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DNA replication in eukaryotes

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

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DNA replication in eukaryotes

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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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