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Archive for the ‘DNA’ Category

DNA polymerase III

DNA pol III is the principal replication enzyme of E. coli. The enzyme is highly complex and has more than 10 types of subunits. These subunits are α, ε, θ, τ, δ, δ’, β, χ, ψ, γ. The α, ε and θ subunits combine to form the core polymerase. The core polymerase has a limited processivity. The two core polymerases are linked in a complex by a dimer of τ subunits. Two γ subunits, one δ, one δ’, one χ and one ψ subunit combine to form a single clamp-loading complex. The dimeric core polymerase and the clamp-loading complex (14 subunits of 9 types) combine to form the DNA pol III enzyme. The α and ε subunits carry out the replication and proofreading activities, respectively. DNA pol III has a low processivity, but addition of β subunits increases the processivity required to replicate the E. coli chromosome. The 4 β subunits associate with the dimeric core and the clamp-loading complex to make the DNA pol III holoenzyme. The β subunits associate in pairs to form a donut-shaped structure. This encircles the DNA and acts like a clamp. This slides along the DNA as replication proceeds and prevents the dissociation of DNA pol III from DNA.


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As mentioned earlier, DNA pol III is the enzyme which carries out the replication of large E. coli chromosome. DNA pol I, because of the following properties, does not qualify as the enzyme for E. coli chromosome replication:

1) The polymerization rate (nucleotides added/sec) of this enzyme is 16-20 nucleotides/sec or approximately 600 nucleotides/min, which is too slow. It is slow by a factor of 100 or more to account for the rate at which the replication fork moves during bacterial chromosome replication.

2) DNA pol I has a very low processivity, 3-200 nucleotides added before polymerase dissociates.

3) A bacterial strain, isolated in 1969 by John Cairns, had a mutant DNA pol I gene, which produced the inactive enzyme. But, surprisingly this bacterial strain was viable. This clearly shows that even in the absence of active DNA pol I, E. coli can survive and replicate its chromosome. It means there is another DNA polymerase present to perform the function of DNA replication.


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DNA pol I: It is a single subunit enzyme with a mol wt of 103,000. It has 3’→5′ exonuclease proofreading activity. The polymerization rate, i.e. nucleotides added per second to a growing DNA molecule, is 16-20. The processivity of DNA pol I is 3-200. Processivity is the number of nucleotides before polymerase dissociates from the nucleic acid. DNA pol I has 5’→3′ exonuclease activity, not found in DNA pol II or DNA pol III.

DNA pol II: The enzyme is composed of more than 4 subunits. It has 3’→5′ exonuclease proofreading activity. The polymerization rate is 40 and processivity is 1500.

DNA pol III: The enzyme is composed of more than 10 subunits. It has 3’→5′ exonuclease proofreading activity. The polymerization rate is 25-1000 and processivity is more than 500,000.


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DNA Polymerases

The enzyme required for DNA replication is DNA polymerase. All living organisms which have DNA as their genetic material require DNA polymerase enzyme.

We will begin our discussion of DNA polymerases with the E. coli enzymes. This prokaryote has 5 different kinds of DNA polymerase: DNA pol I, DNA pol II, DNA pol III, DNA pol IV and DNA pol V. It is DNA pol I, which was discovered by A. Kornberg and also named Kornberg Enzyme. However, DNA pol I is not suited for replication of large E. coli chromosome. The reasons for this would be discussed in a later post. In the early 1970s, DNA pol II and DNA pol III were discovered. DNA pol IV and DNA pol V were discovered much later, in 1999. DNA pol III is the principal replication enzyme in E. coli. DNA pol II is involved in DNA repair. DNA polymerases IV and V are also involved in a special form of DNA repair. “DNA pol I performs a host of clean-up functions during replication, repair and recombination” (Lehninger Principles of Biochemistry, Nelson and Cox, 3rd edition). It is not the primary enzyme of replication in the prokaryote.


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H-DNA

Today I would be writing about a very unusual form of DNA, that is, H-DNA.

The H-DNA is a triple helix. This particularly unusual form of DNA is found in vitro or possibly during recombination and DNA repair. It forms by pairing and interwinding of 3 strands of DNA. Two of the three strands contain pyrimidines and the third contains purines. The three strands show a special base pairing known as Hoogsteen base pairing. Some sequences that can form H-DNA are found within regions involved in the regulation of a number of genes in eukaryotes.

You can read the following article about the H-DNA:

Naturally occurring H-DNA-forming sequences are mutagenic in mammalian cells.

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Z-DNA

Z-DNA was discovered in vitro under high salt conditions. It is know to exist in the interband regions of the giant salivary gland chromosomes of Drosophila and in the transcriptionally active macronucleus of the ciliated protozoan Stylonchia mytilus. The properties of Z-form DNA are listed below:

1) It is a left-handed helix and the helical diameter is 18Å.

2) There are 12 base pairs per turn in the Z-DNA. Because of the maximum number of base pairs per turn found in this form, the Z-DNA has the least twisted structure.

3) It has a zig-zag sugar phosphate backbone.

4) It has a rise of 0.38 nm per base pair.

5) The Z-form DNA occurs in polymers that have a sequence of alternating purines and pyrimidines (especially Gs and Cs). The alternating polymers could be poly d(GC) or poly d(AC).

6) Instead of having both major and minor grooves, the Z-DNA has only a single groove.

Scientists have raised antibodies specific to Z-DNA (E M Lafer, R P Valle, A Möller, A Nordheim, P H Schur, A Rich, and B D Stollar. J Clin Invest. 1983 February; 71(2): 314–321). The antibodies have helped in the detection of Z-DNA under different cellular conditions (M Robert-Nicoud, D J Arndt-Jovin, D A Zarling, and T M Jovin. EMBO J. 1984 April; 3(4): 721–731).

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A-form DNA

The A-DNA is favoured in many solutions that are relatively of water. The properties of A-DNA are:

1) Right-handed helix

2) Helix rise per base pair is 0.23 nm

3) The number of base pairs per helical turn is 11.

4) A-form DNA is shorter and has a greater helical diameter than the B-DNA. The helical diameter of A-DNA is 23 angstrom or 26 angstrom.

5) The rotation per base pair is 34.7°.

6) RNA-DNA and RNA-RNA helices also exist in this form.

7) The A-form is probably very close to the conformation of double stranded regions of RNA, where the presence of the 2′-OH group prevents the adoption of the B-form.

The major groove of A-DNA is less accessible and bases are tilted with regard to the helical axis. The A-DNA is sometimes formed during DNA crystallization because the reagents used to promote DNA crystallization tend to dehydrate it. This leads to a tendency for many DNAs to crystallize in the A-form.

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