Replication in eukaryotes

Replication in eukaryotes

1. DNA REPLICATION IN EUKARYOTES DR. ANU P. ABHIMANNUE ASSISTANT PROFESSOR, DEPARTMENT OF BIOTECHNOLOGY, ST.MARY’S COLLEGE, THRISSUR.

2. REPLICATION Replication is the process by which DNA copies itself to produce identical daughter molecules of DNA. https://www.genome.gov/genetics-glossary/DNA-Replication 2 ANU P A, ST. MARY'S COLLEGE, THRISSUR

3. EUKARYOTIC REPLICATION Eukaryotic genome is relatively complex and their polymerases incorporate nucleotides at much slower rates. To overcome this, replication in eukaryotes occur in small portions, termed replicons . In a human cell, there are about 10,000 to 100,000 replicons . 3 ANU P A, ST. MARY'S COLLEGE, THRISSUR

4. REPLICONS IN EUKARYOTES Replicon was defined by François Jacob; sydney Brenner and François Cuzin . It is the unit in which the cell controls individual acts of replication. 4 https://www.biology-pages.info/D/DNAReplication.html ANU P A, ST. MARY'S COLLEGE, THRISSUR

5. INITIATION 5 ANU P A, ST. MARY'S COLLEGE, THRISSUR

6. ENZYMES INVOLVED Sl no: Protein in initiation Role of protein 1 ORC Proteins (Multi protein complex) Recognition of origin of replication in eukaryotes 2 Cdc6, Cdt1 Assist loading of helicase enzyme onto the DNA 3 Mcm ( Mcm 2 – Mcm 7) DNA helicase enzyme that unwinds the double stranded DNA into single strands. Also called “licensing factors” 4 Replication Protein A (RPA) Binds to the exposed single stranded DNA molecule and prevents its rewinding. 5 Topoisomerase I/II Relieves positive super coils located ahead of the replication fork. 6 ANU P A, ST. MARY'S COLLEGE, THRISSUR

7. AUTONOMOUS REPLICATING SEQUENCES Specific site on DNA that functions like origin of replication Autonomous Replicating Sequences (ARS) consists of a conserved sequence of 11 base pairs. A protein complex called Origin Recognition Complex (ORC) binds DNA at ARS in a specific manner 7 ANU P A, ST. MARY'S COLLEGE, THRISSUR

8. 8 MECHANISM ANU P A, ST. MARY'S COLLEGE, THRISSUR

9. 9 ANU P A, ST. MARY'S COLLEGE, THRISSUR

10. ELONGATION 10 ANU P A, ST. MARY'S COLLEGE, THRISSUR

11. PROTEINS Sl no Protein in elongation Role of protein 1 Primase Synthesizes RNA primers 2 RFC Subunits of DNA polymerase holoenzyme that load the clamp onto the DNA 3 Pol δ/ε Primary replicating enzymes; synthesize entire leading strand and Okazaki fragments; have proofreading capability 4 PCNA Ring-shaped subunit of DNA polymerase holoenzyme that clamps polymerase to DNA 5 pol α Synthesizes short DNA oligonucleotides as part of RNA–DNA primer 11 ANU P A, ST. MARY'S COLLEGE, THRISSUR

12. DNA POLYMERASES Eukaryotic cells contain five DNA polymerases DNA polymerase α DNA polymerase β DNA polymerase γ DNA polymerase δ DNA polymerase ε 12 ANU P A, ST. MARY'S COLLEGE, THRISSUR

13. DNA POLYMERASES - FUNCTIONS Polymerase γ is in mitochondria and replicates mitochondrial DNA. Polymerase β is required for DNA repair. polymerases α, δ and ε have replicative functions. Polymerase α is associated with primase , and initiate synthesis of Okazaki fragment by adding 20 deoxyribonucleotides to the primer. Polymerase δ and ε is the primary DNA-synthesizing enzyme in the lagging strand and leading strand respectively. 13 ANU P A, ST. MARY'S COLLEGE, THRISSUR

14. DNA POLYMERASE The structure of eukaryotic and prokaryotic Polymerase enzyme is quite similar. And consists of Sliding clamp called Proliferating Cell Nuclear Antigen (PCNA). clamp loader that loads PCNA onto DNA is called Replication Factor C (RFC). 14 ANU P A, ST. MARY'S COLLEGE, THRISSUR

15. MECHANISM 15 ANU P A, ST. MARY'S COLLEGE, THRISSUR

16. POLYMERIZATION Eukaryotic polymerases elongate DNA strands in the 5 ' →3 ' direction by the addition of nucleotides to a 3 hydroxyl group, and none of them is able to initiate the synthesis of a DNA chain without a primer. Eukaryotic DNA polymerase is present as a dimer , suggesting that the leading and lagging strands are synthesized in a coordinate manner by a single replicative complex, or replisome 16 ANU P A, ST. MARY'S COLLEGE, THRISSUR

17. SEMI DISCONTINUOUS Leading strand in 5'→3 ‘ is continuous whereas lagging strand in the opposite direction is dis -continuous Okazaki fragments are formed in the lagging strand. Okazaki fragments are smaller than in bacteria, averaging about 150 nucleotides in length. 17 ANU P A, ST. MARY'S COLLEGE, THRISSUR

18. TERMINATION 18 ANU P A, ST. MARY'S COLLEGE, THRISSUR

19. PROTEINS Sl no Protein in termination Role of protein 1 RNase H Cut RNA primers 2 Flap endonuclease 1 (FEN1) Removes RNA primers; functions similar to pol I of E. coli 3 DNA ligase Seals Okazaki fragments into continuous strand 19 ANU P A, ST. MARY'S COLLEGE, THRISSUR

20. MECHANISM 20 ANU P A, ST. MARY'S COLLEGE, THRISSUR

21. REFERENCES Gerald Karp (2010). Cell and molecular biology: concepts and experiments (6 th ed.). John Wiley & sons. ISBN-13 978-0-470-48337-4. https ://bio.libretexts.org/Bookshelves/Genetics/Book%3A_Working_with_Molecular_Genetics_(Hardison)/Unit_II%3A_Replication%2C_Maintenance_and_Alteration_of_the_Genetic_Material/6._DNA_replication_II%3A_Start%2C_stop_and_control/6.1%3A_The_Replicon 21 ANU P A, ST. MARY'S COLLEGE, THRISSUR