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Eukaryotic DNA Replication: Key Similarities and Differences

Explore the intricate process of DNA replication in eukaryotes, comparing it to E. coli replication, highlighting the roles of polymerases, primers, and histones. Discover the importance of telomeres, telomerase, and inhibitors in maintaining DNA integrity.

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Eukaryotic DNA Replication: Key Similarities and Differences

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  1. DNA replication in eukaryotes • Similarities with E.coli replication • Polynucleotide chains are made in the 5’  3’ direction • Require a primer (RNA). • Similarities with the E Coli DNA Pol active site and tertiary structure • Differences • Eukaryotic replication is much slower (only 100 nt/sec). • Many replication origins. • DNA is associated with histones. • DNA Polymerases are more specialized, and their interactions • are more complex. • Chromosomal DNA is linear -> requires special processing of • the ends.

  2. Eukaryotic DNA has many replication origins

  3. Cell Cycle DNA replication (6-8 h)

  4. Eukaryotic DNA polymerases

  5. Analogy between bacterial and eukaryotic proteins involved in DNA replication Bacteria Eukaryotes SSB RPA Pol I polymerase Pol a Pol III polymerase Pol d b2 subunit of Pol III PCNA 3’ exonuclease of Pol I RnaseH + FEN1 g subunit of Pol III RCF RPA = Replication protein A PCNA = proliferating cell nuclear antigen FEN1 = flap endonuclease

  6. Lagging strand synthesis in eukaryotes RNA primer 5’ (a) RPA=Replication protein A RPA Pola/primase 10-30 nt (b) 5’ PCNA RCF RCF = clamp loader PCNA = sliding clamp (c) 5’ Pold 5’ (d) Rnase H/FEN1 RnaseH = 5’-nuclease FEN1 = flap endonuclease (e) ligase (f)

  7. 5‘… 3' 3‘… 5' Telomerase preserves chromosomal ends • The ends of the linear DNA strand cannot be replicated due to the lack of a primer • This would lead to shortening of DNA strands after replication RNA primer • Solution: the chromosomal ends are extended by DNA telomerase This enzyme adds hundreds of tandem repeats of a hexanucleotide (AGGGTT in humans) to the parental strand: 5‘… AGGGTTAGGGTTAGGGTT… 3' 3‘… 5' telomere 5‘… AGGGTTAGGGTTAGGGTT… 3' 3‘… 5' TCCCAATCCCAATCCCAA…

  8. 5‘… 3' 3‘… 5' Circular DNA does not have ends: Upstream Okazaki fragment RNA primer Linear DNA: RNA primer

  9. Telomerase is a reverse transcriptase that uses it own RNA as a template for elongation of the 3’ end of DNA

  10. Telomerase mechanism

  11. Telomerase mechanism - continued

  12. Telomeres form G-tetraplex structures G G G G

  13. Telomerase inhibitors • Telomerase RNA as a target for antisense drugs • G-tetraplexes at chromosomal ends as a drug target. Modified oligonucleotides that hybridize with telomerase RNA, preventing it from being used as a template for telomere synthesis. Porphyrins, anthraquinones: stabilize G-tetraplex structure, inhibit telomerase activity.

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