5 Replication of DNA

5 REPLICATION OF DNA In the laboratory, exposing the two DNA strands of the double helix to high temperatures or to certain chemicals can break the hydrogen bonds between complementary bases, thus separating the strands into two separate single strands of DNA (single-stranded DNA [ssDNA]). This process is called DNA denaturation and is analogous to protein denaturation. The ssDNA strands can also be put back together as double-stranded DNA (dsDNA), through reannealing or renaturing by cooling or removing the chemical denaturants, allowing these hydrogen bonds to reform. The ability to artificially manipulate DNA in this way is the basis for several important techniques in biotechnology. Because of the additional hydrogen bonding between the C = G base pair, DNA with a high GC content is more difficult to denature than DNA with a lower GC content. Fig: In the laboratory, the double helix can be denatured to single-stranded DNA through exposure to heat or chemicals, and then renatured through cooling or removal of chemical denaturants to allow the DNA strands to reanneal. The elucidation of the structure of the double helix by James Watson and Francis Crick in 1953 provided a hint as to how DNA is copied during the process of replication. Separating the strands of the double helix would provide two templates for the synthesis of new complementary strands, but exactly how new DNA molecules were constructed was still unclear. In one model, semiconservative replication, the two strands of the double helix separate during DNA replication, and each strand serves as a template from which the new complementary strand is copied; after replication, each double-stranded DNA includes one parental or “old” strand and one “new” strand. There were two competing models also suggested: conservative and dispersive. Fig: There were three models suggested for DNA replication. In the conservative model, parental DNA strands (blue/gray) remained associated in one DNA molecule while new daughter strands (red/black) remained associated in newly formed DNA molecules. In the semiconservative model, parental strands separated and directed the synthesis of a daughter strand, with each resulting DNA molecule being a hybrid of a parental strand and a daughter strand. In the dispersive model, all resulting DNA strands have regions of doublestranded parental DNA and regions of double-stranded daughter DNA.  The Watson-Crick model of DNA immediately suggested that the two strands of DNA would separate. Fig: Conservation of Sequence by Complementary Base Pairing  Each separated or parent strand serves as a template (model or guide) for the formation of a new but complementary strand.   Thus, the new or daughter DNA molecules formed would be made of one old or parental strand and another newly formed complementary strand. This method of formation of new daughter DNA molecules is called semi-   conservative method of replication. The following experiment suggests that DNA replication is semi-conservative. Messelson and Stahl (1958) conducted experiments using heavy nitrogen (15N) to  determine whether the concept of semi-conservative replication is correct. They used Cesium chloride (CsCl) gradient centrifugation technique for this purpose.   A dense solution of CsCl, on centrifugation, forms density gradient-bands of a solution of lower density at the top that increases gradually towards bottom with highest density. If DNAs of different densities are mixed with CsCl solution, these would separate from  one another and would form a definite density band in the gradient along with CsCl solution. Messelson and Stahl created DNA molecules of different densities by using normal  nitrogen N and its heavy isotope 15N. For this purpose, Escherichia coli was grown in 15NH4Cl containing culture medium 14 for many generations, so that bacterial DNA become completely heavy.  This non-radioactive or heavy DNA (incorporating 15 N) had more density than DNA 14  with normal nitrogen ( N). The bacteria were then transferred to culture medium containing only normal nitrogen (14NH4Cl). The change in density was observed by taking DNA samples periodically. Fig: (Separation of DNA by Centrifugation) Messelson and Stahl's Experiment  If DNA replicates semi-conservatively, then each heavy (15N) DNA strands should separate and each separated strand should acquire a light (14N) partner after one round  of replication. This should be a hybrid DNA made of two strands, i.e., 14N–15N.  Meselson and Stahl observed that such DNA was actually half dense indicating the presence of hybrid DNA molecules.  After second round of replication there would be four DNA molecules.  