Replication in Eukaryotes Lecture Notes PDF

Field of Medicine Medicine And Surgery Program Lecture: Replication in Eukaryotes Dr.Hidi Azmy El Tahawy Date: 27 / 10 /2024  ILOs By the end of the lecture, the student should be able to: 1. Enumerate the difference in DNA replication process between eukaryote...

Field of Medicine Medicine And Surgery Program Lecture: Replication in Eukaryotes Dr.Hidi Azmy El Tahawy Date: 27 / 10 /2024  ILOs By the end of the lecture, the student should be able to: 1. Enumerate the difference in DNA replication process between eukaryotes and prokaryotes : (types of DNA polymerases, …). 2. Comprehend the end replication problem. 3. Describe the molecular mechanism of telomerase.  Replication in Eukaryotes Multiple origins of replication. They are rich in A-T base pairs. Multiple replication forks or multiple replication bubbles. Markedly decreases the time needed for replication. Multiple origin of replication Replication in Eukaryotes  Replication in Eukaryotes 1- Origin Recognition Is achieved by ORC (origin recognition complex). 2- Helicase activity: for unwinding and separation of the two DNA strands. Is achieved by MCM (minichromosome maintenance (complex). 3- Single strand binding (SSB) proteins: to keep the two DNA strands separated and protect the strands from the action of nucleases. Is achieved by RPA (replication protein A).  Replication in Eukaryotes 4- DNA polymerases: DNA polymerase : It contains subunit with primase activity for synthesis of RNA primers & then short DNA stretches connected to the RNA primers. DNA polymerase : for gap filling in DNA repair. DNA polymerase : for mitochondrial DNA synthesis. DNA polymerase : for synthesis of the lagging strand. It has a proofreading activity. In eukaryotes, Okazaki fragments are small (100-200 nucleotides). DNA polymerase : for synthesis of the leading strand. It has proofreading activity. PCNA (proliferating cell nuclear antigen): acts as a sliding clamp.  Replication in Eukaryotes 5- Rnase H and flap endonuclease 1 (FEN1): for removal of RNA primers. 6- DNA Ligase 1: It joins ends of two segments of DNA by catalyzing the formation of phosphodiester bond. 7- DNA Topoisomerases: They catalyze removal of supercoils that are formed during DNA unwinding. 8- Telomerase: for elongation of the 3`-end of telomeres of DNA lagging strand. Replication in Eukaryotes  Replication in Eukaryotes Telomere It is the end of linear chromosome. Consists of a repetitive sequence of T's and G’s. ▪several thousands tandem repeats of non coding sequence 5`-TTAGGG-3` Longer than its complement (single-stranded DNA at the 3'-end of the double helix) Folds back on itself forming a loop that is stabilized by protein Function: 1-protects ends of the chromosomes. 2-Distinguishes the true ends from a break in chromosomes. Replication in Eukaryotes Telomere  Replication in Eukaryotes Problem (Telomere shorting) Replication of 3` end of linear DNA (Eukaryotes) leads to progressive shorting of the chromosome with each cell division. Removing primer leaves gap that can’t be filled by DNA. RNA primer can’t be placed at end of lagging strand. Once telomeres are shortened beyond some critical length, the cell is no longer able to divide, and this may explain cellular aging and death. Cells that do not age (for example, germ-line cells, stem cells and cancer cells) contain an enzyme called telomerase that is responsible for replacing these lost ends. Replication in Eukaryotes End replication problem  Replication in Eukaryotes Telomerase It is a complex that contains a protein (Tert) (Telomerase Reverse Transcriptase) and a short piece of RNA (Terc) (Telomerase RNA Component) that acts as a template(Guide RNA). The RNA molecule is complementary to the telomere sequence at the 3` end of the DNA molecule. Telomerase recognizes single-strand 3` terminus & uses its RNA molecule as a template to elongate parental DNA strand in the usual 5′→3′ direction, extending the already longer 3′-end. Elongated end is used as a template, for completing synthesis of telomere of lagging strand by DNA polymerases (α/primase & δ).  Replication in Eukaryotes Action of Telomerase  Replication in Eukaryotes Telomeres may be viewed as mitotic clocks in that their length in most cells is inversely related to the number of times the cells have divided. The study of telomeres provides insight into the biology of ▪ normal aging, ▪ and cancer.  Which strand would you expect to be shorter at the end of replication?  Summary ▪ Differences between Prokaryotic & Eukaryotic DNA replication. ▪ DNA polymerases in Eukaryotes. ▪ End replication problem in Eukaryotes. ▪ Mechanism of action of telomerase.  References Lippincott's Illustrated Reviews: Biochemistry, 7th Edition. Chapter 30. https://www.youtube.com/watch?v=it8g9RU8KMM