Dna replication

Dna replication presentation

1. Genome Replication By- Sanju Sah St. Xavier’s College, Maitighar, Kathmandu Department of Microbiology

2. What is Replication? In DNA replication two identical daughter DNA molecules are formed from a single DNA molecule This unique feature of DNA no other biological molecule do so.

3. DNA replication  is the process of producing two identical copies of DNA from one original DNA molecule DNA is made up of a double helix of two complementary strands During replication, these strands are separated and each strand of the original DNA molecule serves as a template for the production of its counterpart, a process referred to as  semiconservative replication. As a result of semi-conservative replication, each of the new double helix will be composed of an original DNA strand as well as a newly synthesized strand.

4. DNA Replication First question of discussion was whether the DNA replication is Semiconservative or Conservative? Semiconservative replication Watson and crick model One strand from parent DNA molecule and another is newly formed Conservative replication Both strand are in one molecule are from Parent and newly formed DNA molecule comprising both newly synthesized DNA strand. Dispersive Replication Both strands contains the DNA from parent molecule and newly synthesized DNA.

5. Watson and Crick Model of Replication During DNA replication each of two strand serve as template. DNA replication is semiconservative i.e. Newly formed DNA molecule consist of one strand from parent molecule and another strand is newly synthesized.

6. Messelson-Stahl experimetn to confirm the Semiconservative model . This experiment allowed the differentiation of newly synthesized DNA strand and parental DNA strand. E. coli was grown in the media containing either normal isotope of nitrogen ( 14 N) or heavy isotope ( 15 N). DNA was extracted from both of the condition and was performed density gradient centrifugation. - Heavy DNA was at higher density than normal DNA. Bacteria grown in 15 N containing media were transferred to 14 N containing media. Newly synthesized DNA strand had 14 N DNA strand while parental strand had 15 N DNA

7. Messelson-Stahl experiment contd... They checked the composition of resulting DNA molecule by density gradient centrifugation. - Found an intermediate band - Indicating a hybrid molecule - Containing both 14 N and 15 N DNA molecule

8. Semi-discontinuous model of DNA replication Replication of DNA occurs by forming the complementary strand to both of DNA strand, thus both of the DNA strand acts as the template strand. Two strand of the DNA are anti-parallel to each other, one strand runs in 5’-3’ direction where as complementary strand runs in 3’-5’ direction. Replication is always in 5’-3’ direction. During the replication the DNA strand which runs in 3’-5’ direction is replicated in continuous manner while the DNA strand running in 5’-3’ direction is replicated in discontinuous manner. DNA strand replicated in continuous way is known as the leading strand and DNA strand which is replicated in discontinuous manner is known as lagging strand.

9. Only one complementary strand is formed while replicating leading strand while a number of fragments are formed while replicating lagging strand. These fragments are 1000-2000 nt. long and are known as Okazaki fragments. These Okazaki fragments are joined together later on by DNA ligase to form a complete DNA strand.

10. Cell Cycle

11. Mechanism of replication Replication involves three step: 1)Initiation 2)Elongation 3)Termination

12. Enzymes and proteins of DNA replication Nuclease DNA polymerase DNA ligase Primase Helicase Single strand DNA binding protein (SSBPs) Topoisomerase (DNA gyrase)

13. Nuclease A  nuclease  is an enzyme capable of cleaving the phosphodiester bonds between monomers of nucleic acids There are two primary classifications based on the locus of activity.  Exonucleases digest nucleic acids from the ends.  Endonucleases  act on regions in the  middle  of target molecules. They are further sub divided into deoxyribonucleases (Dnase) and ribonucleases (Rnase). The former acts on DNA, the latter on RNA Exonucleases are of two types 5 l 🡪3 l exonuclease and 3 l 🡪5 l exonuclease

