNA gyrase—a type II DNA topoisomerase. This enzyme catalyzes the transient breaking and rejoining of the phosphodiester bonds of the DNA backbone, to allow the removal of positive supercoils during DNA replication. The other enzyme activities mentioned are not affected. Primase synthesizes RNA primers, helicase breaks hydrogen bonds in front of the replication fork, DNA polymerase I removes RNA primers, and DNA igase joins Okazaki fragments. 

Replication of this lagging strand occurs in segments known as Okazaki fragments... 

In 1968, Okazaki reported that during the time that replication of DNA is taking place bacterial cells contain short fragments of DNA. These are now called Okazaki fragments or replication fragments.260 A... 

Figure 27-20 (A) Hypothetical replisome for concurrent replication of leading and lagging strands by a dimeric polymerase associated with helicase dnaB and a primosome. Open arrows indicate directions of movement of DNA, which is forming a loop as the polymerase fills a gap to complete an Okazaki fragment. The primase will then form a new primer and a new loop. From Komberg and Baker.265 (B) Electron micrograph of the primosome bound to covalently closed ( )X174 duplex replicative form. These enzymatically synthesized duplexes invariably contain a single primosome with one or two associated small DNA loops. From A. Komberg in Hubscher and Spadari,266 pp. 9,10.

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. 

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.

The replication is semiconservative—that is, continuous for the leading strand and fragmentational (Okazaki fragments) for the lagging strand. 

Yes, but in small quantities and only transiently. The nascent (or Okazaki) fragments formed through discontinuous DNA replication contain a short stretch of RNA which serves as a primer for DNA chain growth (Chap. 16). 

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