Prokaryotes DNA polymerases

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... 

DNA synthesis proceeds in the 5 — 3 direction, with the nucleotides being added to the 3 -hydroxyl of the polynucleotide. At the same time, all prokaryotic DNA polymerases have a 3 — 5 exonuclease activity that works in the... 

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... 

In prokaryotes DNA polymerase I has a 5 — 3 DNA polymerase activity as well as a proof reading capacity to chop out nucleotides in either direction through a 5 — 3 and a 3 — 5 direction exonuclease activity DNA polymerases II and III have 5 — 3 ... 

Stover, J.S., Chowdhury, G., Zang, H., Guengerich, F.P., and Rizzo, C.J. (2006) Translesion synthesis past the C8- and N2-deoxyguanosine adducts of the dietary mutagen 2-Amino-3-methylimidazo[4,5-jJquinoline in the Narl recognition sequence by prokaryotic DNA polymerases. Chem. Res. Toxicol, 19, 1506-1517. 

Elenic acid, 7, was isolated from an Indonesian sponge Plakinastrella sp. [25]. It is an enzyme inhibitor of DNA topoisomerase II with an IC50 of 0.1pg/ml [25] and also a potent inhibitor of calf DNA polymerase a and rat DNA polymerases P [250]. Elenic acid was found not to bind to DNA directly and did not affect the activities of plant DNA polymerases I and II, prokaryotic DNA polymerases such as Escherichia coli DNA polymerase I or other DNA metabolic enzymes, for example, HIV reverse transcriptase, T7 RNA polymerase and bovine deoxyribonuclease I. 

Enzymes - Eukaryotic cells contain five DNA polymerases. Three of them (polymerases oi, A, and s) are used during S phase replication. Table 24.2 and Table 24.3 describe the properties of eukaryotic and prokaryotic DNA polymerases. As in prokaryotes, the replication complex also contains other proteins, including helicases and a number of accessory proteins called replication factors. 

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. 

There is a single prokaryotic RNA polymerase that synthesizes all types of RNA in the cell. The core polymerase responsible for making the RNA molecule has the subunit structure Ojpp. A protein factor called sigma (a) is required for the initiation of transcription at a promoter. Sigma factor is released immediately after initiation of transcription. Termination of transcription sometimes requires a protein called rho (p) faaor. This enzyme is inhibited by rifampin. Actinomycin D binds to the DNA preventing transcription. 

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.

Like DNA polymerases, prokaryotic RNA polymerase (RNA pol) is a multiprotein complex that operates only in the 5 to 3 direction as it copies the template. 

Goodman, M.F. (2002) Error-prone repair DNA polymerases in prokaryotes and eukaryotes. Annu. Rev. Biochem. 71, 17-50. Review of a class of DNA polymerases that continues to grow. 

Prokaryotic and eukaryotic DNA polymerases elongate a new ChA strand by adding deoxyribonucleotides, one at a time, to the 3-end of the growing chain (see Figure 29.16). The sequence of nucleotides that are added is dictated by the base sequence of fie Figure 29.15 template strand with which the incoming nucleotides are paired. 

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. 

The size of the genomic DNA in eukaryotic cells (such as the cells of yeast, plants, or mammals) is much larger (up to 10+11 base pairs) than in E. coli (ca. 10+6 base pairs). The rate of the eukaryotic replication fork movement is about fifty nucleotides per second, which is about ten times slower than in E. coli. To complete replication in the relatively short time periods observed, multiple origins of replication are used. In yeast cells, these multiple origins of replication are called autonomous replication sequences (ARSs). As with prokaryotic cells, eukaryotic cells have multiple DNA polymerases. DNA polymerase S, complexed with a protein called proliferating... 

Since the discovery of Escherichia coli DNA polymerase I in 1957 [2], many polymerases have been identified in prokaryotes and eukaryotes, including the recent discovery of several error-prone DNA polymerases [3]. Primary sequence alignments revealed that these polymerases can be cate-... 

When isolated from bacteria, prokaryotic RNA polymerase has two forms The core enzyme and the holoenzyme. The core enzyme is a tetramer whose composition is given as 0C2PP (two alpha subunits, one beta subunit, and one beta-prime subunit). Core RNA polymerase is capable of faithfully copying DNA into RNA but does not initiate at the correct site in a gene. That is, it does not recognize the promoter specifically. Correct promoter recognition is the function of the holoenzyme form of RNA polymerase. 

Reverse transcription (which occurs in both prokaryotes and eukaryotes) is the synthesis of DNA from an RNA template. A class of RNA viruses, called retroviruses, are characterized by the presence of an RNA-dependent DNA polymerase (reverse transcriptase). The vims that causes AIDS, Human Immunodeficiency Virus (HIV), is a retro-vims. Because nuclear cell division doesn t use reverse transcriptase, the most effective anti-HIV drugs target reverse transcriptase, either its synthesis or its activity. Telomerase, discussed in the previous section, is a specialized reverse transcriptase enzyme. See Figure 12-5. 

In prokaryotic cells, with the exception of DNA primers, all types of RNA are transcribed by a single enzyme, a DNA-dependent RNA polymerase. This RNA polymerase has strict requirements for all four nucleotide-5 -triphosphates, a divalent cation, and a DNA template. Unlike DNA polymerase, there is no requirement for a primer. Initiation and elongation of RNA is in the 5 - 3 direction and the product is complementary to one of the two DNA strands, the template strand that runs in the 3 — 5 direction. The reaction may be formulated similarly to that catalyzed by DNA polymerases ... 

Eukaryotic cells contain at least four different DNA-dependent RNA polymerases. Their localization, cellular transcripts, and susceptibility to the cyclic octapeptide a-amanitin (derived from poisonous mushrooms) are shown in Table 11.3. a-Amanitin blocks the elongation phase of RNA synthesis. Although the structures of these enzymes are much more complex than that of the prokaryotic RNA polymerase, the basic mechanism is very similar to that of the prokaryotic enzyme. 

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