PTH241 DNA Replication - Fall 2024 - Galala University PDF

PTH241 Lect 5 DNA Replication Hanan H. Fouad Professor of Medical Biochemistry & Molecular Biology Fall 2024 Galala University gu.edu.eg  G A L A L A U N I V E R S I T...

PTH241 Lect 5 DNA Replication Hanan H. Fouad Professor of Medical Biochemistry & Molecular Biology Fall 2024 Galala University gu.edu.eg  G A L A L A U N I V E R S I T Y T H E F U T U R E S T A R T S H E R E PTH241 DNA Replication Hanan H. Fouad Professor of Medical Biochemistry & Molecular Biology Galala University T H E F U T U R E S T A R T S H E R E 10/28/2024  Intended Learning Outcomes By the end of this Practical Session, you should be able to: 1. Define the flow of genetic information. 2. Understand basic facts of DNA replication. 3. Identify types of enzymes and protein factors involved in DNA replication 4. Explain differences between replication in eukaryotes and prokaryotes 5. Discuss function of telomerase enzyme  Figures to understand and NOT to memorize them Study text associated Prof. Hanan Fouad with figures  Central Dogma of Molecular Biology It describes the flow of information DNA from DNA to DNA RNA to protein. Transcription It is valid in all Semiconservative replication organisms, with mRNAs tRNAs rRNAs the exception of some viruses that Translation have their genetic Protein information stored in RNA 10/28/2024 Prof. Hanan Fouad DNA SYNTHESIS (REPLICATION) Genetic information stored in DNA in the form of nucleotide sequence flows from DNA to DNA (replication) DNA to RNA (transcription). from RNA to protein (translation) The major function of replication is to provide genetic CODE required by daughter cell from parent cell Each parental strand of the DNA molecule acts as a template for the formation of a complementary new DNA strand.  Therefore, each of the daughter DNA molecules is composed of one original (conserved) strand and one newly synthesized strand. This is called semi-conservative replication 10/28/2024  10/28/2024 Prof. Hanan Fouad  Overall direction of replication From the origin in both directions towards the replication fork Prof. Hanan Fouad  In prokaryotes single replication fork 10/28/2024 Prof. Hanan Fouad  There are four origins of replication in the fork and all of them move towards the replication fork 10/28/2024 Prof. Hanan Fouad  Replication in Prokaryotes (E- Coli)  Replication in E. Coli starts at a unique origin (REPLICATION FORK, proceeds in opposite directions simultaneously Enzymes of DNA replication  1. DNA Polymerases DNA polymerases catalyze the formation of polynucleotide chains, synthesize the new DNA strand in 5` to 3` direcrion DNA polymerase needs RNA primer  2. DNA Ligase It joins ends of two segments of DNA  3. DNA Helicase It catalyzes unwinding of DNA double helix 10/28/2024 (removal of helix) Prof. Hanan Fouad Prof. Hanan H. Fouad 10/28/2024  4. DNA Topoisomerases Removes positive supercoils. Topoisomerases I make transient break in one DNA strand Topoisomerases II make transient break in two DNA strands Steps of Prokaryotic Replication I- Origin of replication is designated as ori C. It acts as binding sites for DNA binding proteins (dna A) Binding of dna A protein to ori C produces local opening and unwinding of DNA double helix 10/28/2024 Prof. Hanan Fouad  II- Formation of Two Replication forks  A complex of dna B and dna C also binds to ori C to open the duplex DNA, dna B is a helicase  Single strand binding proteins (SSBP) bind to the single strands of unwound portion of DNA duplex and stabilize the single strands  By the action of helicase and SSBP, a replication fork is created  Removal of positive supercoils by type I DNA topoisomerase  III- Synthesis of Both DNA Strands (Leading and Lagging Strands)  At the replication fork, both strands of parental DNA serve as templates for the DNA synthesis  To initiate DNA synthesis, RNA primer is formed by the action of primase, a DNA-dependent-RNA-polymerase  These primers are elongated by DNA polymerase III  DNA-polymerase Prof. Hanan H. Fouad III synthesizes both strands of DNA at replication fork 10/28/2024 connected to 3` end of RNA primers. Proteins Involved in the Replication Fork Functions of the Proteins involved Unwinding the double helix ahead of the advancing replication fork. Maintaining the separation of the parental strands * DnaA protein & DNA Helicase Recognize the origin of replication and unwind the double helix * Single stranded DNA-binding proteins 1. Keep the 2 strands of DNA separate 2. Protect the DNA from nucleases that degrade ssDNA Proteins Involved in the Replication Fork DNA polymerase I Degrades RNA primers Replaces with DNA DNA polymerase II Proof reading Repair any defect in replication DNA polymerase III Synthesizes leading and lagging strands by adding nucleotides to 3’ ends of deoxyribose 10/28/2024 Prof. Hanan Fouad  Pre-priming complex DnaA protein, SSB proteins and DNA helicases form Pre-priming complex which is responsible for initiation and maintaining the separation of the two DNA strands DnaA Helicase 10/28/2024 Prof. Hanan Fouad  10/28/2024 Prof. Hanan Fouad   DNA-polymerase III is only Origin of replication Origin of replication able to synthesize the new Lagging strand Leading strand strand in the direction 5` to 3` 3` 5` 3` 3` 5` 5` 5` 3`, this can be completed by 5` 3` 5` 3` 5` 3` 5` 3` two different mechanisms on each strand Two replication forks Leading strand Lagging strand  Leading strand: It is copied Synthesis of lagging strand in the direction of the 3` 5` advancing replication fork and 3` Synthesis of RNA 5` it is synthesized in a Leading strand 3` primer by primase 5` 5` 3` continuous manner DNA helicase Synthesis of new DNA Primase  Lagging strand: The other 3` 3` by DNA polymerase III 5` RNA primer 5` 3` strand is copied DNA Replication 5` Synthesis of a new primer discontinuously in the polymerase III Fork 3` and a new DNA strand opposite direction of the Okazaki 5` 3` 5` 5` 3` advancing replication fork in fragment RNA primer are removed and gaps are filled with DNA by the form of Okazaki Single stranded DNA DNA polymerase I fragments. It is known as 3` binding proteins Lagging strand 3` 5` 5` 3` lagging strand. The length of 5` RNA primer DNA fragments are joined these segments range from by DNA ligase Parental DNA template 3` 1000 to 2000 bases. 