Polymerase eukaryotic cells

Unlike prokaryotes, in which all major types of RNA are synthesized by one RNA polymerase, eukaryotic cells contain three nuclear DNA-dependent RNA polymerases, each responsible for synthesizing a different class of RNAs. 

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. 

Deslongchamps and coworkers [26] used a combination of a transannular Diels-Alder cycloaddition and an intramolecular aldol reaction in the synthesis of the unnatural enantiomer of a derivative of the (+)-aphidicolin (4-74), which is a diterpe-noic tetraol isolated from the fungus Cephalosporium aphidicolia. This compound is an inhibitor of DNA polymerase, and is also known to act against the herpes simplex type I virus. In addition, it slows down eukaryotic cell proliferation, which makes it an interesting target as an anticancer agent... 

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. 

In eukaryotic cells, there are three classes of RNA polymerases (I, II and III) which synthesise different classes of RNA, as follows ... 

Transcription is catalyzed by DNA-dependent RNA polymerases. These act in a similar way to DNA polymerases (see p. 240), except that they incorporate ribonucleotides instead of deoxyribonucleotides into the newly synthesized strand also, they do not require a primer. Eukaryotic cells contain at least three different types of RNA polymerase. RNA polymerase I synthesizes an RNA with a sedimentation coef cient (see p. 200) of 45 S, which serves as precursor for three ribosomal RNAs. The products of RNA polymerase II are hnRNAs, from which mRNAs later develop, as well as precursors for snRNAs. Finally, RNA polymerase III transcribes genes that code for tRNAs, 5S rRNA, and certain snRNAs. These precursors give rise to functional RNA molecules by a process called RNA maturation (see p. 246). Polymerases II and III are inhibited by a-amanitin, a toxin in the Amanita phalloides mushroom. 

One area of basic biochemical research that has paid unexpected dividends is DNA replication. Enzymological work here has characterized the various DNA polymerases in bacterial and eukaryotic cells. With progress in the biochemical characterization of these enzymes, new applications have been found for them in research... 

Prelich, G. RNA polymerase II carboxy-terminal domain kinases emerging clues to their function. Eukaryot. Cell, 1, 153-162 (2002)... 

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... 

Eukaryotic Cells Have Three Kinds of Nuclear RNA Polymerases... 

The transcriptional machinery in the nucleus of a eukaryotic cell is much more complex than that in bacteria. Eukaryotes have three RNA polymerases, designated I, II, and III, which are distinct complexes but have certain subunits in common. Each polymerase has a specific function and is recruited to a specific promoter sequence. 

Rifampin binds to RNA polymerase and changes its conformation so that it cannot initiate RNA synthesis. RNA polymerase from eukaryotic cells does not bind rifampin, and RNA synthesis is unaffected. 

There are three distinct classes of RNA polymerase in the nucleus of eukaryotic cells. All are large enzymes with multiple subunits. Each class of RNA polymerase recognizes particular types of genes. 

Ribosomal RNAs of both prokaryotic and eukaryotic cells are synthesized from long precursor molecules called preribosomal RNAs. The 23S, 16S, and 5S ribosomal RNAs of prokaryotes are produced from a single RNA precursor molecule, as are the 28S, 18S, and 5.8S rRNAs of eukaryotes (Figure 30.15). [Note Eukaryotic 5S rRNA is synthesized by RNA polymerase III and modified sepa-j rately.] The preribosomal RNAs are cleaved by ribonucleases to yield intermediate-sized pieces of rRNA, which are further "trimmed"... 

Other DNA polymerases. Reverse transcriptases synthesize DNA using an RNA template strand. They are best known for their function in retroviruses (Chapter 28). The HIV reverse transcriptase is a heterodimer of 51- and 66-kDa subunits. The larger subunit contains a ribonuclease H domain.288-2893 The enzyme is a prime target for drugs such as AZT and others.290 291 A different reverse transcriptase is found in all eukaryotic cells in telom-erase, an enzyme essential for replication of chromosome ends. Reverse transcriptases have also been found in rare LI sequences that are functioning ret-rotransposons (Section D).292... 

Compare systhesis of the leading and lagging strands in the elongation phase of DNA replication. Explain why DNA polymerases may have difficulty in replicating the 3 -end of the lagging strand of linear DNA. How has this problem been solved in many bacterial and viral systems In eukaryotic cells ... 

