DNA Replication PDF

III-DNA SYNTHESIS (REPLICATION)  Time of DNA replication: S phase of cell cycle  Function of replication: transfer genetic information to the daughter cells  Def of Replication: The production of two daughter DNA molecules.  The newly formed DNA molecules contain one of t...

III-DNA SYNTHESIS (REPLICATION)  Time of DNA replication: S phase of cell cycle  Function of replication: transfer genetic information to the daughter cells  Def of Replication: The production of two daughter DNA molecules.  The newly formed DNA molecules contain one of the original strands and a new complementary strand. This process is called semi conservative replication. Steps of eukaryotic replication I- Separation of the Two DNA Strands A- Opening of DNA at multiple Origin of replication:  Starting points are many origins across the chromosomes (rich in AT base pairs).  These origins are recognized by origin recognition complex (ORC) proteins.  ORC proteins bind to the replication origins and produce local opening and unwinding of DNA double helix.  The DNA segments between any two origins of replication are termed replicons.  The presence of multiple replication origins markedly decreases the time needed for replication from a month to a few hours. B- Formation of Two Replication Forks in each replication origin  Helicase enzyme unwind DNA by breaking the hydrogen bonds between the nitrogenous base pairs forming (replication bubbles).  Single strand binding (SSB) proteins - They bind to the single strand of DNA and stabilize the single strands. - They bind cooperatively and prevent rewinding of DNA - Also, they protect the single strand from nucleases that cleave single- stranded DNA.  By the action of helicase and SSB proteins, a replication fork is created. II- Initiation of DNA Synthesis: Formation of RNA Primers  At the replication fork, both strands of parental DNA serve as templates for DNA synthesis.  To initiate DNA synthesis, this requires an RNA primer (DNA polymerases cannot start from scratch).  RNA primer is formed by the action of DNA polymerase α-primase complex.  The 5'- end of the primer is attached to the 3'- end of the parental strand, and the 3'- end of the primer will accept the first deoxynucleotide. 15  III- Elongation: Synthesis of Both DNA Leading and Lagging Strands  DNA polymerases are template-directed enzymes.  They read the parental nucleotide sequence in 3'to 5 direction, thus synthesize the new strands in 5 to 3 direction.  Synthesis of the leading and lagging strand A- Leading strand B- Lagging strand It is synthesized by DNA polymerase It is synthesized by DNA polymerase ε δ It is copied in the same direction of the It is synthesized in the opposite replication fork. direction of replication fork It requires only one primer It requires multiple RNA primers It is synthesized continuously It is synthesized discontinuously in the form of short Okazaki fragments The length of these Okazaki fragments ranges from 100 to 200 bases C- Proofreading of newly synthesized DNA strands:  DNA polymerases ε and δ have proof-reading activity.  They hydrolytically remove the misplaced nucleotide (act as exonuclease) and replace it with the correct nucleotide. D- Removal of DNA supercoiling  When DNA unwinds, supercoils are formed during DNA replication. They are removed by DNA topoisomerases. Topoisomerase I Topoisomerase II It breaks phosphodiester bond in one It makes transient break in both DNA strand (produce cut or nick) strands And reseals the break It allows DNA to rotate around the other strand, then reforms phosphodiester bond IV- Termination: Removal of Primers and Joining the Fragments A- Removal of the primers:  RNase H removes the RNA primers by its exonuclease activity then it fills the gaps between Okazaki fragments by DNA polymerase δ. B- Joining of DNA fragments DNA ligase  DNA ligase joins the ends of adjacent fragments Restoring the chromosomal end by telomerase enzyme  Telomeres are the ends of the eukaryotic linear chromosomes.  Telomere consist of thousands copies of 5`-TTAGGG-3` repeats of the parent strand. 16   DNA at the parent 3'-end is a few hundred nucleotides longer than the complement lagging strand forming single stranded region.  The single-stranded region folds back on itself forming a structure that is stabilized by protein to prevent stacking of chromosomes and protect the ends of the chromosomes from nucleases  During the process of replication, the leading strand can be synthesized to the end of the parent strand while the lagging strand cannot because: 1) The primase cannot act at the 3'end of the strand due to the narrow space. 2) Also, if primase acts, the removal of the RNA primer would leave a short gap.  Cellular aging and death in differentiated cells occur when the ends of their chromosomes get slightly shorter with each cell division until the telomeres are gone, and DNA is degraded.  Cells that do not age (for example, germ-line cells and cancer cells) contain an enzyme called telomerase that maintain telomere ends.  Telomerase enzymes restore chromosomal length.  Telomerase is a reverse transcriptase enzyme that can synthesize DNA complementary to the short RNA molecule present in its structure that act as a template.  Telomerase recognizes elongate the 3' end of parent strand by about 100 nucleotides, so that it allows the binding of RNA primer and complete synthesis of the lagging strand to the end N.B: a- Cells that have differentiated and no longer divide do not express telomerase. b- Cells that do not age (e.g., germ-line cells and cancer cells) contain telomerase that is responsible for replacing these lost ends. c- Telomerase expression is reactivated in tumor cells. This makes telomerase an attractive target in cancer chemotherapy. Summary of Enzymes of Eukaryotic Replication 1- DNA helicases: for unwinding and separation of the two DNA strands. 2- Single strand binding proteins: to keep the two DNA strands separated. 3- DNA polymerases: - DNA polymerase -primase complex: for synthesis of RNA primers and short DNA part connected to the RNA primers. - DNA polymerase : for DNA repair. - DNA polymerase : for mitochondrial DNA synthesis. - DNA polymerase : for synthesis of the lagging strand. - DNA polymerase ƹ: for synthesis of the leading strand 4- RnaseH: for removal of RNA primers 5- Topoisomerases: for removal of positive supercoils. 6- Telomerase: for elongation of the 3`-end of telomeres of DNA. 17  DNA Repair  DNA replication is very accurate but error can occur for every 30,000 bases, or it may be subjected to radiations and chemical that cause damage to DNA  If the damage not repaired mutation occurs and result in hereditary diseases.  Steps of repair:  Recognition of lesion by endonuclease which form a cut in the damaged strand  Removal of damaged part by excision exonuclease  Fill the gap with repair DNA polymerase  Ligation by DNA ligase hereditary DNA repair disorders: 1. Xeroderma pigmentosum: hypersensitivity to sunlight (UV light) leading to increased skin cancer, premature aging and death. 2. Ataxia telangiectasia: sensitivity to ionizing radiation and some chemical agents. 3. Werner's syndrome: premature aging and retarded growth 3` 5` 3` 5` Leading strand Elongated leading strand DNA helicase DNA helicase RNA primer 3` Progress of 3` DNA Replication replication fork polymerase III Fork New Okazaki New RNA primer Okazaki 5` fragment 5` fragment Single stranded DNA binding proteins 3` Lagging strand 5` Lagging strand RNA primer 3` Removal of primer and filling of gaps between 5` two fragments by DNA polymerase I and ligation of the two fragments by DNA ligase 18