5` Newly formed DNA strand 5` 10/28/20243` College of Pharmacy, UOH Prof. Hanan H. Fouad  DNA topoisomerases remove positive supercoils in the helix 10/28/2024 Prof. Hanan Fouad  Replication in Eukaryotes Enzymes of Eukaryotic Replication DNA replication in eukaryotes is a complicated process because of the bound histones. The process requires the following: 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: 4) DNA polymerase -primase complex: for synthesis of RNA primers and short DNA stretches connected to the RNA primers. 5) DNA polymerase : for DNA repair. 6) DNA polymerase : for mitochondrial DNA synthesis. 7) DNA polymerase : for synthesis of the lagging strand. In eukaryotes, Okazaki fragments are small (100-200 nucleotides). 8) DNA polymerase : for synthesis of the leading strand. 9) RnaseH: for removal of RNA primers 10) DNA ligase to join the ends of adjacent DNA segments 11) Topoisomerases: for removal of positive supercoils in front of the replication fork. 12) Telomerase: for elongation of the 3`-end of telomeres of DNA lagging strand. 10/28/2024 Prof. Hanan Fouad  Replication in Eukaryotes The process of eukaryotic DNA replication closely follows that of prokaryotic DNA synthesis. Some differences, such as the multiple origins of replication in eukaryotic cells versus single origin of replication in prokaryotes. This reduces markedly the time needed for replication (the entire mammalian genome replicates in approximately 9h). If a mammalian genome (3X109 bp) is replicated at the same rate as bacteria (3X105 bp/min) but from a single ori, replication would take over 150 h. 10/28/2024 Prof. Hanan Fouad  Please watch this video explaining DNA replication by animation. Semidiscontinuous DNA replication https://www.youtube.com/watch?v=0Ha9nppnwOc https://www.youtube.com/watch?v=QMX7IpME7X8 10/28/2024 Prof. Hanan Fouad What are the functions of the following enzymes?  Helicases  Endonucleases  Exonucleases  SSBP  Topoisomerases (I , II)  DNA polymerase I, II, III  Primase  DNA polymerase , , , ,   RnaseH  Telomerase Ch. 16 – (DNA Replication and Repair) 10/28/2024 Prof. Hanan H. Fouad (slideplayer.com) TELOMERASE To protect ends of the chromosomes, prevent shortening of the 3` ends of the lagging DNA strands Eukaryotic cells face a special problem in replicating the 3`ends of their linear DNA molecules. Following removal of the RNA primer during copying of the end of lagging strand, there is no way to fill in the remaining gap with DNA. To solve this problem, and to protect the ends of the chromosomes from attack by nucleases, noncoding sequences of DNA are complexed with proteins are found at these ends called telomeres. 10/28/2024 Prof. Hanan H. Fouad  Telomerase 26 Telomere end consists of a repetitive sequence of T's and G's. In humans, telomeres consist of many (650 up to 2500) copies of 5`-TTAGGG-3` repeats. The TG strand is longer than its complement, leaving a region of single-stranded DNA at the 3'-end of the double helix that is a few hundred nucleotides long. The single-stranded region folds back on itself forming a structure that is stabilized by protein. This complex protects the ends of the chromosomes. Cells undergoing the aging process, the ends of their chromosomes get slightly shorter with each cell division until the telomeres are gone, and DNA essential for cell function is degraded, a phenomenon related to cellular aging and death. Cells that do not age (for example, germ-line cells and cancer cells) contain an enzyme called 10/28/2024 telomerase that is responsible for replacing these lost ends. Prof. Hanan H. Fouad  Telomerase is a special kind of reverse transcriptase that carries its own RNA molecule of about 150 nucleotides long. (it uses its RNA strand as a template for synthesis of a complementary DNA strand). In that RNA are copies of the A/C sequence that is complimentary to the T/G repeat sequence. The RNA base-pairs with the terminal nucleotides at the single- stranded 3'-end of the DNA strand. Telomerase recognizes the single-strand 3` terminus and uses its RNA molecule as a template to elongate the parental strand by about 100 nucleotides (the process may be repeated). The elongated end is used as a template, for completing synthesis of the telomere of the lagging strand by DNA polymerase, RNA primer is removed, and ends are joined by DNA ligase 10/28/2024 Prof. Hanan H. Fouad Prof. Hanan H. Fouad 10/28/2024  10/28/2024 Prof. Hanan H. Fouad Watch these videos of telomerase enzyme https://www.youtube.com/watch?v=i6nE6gUp2cw https://www.youtube.com/watch?v=2NS0jBPurWQ Watch this simplified video of DNA replication https://www.youtube.com/watch?v=igFfMd6cgbA Prof. Hanan H. Fouad 10/28/2024 There is a mistake in this picture. What is it ???? 25 Prof. Hanan H. Fouad 10/28/2024  26 10/28/2024 Prof. Hanan H. Fouad  27 10/28/2024 Prof. Hanan H. Fouad Thank You gu.edu.eg T H E F U T U R E S T A R T S H E R E