Deoxyribonucleic acid (DNA) contains the genetic instructions for the biological development of a cellular form of life including some viruses. DNA is an antiparallel double helix molecule with sugar-phosphate backbone on the outer side and nitrogen bases in the inner side. The bases are paired specifically, also known as complementary pairing, Adenine (A) with Thymine (T), and Guanine (G) with Cytosine (C) by two and three hydrogen bonds, respectively. DNA is a long polymer of nucleotides (a polynucleotide) that transfers the information to RNA (mRNA) that in turn controls the sequence of amino acid residues in proteins, using the genetic code. James Watson and Francis Crick proposed the DNA double helix structure in 1953 and shared the the Noble Prize in 1962 with Maurice Wilkins. Models for DNA Replication
While proposing their double helix model, Watson and Crick were particularly excited because the complementary nature of the DNA molecule suggested a self-replicating mechanism, a property which would be crucial for a genetic material. Three hypothesis were in contention regarding the mode of self-replication of DNA and these were (Fig. 1): i)
Conservative replication according to which the original strand is unchanged and a completely new DNA molecule is synthesized.
Semi-conservative replication describes a way in which each DNA strand of a double helical molecule serves as a template for the synthesis of two new DNA molecules each with one new and one old strand.
iii) Dispersive replication suggests more-or-less random interspersion of parental and new segments in daughter DNA molecules.
In 1957, Matthew Meselson and Franklin Stahl, proved that DNA is replicated by semiconservative mode of replication. Their experiment involved growing a culture of Escherichia coli in a medium containing 15NH4Cl (ammonium chloride labeled with the heavy isotope of nitrogen) for several generations. A small aliquot of culture was taken out, it’s DNA extracted and was run on CsCl density gradient centrifugation. All the DNA was seen to settle at a point as a single band. From this culture, some cells were transferred to a normal growth medium (containing non- labeled 14NH4Cl). Samples were taken after 20 minutes (i.e. after one cycle of cell division) and 40 minutes (i.e. after two cell divisions) of incubation. DNA was extracted from each sample and subjected to density gradient centrifugation. The results are depicted in Fig.2. After 20 minutes all the DNA contained similar amounts of 14N and 15N, but after 40 minutes two bands were seen, one corresponding to hybrid 14N-15N-DNA, and the other to DNA molecules made entirely of 14N. This was inferred from the different patterns of DNA band(s) after density gradient centrifugation.
The banding pattern seen after 20 minutes enables conservative replication to be discounted because this scheme predicts that after one round of replication there will be two different types of double helix, one containing just 15N and the other containing just 14N. The single 14
N-15N-DNA band that was actually seen after 20 minutes is compatible with both dispersive and semi-conservative replication, but the two bands seen after 40 minutes are consistent only, with semi-conservative replication. Dispersive replication should continue to give rise to hybrid 14N-15N molecules after two rounds of replication, whereas the granddaughter molecules produced at this stage by semiconservative replication include two that are made entirely of 14N-DNA and 14N /15N-DNA.
Bidirectional Nature of DNA Replication
Once the semi-conservative mode of replication of DNA was confirmed, scientists started asking new questions, e.g. a) is there a discreet region on DNA for initiating DNA replication?, b) is replication unidirectional or bidirectional? and c) Does both DNA strands unwind partially or completely during replication? Answer to these problems were provided by J. Cairns...
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