DNA eukaryotic polymerases

DNA Replication (from Chapter 4), E. coli DNA Polymerases, Eukaryotic DNA Polymerases, Other Replication Proteins, Topoisomerases... 

DNA Replication Overview Replication Fork E. coli DNA Polymerases Eukaryotic DNA Polymerases Other Replication Proteins Topoisomerases Uracil-DNA N-Glycosylase Replication Complexes Initiation of DNA Replication Replication of Linear Genomes Fidelity of DNA Replication... 

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... 

The RNA oligonucleotides are complementary to a sequence on one of the strands of the DNA template and base pair with a portion of the DNA molecule. Subsequently, deoxyribonucleotides are covalently attached to the RNA primer. The synthesis of the primer itself is catalyzed by a special RNA polymerase called primase. Similar RNA polymerase-like enzymes are used to prime the synthesis of certain viral DNAs and eukaryotic DNA. 

Since all DNA polymerases require a primer and work only in the 5 to 3 direction, there s a problem with replicating the 5 ends of the DNA. If an RNA primer has to be laid down and later removed, these ends can t get replicated. For bacteria with a circular genome, this isn t a problem. Eukaryotes have specialized structures called telomeres at the... 

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. 

Telomeres are r etitive sequences at the ends of linear DNA molecules in eukaryotic chromosomes. With each round of replication in most normal cells, the telomeres are shortened because DNA polymerase cannot complete synthesis of the 5 end of each strand. This contributes to the aging of cells, because eventually the telomeres become so short that the chromosomes cannot function properly and the cells die. 

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... 

A third alternative starts with an extract of RNA, not DNA. Mature eukaryotic mRNA contains a long run or tail of adenine residues at its 3 end. The poly(rA) tail can be hybridized with an oligomer of thymine residues, and the oligo(dT) can then be used as a primer for a particular kind of DNA polymerase known as reverse transcriptase. This enzyme, a polymerase associated with retroviruses, will use RNA as a template to make a complementary DNA copy of the RNA, creating a DNA-RNA double-stranded hybrid. In another round of synthesis, the enzyme can replace the RNA strand entirely with DNA, so that the RNA-DNA hybrid is completely converted to double-stranded DNA containing an exact copy of the original RNA sequence. This DNA molecule is known as cDNA because it has a strand that is complementary to (or a copy of) the original RNA. 

S ATP -I- [DNA-directed eukaryotic RNA polymerase II subunit Ila] (<4> distinct from other protein phosphokinases, transfers about 20 phosphates to the heptapeptide repeats Pro-Thr-Ser-Pro-Ser-Tyr-Ser in C-terminal domain of MW 220000 subunit of RNA-polymerase II [7] <4> substrates are RNA-polymerase II subunits of wheat germ, soy bean, pea and human [7] phosphorylates predominantly Ser-residues [1-3,5,7] <1> kinase CTDKl almost exclusively phosphorylates Ser-residues [5] <1> kinase CTDK2 phosphorylates to a lesser extent Thr-resi-dues [1] <3-5> phosphorylates to a lesser extent Thr-residues [1,5,7] <1> phosphorylates Ser- and Thr-residues equally [6] <1,3,5> phosphorylates not Tyr-residues [1,6] <1> kinase CTDKl 33 mol phosphate per mol IIA-subunit [5] <1> kinase CTDK2 40-50 mol phosphate per mol IIA-subunit, i.e. 1 phosphate per heptapeptide repeat [5] <4> no substrate is GTP [7] <2,4> no substrates are CTP and UTP [3,7] <2> no substrates are dTTP and AMP-PNP [3] <4> no substrates are bovine serum albumin and calf thymus histone [7] <5> no substrate is phosvitin... 

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... 

In bacteria, error-prone translesion DNA synthesis, involving TLS DNA polymerases, occurs in response to very heavy DNA damage. In eukaryotes, similar polymerases have specialized roles in DNA repair that minimize the introduction of mutations. 

Good summary of the properties and roles of the more than one dozen known eukaryotic DNA polymerases. 

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. 

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