Histone DNA
In eukaryotic cells, DNA coils around proteins called histones and then further package to form nucleosomes. This allows the very long DNA to be compacted inside a cell. Each nucleosome consists of two copies each of H2A, H2B, H3 and H4 histone proteins. The complex with eight histone proteins is called an octamer. Adjacent nucleosomes are typically around 200bp apart. 146bp of DNA are wrapped around the octamer and the remaining DNA acts as linker DNA between nucleosomes. Histones are small in size with molecular weight ranging from 11,000 to 21,000. The amino acid sequence of histone contains large amount of arginine and lysine so that histones are basic and cationic. The positive charges possessed by histones enable them to interact with the polyanionic phosphate backbone in DNA.
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DNA polymerase
The synthesis of DNA using DNA or RNA as template is catalysed by DNA polymerase. A primer is necessary to initiate the polymerization of DNA. DNA polymerase I is common in prokaryotes (e.g. E. coli). The functions of DNA polymerase I include the following: firstly, it carries out 5’ to 3’ polymerase activity with the presence of magnesium ion, dNTPs and primers. Secondly, it possesses proofreading 3’ to 5’ exonuclease activity for single-stranded or double-stranded DNA. It cleaves DNA when there is no dNTP. However, this activity is inhibited by 5’ to 3’ polymerase activity. Lastly, DNA polymerase I has 5’ to 3’ exonuclease activity. It digests one strand of DNA in double-stranded DNA or RNA/DNA hybrid from 5’ direction. Klenow fragment only has 3’ to 5’ exonuclease and polymerase activities. DNA polymerase I is widely used in recombinant DNA technology. Applications include removal of 3’ overhang to generate a blunt end and DNA labelling by filling in 5’ overhang with radioactive dNTPs, nick translation and replacement reaction. In polymerase chain reaction, thermophilic Taq DNA polymerase from Thermus aquaticus is used. It is resistant to heat but does not have 3’to 5’ proofreading activity.
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Helicase
Helicase is an important enzyme in organisms since it is involved in many processes, such as DNA replication, transcription, translation, DNA repair, recombination etc. The main function of helicase is to separate the double-stranded DNA into single strands using the energy of ATP hydrolysis at the replication fork. dnaB is an helicase in bacteria, for example E. coli. In eukaryotic cells, to prepare for replication, origin of replication complex (ORC), additional proteins Cdc6 (cell division cycle) and Cdt1 (CDC10-dependent transcript 1) bind to the origin of replication. Six subunits called Mcm2-7 (minichromosome maintenance proteins) assemble to form the ring-shaped helicase. Mcm2-7 separates the double-stranded DNA, and the single strands are stabilized by the binding of replication protein A, a single-stranded DNA binding protein.
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RNA polymerase
RNA polymerase is present in many organisms and viruses (e.g. T7 bacteriophage). There are three kinds of RNA polymerase in the cytoplasm of eukaryotes while there is only one type in bacteria. It is responsible for the synthesis of RNA in transcription. Similar to DNA polymerase, the direction of synthesis is from 5’ to 3’. Unlike DNA polymerase, RNA polymerase however does not require a primer at the starting point of synthesis. RNA strand can be made when there is DNA template, NTPs and magnesium ions. In addition, RNA polymerase has helicase activity so it can unwound without the help of extra enzymes. The error rate of RNA polymerase is around 10-5, which is higher than that of DNA polymerase. It is because RNA has shorter lifespan in the cell than DNA.
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DNA ligase
DNA ligase is an enzyme which catalyses the joining of DNA in DNA replication, DNA repair and recombinant DNA experiments. DNA with either blunt ends or cohesive ends can be ligated together by DNA ligase, but the conditions and enzyme concentration for joining blunt end DNA need to be adjusted to increase the efficiency. The ligation reaction takes three steps: firstly, the lysine residue of DNA ligase is self-adenylated by free ATP or NAD. A pyrophosphate group is released. The adenyl group is then transferred to the 5’ phosphate group, which acts as a donor. Finally, phosphodiester bond is formed between adenylated 5’ phosphate donor and the 3’ hydroxyl acceptor together with one AMP produced in the reaction. DNA ligases are also useful in recombinant DNA technology. T4 DNA ligase from T4 bacteriophage is the most commonly used in laboratories.
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