Replication

Replication is the formation of  the exact carbon copy of a substance. It occurs in case of DNA. Rather replication is an autocatalytic activities of DNA. It takes place during S-phase of cell cycle. Both DNA replication and cell division cycle are highly coordinated. Failure of cell division after DNA replication causes chromosomal doubling.

The phenomenon is called polyploidy. It is a chromosomal anomaly. DNA replication can occur by three possible methods - conservative, disruptive and semiconservative. In conservative replication the parent structure remains intact. The replica is a completely new structure. In disruptive or dispersive replication the parent structure fragments and two new structures are formed afresh. In semiconservative replication, one half of the parent structure passes into each replica while the second half is built anew. The DNA structure was proposed by Watson and Crick in (1953) was based on its semiconservative replications with both the strand of DNA functioning as the templates and producing new complementary chain. The new chains get permanently associated with the templates giving rise to two double stranded replicas.

Context 

1. Replication is Semiconservative

2. Replication is Semidiscontinous

1. Replication is Semiconservative

M. Meselson and F.W. Stahl (1958) grew colonies of Escherichia coli for several generations on a culture medium having ammonium chloride with heavy isotope of nitrogen, 15N. The heavy isotope got incorporated in nitrogen containing compounds including DNA. When the whole DNA was found to contain heavy isotope of nitrogen, bacteria were shifted to culture medium having normal nitrogen, 14N. Generation time is 20 minutes in E.coli. DNA was isolated from the cells of each generation. It was tested for the presence of heavy DNA and normal DNA. For this the isolated DNA was added to tubes having caesium chloride (CsCl). The tubes were provided with centrifugal force of 50,000 revolutions per minute for many hours till DNA came to lie at a fixed position in

the tube. Comparing the heaviness of DNA of each generation, it was found that DNA of the generation having completely heavy isotope was the heaviest. In the first generation after bringing the bacteria on culture having normal nitrogen, DNA settled at a slightly higher level indicating that it has become lighter as compared to DNA of its parent generation. In the second generation (after 40 minutes), DNA formed two sediments, one at a level of DNA of first generation and the second at a still higher level. It is clear that DNA of first generation is intermediate. It contains both 15N and 14N. In the second generation there were two types of DNA, one of intermediate heaviness having 15N/14N and second fraction of lighter DNA having 14N/14N. The ratio of the two types of DNA was 50: 50 in the second generation. In third generation (after 60 minutes) again there were two types of DNA, intermediate and light. However, percentage of intermediate was reduced from 50% to 25% while that of lighter fraction increased from 50% to 75%. In fourth generation (after 80 minutes) the percentage of intermediate fraction decreased further to 12.5% while that of light fraction increased from 75% to 87-5%. All this could happen when in each generation, out of the two strands of DNA, one was obtained from the parent double strand while the second was formed fresh from nucleotides. This proves semiconservative nature of replication.

2. Replication is Semidiscontinous

1. Initiation-Orgin of replication is recognised by origin recognition complex.

It attracts enzymes. Enzyme helicase unwinds the DNA helix and unzips the two strands of DNA by breaking hydrogen bonds. The separated strands become established in this condition with the help of single strand binding proteins or SSBPs.

Unwinding creates tension which is released by cutting and resealing enzymes topoisomerases I and II. Topoisomerase II of prokaryotes is also called gyrase. It functions both as helicase and topoisomerase.

Unzipping creates a Y-shaped configuration called replication fork.

2. Priming. Replication fork exposes two different ends of the two DNA strands, 3' end and 5' end. At the free 3' end of one strand and fork end of the second strand (with free 5' end) a small RNA is synthesised with the help of DNA-dependent enzyme RNA polymerase or primase. The synthesize RNA is called RNA primer. It is 4-12 nucleotide long. RNA primer functions as 5'end of the new strands. It provides 3-OH group for joining of DNA nucleotides. 

3. New Strand Formation. It requires enzyme polymerase III in prokaryotes and polymerase 8 in eukaryotes alongwith source of energy ATP/GTP/TTP and Mg2+. The enzyme helps in establishing phospho-diester linkages between successive nucleotides. This produces polynucleotide chain. Polymerase III or 8 adds nucleotides only at 3' end of the chain so that DNA strand formation occurs in 5' 3' direction. Since 3' ends of the new chains would lie in opposite directions of the separated DNA template strands, chain elongation occurs in opposite directions. The parent DNA strand unzippers further to expose new regions for chain building. However, while DNA synthesis is continuous in one strand, it is formed in small stitches in the other strand because of the opposite arrangement of nucleotides. Where new DNA strand is built in small segments, a new RNA primer is formed everytime in the region of opening of the chain. RNA primers are removed by polymerase I in prokaryotes and polymerase a in eukaryotes. The same enzyme also extends DNA strand in the region exposed by dissolution of RNA primer. Replication is discontinuous over the strand over which only small streches of DNA are built due to opposite running of DNA template. The small streches of DNA are called Okazaki fragments named after the scientist Okazaki who discovered them in 1968. Okazaki fragments are joined together by enzyme DNA ligase (Khorana 1967). New DNA strand built of Okazaki fragments is called lagging strand because it takes longer time to develop. The other replicated strand grows continuously and is called leading strand. Since replication is continuous over one strand and discontinuous over the other, it is called semicontinuous.