Prokaryotes DNA replication

B. Prokaryotic DNA replication is accomplished by DNA polymerases, large multienzyme complexes that move out bidirectionally from the origin of replication. 

Figure 11-2. The prokaryotic DNA replication fork. A schematic representation of semi-conservative replication of DNA by different mechanisms on the leading and lagging strands by DNA polymerase III (DNA pol III) is shown. Other enzymes and accessory proteins that participate in initiation, elongation, and ligation phases of the process are indicated, with DNA pol I depicted as having just dissociated from a completed Okasaki fragment. SSBs, single-stranded DNA binding proteins.

The above description of prokaryotic DNA replication has been pieced together by the enormous efforts of a large number of laboratories. The replication of eukaryotic DNA appears to be much more complex, and therefore much less is known about it. 

DNA SYNTHESIS IN PROKARYOTES DNA replication in E. coli has proven to be a complex process that consists of several basic steps. Each step requires certain enzyme activities. 

Mechanisms - The basic mechanisms of DNA replication are quite similar in eukaryotes and prokaryotes. DNA replication is semiconservative and is continuous on one strand and discontinuous on the other. As in prokaryotes, eukaryotic replication entails the assembly of short RNA primer molecules, elongation from the primers by a DNA polymerase, and (on the discontinuous strand) ligation of Okazaki fragments. A significant difference in eukaryotic and prokaryotic DNA replication is in the smaller size of the Okazaki fragments in eukaryotic cells - about 135 bases long, or about the size of the DNA on a nucleosome. 

Eukaryotic cell cycle - The processes by which cells divide and DNA is replicated (see here) are somewhat more complicated in eukaryotes than in prokaryotes. DNA replication in bacteria is an almost continuous process, at least during exponential growth. The somatic cells of eukaryotes, on the other hand, typically divide much less frequently, and some, in certain types of mature tissue, do not divide at all. Eukaryotic cells that are dividing in growing tissues exhibit a well-defined cell cycle, which is almost always separated into several distinct phases, as shown in Figure 28.14, Figure 28.15, and Figure 28.16. 

The general features of DNA replication in eukaryotes are similar to those in prokaryotes. Table 10.5 summarizes the differences. As with prokaryotes, DNA replication in eukaryotes is semiconservative. There is a leading strand with continuous synthesis in the 5 3 direction and a lagging strand with discontinuous synthesis in the 5 3 direction. An RNA primer is formed hy a specihc enzyme in eukaryotic DNA replication, as is the case with prokaryotes, hut in this case the primase activity is associated with Pol a. The structures... 

There s not just one DNA polymerase there s a whole army. DNA replication actually occurs in large complexes containing many proteins and sometimes many polymerases. In eukaryotic cells we have to replicate both mitochondrial and nuclear DNA, and there are specific DNA polymerases for each. In addition to DNA replication, you have to make new DNA when you repair. Consequently, the function may be specialized for repair or replication. There can also be specialization for making the leading or lagging strand. Some of the activities of DNA polymerases from eukaryotes and prokaryotes are shown in the table on the next page. 

Prokaryote cells divide and grow into two daughter cells. In the division process, the DNA replicates and each daughter cell receives one copy. 

The overall process of DNA replication in prokaryotes and eukaryotes is compared in Figure 1-2-1. 

The mechanism of replication in eukaryotes is believed to be very similar to this. However, the details have not yet been completely worked out. The steps and proteins involved in DNA replication in prokaryotes are compared with those used in eukaryotes in Table 1-2-2. 

Quinolones and fluoroquinolones inhibit DIMA gyrase (prokaryotic topoisomerase II), preventing DNA replication and transcription. These dmgs, which are most active against aerobic gram-negative bacteria, indude ... 

E. Eukaryotic DNA replication is similar to that of prokaryotes but more complex in scale, and the process is coordinated with the cell cycle. 

Typical classes and examples within these categories as they apply to what is currently most prescribed on the U.S. market are summarized in Table 1.8. The targets in groups 1 and 4 are unique in bacteria and absent in humans and other animals, whereas groups 2, 3, and 5 have human counterparts that are structurally different between prokaryotes and eukaryotes. These differences in targets make the use of antibiotics selective for bacteria with little or no effect on eukaryotic cells from a therapeutic perspective. However, that does not mean that antimicrobial compounds are completely inert to eukaryotes. The mechanisms that block bacterial protein synthesis, block DNA replication, and those that disrupt membrane integrity affect membrane pores. 