Of these, two molecules would be hybrid (14N–15N) showing half density as earlier and  the remaining two molecules would be made of light strands (14N–14N) Thus, after second generation, the same half dense band (14N–15N) was seen but the density of light bands (14N–14N) increased. Messelson and Stahl's work as such provided confirmation of Watson-Crick model of   DNA and its semi-conservative replication. Taylor proved semiconservative mode of chromosome replication in eukaryotes using tritiated thymidine in the root of Vicia faba (faba beans). Cairns proved semiconservative mode of replication in E. coli by using tritiated thymidine (H3–tdR) in autoradiography experiment.  He proposed model for replication in circular DNA. Mechanism of DNA Replication: DNA replication involves following four major steps: (1) Initiation of DNA replication (2) Unwinding of helix (3) Formation of primer strand (4) Elongation of new strand 1. Initiation of replication.  Replication of DNA always begins at a definite site called origin of replication.  Prokaryotes have single origin of replication.  It is called ori-c in E.coli.  On the other hand, eukaryotes have several thousand origins of replication. 2. Unwinding of helix.      DNA replication requires that a double helical parental molecule is unwound so that its internal bases are available to the replication enzymes. Unwinding is brought about by enzyme helicase which is ATP dependent. Unwinding of DNA molecule into two strands results in the formation of Y-shaped structure, called replication fork. These exposed single strands are stabilised by a protein known as Single-strand binding protein (SSB). Due to unwinding, a supercoiling develops on the end of DNA opposite to replicating fork. This tension is released by enzyme topoisomerase. 3. Formation of primer strand.   A new strand is to be synthesized opposite to the parental strands DNA polymerase III is the true replicase in E. coli, which is incapable of initiating DNA synthesis, i.e., it is unable to deposit the first nucleotide in a daughter (new) strand without the Primer. Another enzyme, known as primase, strand synthesizes a short primer strand of RNA.  The primer strand then serves as a stepping stone (to start errorless replication).  Once the initiation of DNA synthesis is completed, this primer RNA strand is then removed enzymatically. 4. Elongation of new strand.  Once the primer strand is formed, DNA replication occurs in 53 direction, i.e., during synthesis of a new strand, deoxyribonucleoside triphosphates (dATP. dGTP, dTTP, dCTP) are added only to the free 3OH end. Thus, the nucleotide at 3 end is always the most recently added nucleotide to the chain.  As the DNA replication proceeds on the two parental strands, synthesis of daughter or new strand occurs continuously along the parent 3'5'strand. It is now known as leading daughter strand.  Fig: At the origin of replication, topoisomerase II relaxes the supercoiled chromosome. Two replication forks are formed by the opening of the double-stranded DNA at the origin, and helicase separates the DNA strands, which are coated by single-stranded binding proteins to keep the strands separated. DNA replication occurs in both directions. An RNA primer complementary to the parental strand is synthesized by RNA primase and is elongated by DNA polymerase III through the addition of nucleotides to the 3’-OH end. On the leading strand, DNA is synthesized continuously, whereas on the lagging strand, DNA is synthesized in short stretches called Okazaki fragments. RNA primers within the lagging strand are removed by the exonuclease activity of DNA polymerase I, and the Okazaki fragments are joined by DNA ligase.  Synthesis of another daughter strand along the other parental strand, however, takes place in the form of short pieces.   This is called lagging daughter strand. These short pieces of DNA are known as Okazaki fragments, these segments are about 1,000-2,000 nucleotides long in prokaryotes. Hense, DNA replication is semidiscontinuous. Discontinuous pieces of the lagging strand are joined together by the enzyme DNA ligase (after removal of primer) to form   continuous daughter strand. Thus two DNA molecules are now formed from one molecule. Each of these daughter DNA molecules is made of two strands, of which one is old (parental) and other one is new or complementary strand.   DNA polymerase is the most important enzyme of DNA replication. DNA polymerases are of three types i.e. DNA polymerase I, II and III in prokaryotes. Fig: DNA Replication in Eukaryotes : Eukaryotic chromosomes are typically linear, and each contains multiple origins of replication.