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 are readily accessible to RNA polymerase binding and transcription. By contrast, most DNA in eukaryotic cells exists in a condensed form (chromatin), which is not readily accessible to transcription. The small fraction of DNA accessible to the RNA polymerase in any given cell type is especially sensitive to cleavage by mild treatment with bovine pancreatic DNase I. These regions of the DNA often contain bound RNA polymerase, modified histones, and additional nonhistone proteins. Active regions are often undermethylated compared with the total DNA. Most of the methylated groups in DNA are on the C residues in the CG sequence. 

The enzyme controlled by the E. coli genes recB and recC has several activities in vivo associated with substrate DNA, notably the degradation of single- and double-stranded DNA. Activity depends upon the presence of K+, which is suggested to maintain the conformation of the enzyme in the active form.98 Many DNA polymerases are stimulated as much as five-fold by cations, particularly K+ and NH4+, at concentrations up to 50 mM.99 At higher concentrations of M+, most DNA polymerases are inhibited. Inhibition by monovalent cations has been used to distinguish between DNA polymerase-a and -j8 from eukaryotic cells, since the latter enzyme is not inhibited by concentrations as high as 300 mM. A DNA polymerase coded for by herpes virus is uniquely stimulated by both Na+ and K+. Little is known about the mechanism of action of the IA cations in these cases. 

In eukaryotic cells, a gene consists of the transcribed portion, as well as the 5 and 3 flanking regulatory DNA sequences. Transcription initiation is usually regulated by the 5 regulatory sequences, which include some common DNA elements. One of these elements, the TATA box (TATAAA), is located 25-35 base pairs upstream of the transcription start site for RNA polymerase II, and its function is to direct the transcription at the so-called cap site (Breathnach and Chambon, 1981). The CAAT box (GGCCAATCT) is known to be present upstream of the TATA box. Transcription is initiated by an interaction between these DNA elements and regulatory proteins. 

Pretazettine (395) has been the subject of numerous biological studies, and it has been shown to exhibit a number of interesting activities (96,97,101,178-187). For example, 395 was found to inhibit HeLa cell growth as well as protein synthesis in eukaryotic cells by interfering with the peptide bond formation step (97,101). Furthermore, pretazettine inhibited the purified RNA-dependent DNA polymerase (reverse transcriptase) from avian myeloblastosis virus, a typical C-type virus (178), in an unusual fashion since it physically combined with the polymerase enzyme itself rather than interacted with the nucleic acid template. Pretazettine also exhibited antiviral activity against the Rauscher leukemia virus in mouse embryo cell cultures by suppressing viral replication (179). 

Eukaryotic cells contain five different DNA polymerases a, (3, y, 8 and e. The DNA polymerases involved in replication of chromosomal DNA are a and 8. DNA polymerases (8 and e are involved in DNA repair. All of these polymerases except DNA polymerase y are located in the nucleus DNA polymerase y is found in mitochondria and replicates mitochondrial DNA. 

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... 

The end of a linear chromosome is called a telomere. Telomeres require a special mechanism, because the ends of a linear chromosome can t be replicated by the standard DNA polymerases. Replication requires both a template and a primer at whose 3 end synthesis begins. The primer can t be copied by the polymerase it primes. What copies the DNA complementary to the primer In a circular chromosome, the primer site is to the 3 direction of another polymerase, but in a linear chromosome, no place exists for that polymerase to bind. As a result, unless a special mechanism for copying the ends of chromosomes is used, there will be a progressive loss of information from the end of the linear chromosome. Two characteristics about telomeres help avoid this situation. First, they consist of a short sequence—for example, AGGGTT—repeated many times at the end of each chromosome. Telomeres, therefore, are part of the highly repetitive DNA complement of a eukaryotic cell. Secondly, a specific enzyme, telomerase, carries out the synthesis of this reiterated DNA. Telomerase contains a small RNA subunit that provides the template for the sequence of the telomeric DNA. Eukaryotic somatic cells have a lifespan of only about 50 doublings, unless they are cancerous. One theory holds that a lack of telomerase in cells outside the germ line causes this limitation. 

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