The DNA molecules in eukaryotic cells are considerably larger than those in bacteria and are organized into complex nucleoprotein structures (chromatin p. 938). The essential features of DNA replication are the same in eukaryotes and prokaryotes, and many of the protein complexes are functionally and structurally conserved. However, some interesting variations on the general principles discussed above promise new insights into the regulation of replication and its link with the cell cycle. 

The process of eukaryotic DNA replication closely follows that of prokaryotic DNA synthesis. Some differences, such as the multiple origins of replication in eukaryotic cells versus single origins of replication in prokaryotes, have already been discussed. Eukaryotic single-stranded DNA-binding proteins and ATP-dependent DNA helicases have been identified, whose functions are analogous to those of the prokaryotic enzymes previously discussed. In contrast, RNA primers are removed by RNase H. 

DNA replication begins at the origin of replication (one in prokaryotes, multiple in eukary otes). The strands are separated locally, forming two replication forks. Replication of double-stranded DNA is bidirectional. 

Prokaryotic DNA polymerases are so accurate that special kinetic assays have had to be introduced to detect errors in vitro. These depend on replicating under controlled conditions the circular DNA of a small bacteriophage that contains a... 

Postreplicational mismatch repair has been found to correct errors in base substitution occurring during DNA replication in prokaryotes.48 This lowers the error rate for the polymerase from 1 in 106 to 107 to the observed range of values of 1 in 108 to 1010 in E. coli. How does the repair system know in this case which base in a mispair is the incorrect one The answer appears to be that the parent strand is tagged by methylation. A small proportion, some 0.2%, of the cytosine residues are methylated at the 5 position, and a similar proportion of the adenine residues are methylated at the 6 position. As methylation is a postreplicative event, the daughter strand is temporarily undermethylated after replication. 

There are many different types of DNA polymerases, and they vary greatly in their activities and in the nature of the reactions they catalyze. Some polymerases are involved mainly in the replication of DNA. Others are used for the repair of damaged DNA. There is also an important difference between the enzymes isolated from eukaryotes and those isolated from prokaryotes. Most of the eukaryotic DNA polymerases that have been isolated so far have just the simple 5 —> 3 polymerization activity shown in equation 14.1. Prokaryotic DNA polymerases, however, are multifunctional. In addition to their 5 — 3 polymerase activity, they possess a 3 5 exonuclease activity that can excise incorporated... 

Many important details have emerged concerning the mechanisms of DNA replication in both bacteria (Prokaryotes) and higher cells (Eukaryotes). These mechanisms are vital in understanding how a cell duplicates its genetic material (DNA), and how this duplication is related to cell division. For these reasons, cells have evolved elaborate mechanisms to ensure that the process of duplication (DNA replication) is error free. This level of control is so important that cells will actually cease cell division if errors become too frequent and wait until the DNA is repaired. 

TWn relatively recent developments have added to our knowledge significantly concerning how DNA replication occurs with fidelity or in what molecular biologists and biochemists call a processive polymerase activity. DNA polymerase is the enzyme which actually polymerizes (adds DNA precursors or building blocks) DNA. There are many such DNA polymerases in pro- and eukaryotic cells that have different functions but the main enzyme in prokaryotes is DNA polymerase 111 and in Eukaryotes. DNA polymerases alpha, delta, and epsilon. All four of these DNA polymerases are made of subunits. 

DNA replication proceeds by the synthesis of one new strand on each of the parental strands. This mode of replication is called semiconservative, and it appears to be universal. DNA synthesis initiates from a primer at a unique point on a prokaryotic template such as the E. coli chromosome. From the initiation point, DNA synthesis proceeds bidirectionally on the circular bacterial chromosome. The bidirectional mode of synthesis is not followed by all chromosomes. For some chromosomes, usually small in size, replication is unidirectional. 

Eukaryotic DNA is replicated at a slower rate than prokaryotic DNA. One reason may be the requirement for the deposition of histone proteins on DNA (histone synthesis and DNA replication are coupled). Describe a model for the replication of eukaryotic DNA and nucleosome formation. 

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. 

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