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Archive for the ‘DNA replication’ Category

DNA replication is carried out by DNA polymerase. During the replication, one DNA strand synthesized as leading strand and the other as lagging strand. The leading strand synthesis is continuous, whereas lagging strand synthesis is discontinuous. DNA replication at the DNA ends i.e. telomeres is an altogether different story. If a primer forms at the end of a linear DNA molecule to initiate replication of the leading strand, a single-stranded gap remains at that end after DNA has been synthesized and the primer has been removed. DNA pol cannot fill his gap as no 3-OH group is available onto which nucleotides can be added. If this gap were left unfilled a linear DNA molecule would shorten at each end by the length of the primer with each replication cycle. After many rounds of replication, the molecule would disappear. This problem is known as the linear DNA replication paradox. Besides, as the lagging strand is replicated in the discontinuous manner, it cannot be replicated in its entirety. When the RNA primer is removed, there is no upstream strand onto which DNA pol can build to fill the resulting gap. Without some special mechanism, the daughter DNA strand resulting from lagging strand synthesis would be shortened from the telomeric ends at each cell division.

The enzyme that prevents this shortening of DNA strands is known as telomerase. The enzyme functions as a reverse transcriptase (RNA-dependent DNA polymerase). It holds a small RNA molecule as its component. It was discovered by Elizabeth H. Blackburn et al. The enzyme contains a catalytic site that polymerizes the nucleotides using RNA as the template. The RNA template is part of the enzyme and is approximately 160 bases long. The enzyme uses this RNA as template for adding telomeric repeats to the chromosome ends. First, the 3’ end of the telomere hybridizes with the RNA component of telomerase. Within the RNA sequence is the 6-nucleotide repeated sequence 5’CCCCAA3’ (Tetrahymena) which is complementary to the telomere repeat sequence. Telomerase binds to the 3’ end of to the repeated region. Telomerase binds to the 3’ end of the ssDNA, adding new copies of the TTGGGG repeat sequence. These repeats further extend the single stranded gap left after primer removal. Primers may be added to the repeated segments of DNA and a DNA pol may now fill in much of the gap with DNA.

The repetitive sequence added by telomerase is determined by the RNA associated with the enzyme, which differs among telomerases from different organisms. This extends the dsDNA till it is at least as long as, or longer, than the parent molecule. The single stranded end left after several rounds of addition can be trimmed. The extension and trimming of telomeric DNA is not precise and therefore the number of telomeric repeats is highly variable. Certain proteins, like Rap1 in S. cerevisiae, TRF1 in human beings, keep track of the number of telomeric repeats. It is actually the number of protein molecules attached to the telomere ends that is counted, and not the number of repeats, to decide if telomeres should be added.

The activity of telomerase gradually declines indicating that the DNA molecules must grow shorter with each cell division. This is of little consequence for most cells as they undergo a limited number of cell divisions and then stop dividing: This is the normal ageing process which occurs in all body cells, end at a certain telomeric length, and cells stop dividing. However, if telomerase starts behaving erratically, the chromosomes will not be shortened and cells will continue to divide leading to continuous growth. When cancer cells were examined for the presence of telomerase, the active enzyme was found in abundance, indicating that the linear DNA molecules were being fully replicated. Thus telomerase is found to be essential for cancer cells to continue dividing. Thus, the attention is now turning to the possible clinical application of this knowledge. Research is now under way to determine if blocking the activity of telomerase may be an effective treatment for cancer. Further, studying normal telomere shortening, which acts as a biological clock, may help understand senescence.

 


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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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This post is a tribute to Arthur Kornberg. He died of respiratory failure on Friday in Stanford, California at the age of 89. He discovered the enzyme DNA polymerase, which replicates the DNA molecule. The search for an enzyme that could synthesize DNA started in 1955, which finally culminated in 1959 with a Nobel Prize to Arthur Kornberg in medicine. He and his co-workers purified and characterized DNA polymerase from E. coli. The enzyme they purified is named DNA polymerase I. Four other DNA ploymerases are found in E. coli. The enzyme has also been named Kornberg Enzyme in honour of the scientist. The enzyme is made of a single polypeptide and has a mol wt of 103,000. Read the full story here.


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