14. A  restriction enzyme  or  restriction endonuclease  is an enzyme that cleaves DNA into fragments at or near specific recognition sites within the molecule known as restriction sites To cut DNA, all restriction enzymes make two incisions, once through each sugar-phosphate backbone (i.e. each strand) of the DNA double helix Restriction enzymes recognize a specific sequence of nucleotides and produce a double-stranded cut in the DNA. The recognition sequences can also be classified by the number of bases in its recognition site, usually between 4 and 8 bases

15. DNA polymerase DNA polymerases  are enzymes that carry out synthesis of DNA molecules using deoxyribonucleotides Prokaryotic DNA polymerase Three major DNA polymerases are known in E.coli and includes DNA polymerase I, II and III DNA polymerase I is repair polymerase with 5‘🡪3' polymerase and both 3‘🡪5' and 5‘🡪3' exonuclease activity. It is involved in processing of  Okazaki fragments  generated during lagging strand synthesis DNA polymerase II has 3‘🡪5' exonuclease activity and participates in DNA repair with 5‘🡪3' polymerase activity

16. DNA polymerase III is a multimeric  holoenzyme having 10 subunits and is the primary enzyme involved in DNA replication in  E. coli. It has 3‘🡪5' exonuclease activity and 5‘🡪3' polymerase activity Prokaryotic DNA is replicated by DNA polymerase III in the 5′ to 3′ direction at a rate of 1000 nucleotides per second. Subunits includes alpha (α), beta (β), epsilon (ε), theta (θ), tau (τ), gamma(γ), delta (δ), delta dash (δ l ), chi (χ) and psi (ψ) Core enzyme includes alpha (α), beta (β) and theta (θ)

17. Eukaryotic DNA polymerases

18. DNA helicase They are motor proteins that move directionally along a nucleic acid phosphodiester backbone, separating two annealed nucleic acid strands (i.e., DNA, RNA, or RNA-DNA hybrid) and establish replication fork using energy derived from ATP hydrolysis, a process characterized by the breaking of hydrogen bonds between annealed nucleotide bases

19. DNA Topoisomerases DNA Topoisomerases are a class of enzymes that release helical tension during transcription and replication, by creating transient nicks within the phosphate backbone on one or both strands of the DNA  Class I DNA topoisomerase break one strand of DNA helix Produce temporary single strand breaks in DNA one at a time Class II DNA topoisomerase break both strand of DNA helix

20. DNA primase DNA primase  is an enzyme involved in the replication of DNA and is a type of RNA polymerase. Primase catalyzes the synthesis of a short RNA (or DNA in some organisms) segment called a primer complementary to a ssDNA template. Primase is of key importance in DNA replication because no known replicative DNA polymerases can initiate the synthesis of a DNA strand without an initial RNA or DNA primer (for temporary DNA elongation). After this elongation the RNA piece is removed by a 5' to 3' exonuclease and refilled with DNA. In bacteria, primase binds to the DNA helicase forming a complex called the primosome. Primase is activated by DNA helicase where it then synthesizes a short RNA primer approximately 11 ±1 nucleotides long, to which new nucleotides can be added by DNA polymerase.

21. DNA ligase DNA ligase  is a specific type of enzyme, a ligase, that facilitates the joining of DNA strands together by catalyzing the formation of a phosphodiester bond. The mechanism of DNA ligase is to form two covalent phosphodiester bonds between 3' hydroxyl ends of one nucleotide ("acceptor"), with the 5' phosphate end of another ("donor") at expense of ATP

22. Terms used in DNA replication Replicon  is a DNA molecule, or a region of DNA, that replicates from a single origin of replication For most prokaryotic chromosomes, the replicon is the entire chromosome For eukaryotic chromosomes, there are multiple replicons per chromosome Replisome  is a complex molecular machine that carries out replication of DNA and includes several enzymes and proteins such as  helicase, topoisomerase, SSB, primase, DNA polymerase, RNAse, ligase etc

23. DNA replication in Prokaryotes In Prokaryotes, DNA replication occurs principally by three methods Theta mode Sigma mode Linear mode

24. References: Bruce Albert: Molecular biology of cell. Lodish et al, Molecular cell Biology, 5 th edition R. Weaver, Molecular biology, 2 nd edition