Replication_1 ( LECTURE ON REPLICAATION)

Mechanism of Replication

1. Central Dogma of Molecular Biology DNA codes for RNA, which codes for proteins. 

2. DEFINITION OF GENE https://en.wikipedia.org/wiki/Gene

3. Figure 15.1 Genomes 3 (© Garland Science 2007) DNA replication, as predicted by Watson and Crick. 1. The topological problem was the primary concern During replication, the double helix need to unwind This is the main stumbling block of accepting the double helix model of DNA. Meselson -Stahl confirmed semi-conservative replication predicted by Watson-Crick. The problem was solved when mode of action of DNA Topoisomerase was understood. Three Major Concern of DNA Replication DNA replication research has been driven by three issues

4. Figure 15.1 Genomes 3 (© Garland Science 2007) DNA replication, as predicted by Watson and Crick. 2. Revealed DNA Replication Function of enzymes and proteins involved in replication in E. coli were identified. Understand details of eukaryotic DNA replication Ongoing research on - Initiation of replication - Mode of action of proteins involved in replication fork - Three Major Concern of DNA Replication

5. Figure 15.1 Genomes 3 (© Garland Science 2007) DNA replication, as predicted by Watson and Crick. 3. Regulation of Genome Replication Cell cycle Initiation is the key control point Three Major Concern of DNA Replication

6. Figure 15.2 Genomes 3 (© Garland Science 2007) Three possible schemes for DNA replication The Watson-Crick’s model of DNA structure and replication suggested that once DNA replication is initiated, the two original polynucleotide strands of the duplex or helix will unwind, at least locally, so that each can serve as a template for a new strand. According to this proposal, both duplexes that result from replication should be hybrid in nature , each containing an old strand derived from the original molecule and a new strand which has been formed during the replication process. Since each of the two double helices conserves only one of the parent polynucleotide strands , the process is said to be semiconservative. Conservative replication, in which both strands of parent double helix would be conserved and the new DNA molecule would consist of two newly synthesized strands; and Dispersive replication, in which replication would involve fragmentation of the parent double helix and the intermixing of pieces of the parent strands with newly synthesized pieces , thereby forming the two new double helices .

7. Meselson and F.W. Stahl (1958) verified the semiconservative nature of DNA replication in a series of elegant experiments using isotopically labelled DNA and a form of isopycnic density gradient centrifugation. They cultured Escherichai coli cells in a medium in which the nitrogen was 15N (a ‘heavy’ isotope of nitrogen3333) instead of commonly occurring and lighter 14N. In time, the purines and pyrimidines of DNA in new cells contained 15N (where 14N normally occurs) and, thus, the DNA molecules were denser. DNA in which the nitrogen atoms are heavy (15N) can be distinguished from DNA containing light nitrogen (14N), because during isopycnic centrifugation, the two different DNAs band at different density positions in the centrifuge tube. Meselson and Stahl’s experiment.

8. Figure 15.3a Genomes 3 (© Garland Science 2007) The Meselson–Stahl experiment

9. Figure 15.3b Genomes 3 (© Garland Science 2007) The Meselson–Stahl experiment

10. J.H . Taylor and P. Woods (1957) provided evidence in support of semiconservative mode of DNA replication in eukaryotes by using the technique of autoradiography and light microscopy in dividing root tip cells of the bean, Vicia faba . After incorporation of tritiated thymidine, when root tips were transferred to unlabelled culture medium ( and colchicine was added to the medium to prevent anaphase separation of sister chromatids), in the first generation of duplication both chromatids were labelled (this is interpreted as one DNA double helix in each chromatid and only one of the two strands labelled). In the second cycle of duplication (in the unlabeled medium ) in each chromosome, one of the two chromatids was found to be labelled (Fig. 4.5). This was interpreted as showing semiconservative mode of duplication 2. Evidence for Semi conservative Replication of Chromosomes (or DNA) in Eukaryotes 31/684

11. 3. Semidiscontinuous DNA Replication DNA synthesis is continuous on one strand ( 3' to 5' strand), called leading strand and discontinuous on the other strand (5' to 3' strand), called lagging strand. Since DNA synthesis always proceeds in 5' to 3' direction, so, on the lagging strand , synthesis takes place discontinuously in pieces, called okazaki fragments ( R . Okazaki , 1968). Later on, these pieces are fused with the help of ligase enzyme to form an intact lagging strand. Such a DNA replication, where the leading strand is synthesized continuously and the lagging strand is synthesized discontinuously, is called semidiscontinuous replication.

12. Enzymes of DNA Metabolism Different prokaryotic and eukaryotic cells contain three kinds of nuclear enzymes or enzymatic activities that act on DNA , namely nuclease, polymerases and ligases . Nuclease enzymes The nuclease enzymes act to hydrolyze or break down a polynucleotide chain into its component nucleotides . A polynucleotide is held together by 3´, 5 ´ phosphodiester bonds and a nuclease enzyme will attack either the 3´ or the 5´ end of this linkage .

13. Enzymes of DNA Metabolism The nuclease enzymes may be of the following two kinds : Exonuclease enzymes. A nuclease enzyme which begins its attack from a free end of a polynucleotide is called exonuclease . Depending on the specificity of the enzyme, an exonuclease will either begin at a free 3'-OH end of a polynucleotide and progressively cleave the bonds on the 3'-OH side of the phosphodiester backbone or it will begin at a free 5'-P end and digest the polynucleotide in a 5' →3‘ direction. In both cases the enzyme travels along the chain in a stepwise manner, liberating single nucleoside monophosphate molecules and eventually digesting the entire polymer.

14. (b) Endonuclease enzyme. Endonuclease enzyme also attacks one of the two sides of phosphodiester linkages, but they react only with those bonds that occur within the interior of a polynucleotide chain. If the polynucleotide chain is single stranded ( e.g., viral DNA), such an attack will obviously cut the chain into two pieces. If , however, the polynucleotide strand is a member of a DNA helix (e.g., prokaryotic and eukaryotic DNA ), a single endonucleolytic cut will create a nick in the helix ; the helix remains in one piece but it now possesses a gap that contains two tree ends, which can serve as substrates for exonucleases . A nicked double helix suffers a localized disruption of its secondary structure.

15. Figure 15.4 Genomes 3 (© Garland Science 2007) Mode of action: DNA Topoisomerase Figure 15.4 The mode of action of a Type I DNA Topoisomerase)

16. Table 15.1 Genomes 3 (© Garland Science 2007) DNA Topoisomerases

17. Figure 15.5 Genomes 3 (© Garland Science 2007) Unzipping the Double Helix

18. Figure 15.6 Genomes 3 (© Garland Science 2007) Displacement Replication D-loop contains a short RNA molecule that primes DNA synthesis After completion of the first strand synthesis a second RNA primer attaches to the displaced strand and initiates replication of this molecule. In this diagram, newly synthesized DNA is shown in red.

19. Figure 15.7 Genomes 3 (© Garland Science 2007) Used for replication of a circular genome. E.g., λ and various bacteriophages. It initiates at a nick which is made in one of the parent polynucleotides. The free 3´ end that results is extended, displacing the 5´ end. Continued DNA synthesis “rolls off” a complete copy of the genome, and further synthesis eventually results in a series of genomes linked head to tail These genomes are single stranded and linear, but can easily be converted to double-stranded circular molecules by complementary strand synthesis. The Rolling Circle Replication

20. Figure 15.8a Genomes 3 (© Garland Science 2007) The Replication Process Initiation: Recognition the site of DNA Elongation: Parental Nucleotides copied Termination: Replication completed