BMS 141 Lecture 3 DNA Replication PDF

BMS: 141 Lecture No: 3 Title: DNA Replication Instructor Name: Dr Amira Abdel Haleem Medicine and Surgery Program Fall 2024  Intended Learning Outcomes of lecture At the end of this lecture, the student...

BMS: 141 Lecture No: 3 Title: DNA Replication Instructor Name: Dr Amira Abdel Haleem Medicine and Surgery Program Fall 2024  Intended Learning Outcomes of lecture At the end of this lecture, the students will be able to: 1. Define the process of replication 2. Define replication fork 3. Realize that DNA replication is semiconservative, semi-continuous and bidirectional 4. List the proteins involved in replication and outline their functions  Intended Learning Outcomes of lecture At the end of this lecture, the students will be able to: 5. Name the components needed for DNA synthesis and match each component with chronological steps in prokaryotic DNA synthesis 6. Compare leading and lagging strands 7. Explain the need for RNA primer in DNA synthesis 8. Label DNA replication fork drawing  DNA Replication Def.: Transmission of the genetic information found in parent DNA to daughter cells Or Synthesis of daughter DNA from parent one Or Replication is a process in which DNA copies itself to produce identical daughter molecules of DNA  Character of Replication DNA replication is semiconservative : [The parent DNA has two strands complementary to each other. Both the strands undergo simultaneous replication to produce two daughter molecules. Each one of the newly synthesized DNA has one- half of the parental DNA (one strand from original) and one- half of new DNA]  Overview 1- Separation of the two complementary strands of the parental DNA 2- Formation of the replication fork 3- Formation of RNA primer 4- Chain elongation (a new strand is formed by base pairing complementary with the parent strand) 5- Excision of RNA primers and their replacement by DNA 6- Two molecules are made, each has one new and one old DNA strand  Steps of DNA Replication in Prokaryotes A- Separation of the parental 2 DNA strands (unwinding): 1- The initiation of DNA synthesis occurs at a site called origin of replication. In case of prokaryotes, there is a single site whereas in eukaryotes, there are multiple sites of origin. 2- These sites mostly consist of a short sequence of A-T base pairs. A specific protein called dnaA binds with the site of origin for replication. 39  Steps of DNA Replication in Prokaryotes (Cont.,) 3- The two complementary strands of DNA separate at the site of replication by DNA helicase to form a bubble. Multiple replication bubbles are formed in eukaryotic DNA molecules, which is essential for a rapid replication process. 4-These enzymes (DNA helicases) separate the double helix (they cleave the hydrogen bonds between the two DNA strands).  Steps of DNA Replication in Prokaryotes (Cont.,) 4- Prevention of restacking of the 2 DNA strands by single strand DNA binding proteins (SSBP) which prevent the 2 DNA strand from rejoining and protect the template from nucleases that cleave single stranded DNA. 5- As the 2 strands are separated from each other, this creates coils in front of the separated part (supercoils) which prevents further separation of the helix. - This can be solved by DNA topoisomerases which remove supercoils.  Steps of DNA Replication in Prokaryotes (Cont.,) Function of topoisomerases: ✓Topoisomerases have both nuclease(strand cutting) and ligase(strand resealing) activities. ✓ They make transient cut (in the phosphodiester bond) in one strand (topoisomerase I) or both strands (topoisomerase II). ✓ This cut relaxes the supercoils and then topoisomerase reseals the cut (reform the phosphodiester bond).  Steps of DNA Replication in Prokaryotes (Cont.,) N.B: DNA gyrase, a type II topoisomerase found in bacteria and plants.  Steps of DNA Replication in Prokaryotes (Cont.,) B- Direction of DNA replication: DNA replication is bidirectional which means that the synthesis of two new DNA strands, simultaneously, takes place in the opposite direction [one is in a direction (5→3') towards the replication fork which is continuous, the other in a direction (5'→3') away from the replication fork which is discontinuous  Steps of DNA Replication in Prokaryotes (Cont.,) C- Synthesis of new DNA strands by DNA polymerase enzyme: Notes on DNA ploymerases: 1- They cannot initiate DNA synthesis; they are able to add a new complementary deoxynucleotide to a preexisting nucleic acid chain (RNA primer) 2- They can only synthesize DNA in one direction from 5’ to 3’ (as they are able to read the DNA template in one direction 3’ to 5’). Since the two DNA strands are antiparallel, one strand can be synthesized in the 5’→3’direction toward the replication fork and the 2nd strand is synthesized in the 5’→3’direction away from the replication fork.  Steps of DNA Replication in Prokaryotes (Cont.,) 3- There are three types of DNA polymerases in prokaryotes (Pol I, Pol II, Pol III) and many types in eukaryotes (e.g.: α, β, δ...)  Steps of DNA Replication in Prokaryotes (Cont.,) C- Synthesis of new DNA strands: 1) RNA primer synthesis Primase (a specific RNA polymerase) synthesize a segment of RNA (RNA primer). The primer acts as an acceptor of the new incoming DNA nucleotide provided by DNA polymerase III.  Steps of DNA Replication in Prokaryotes (Cont.,) 2) Chain elongation DNA polymerase III brings a deoxynucleotide (dATP, dCTP, dGTP and dTTP) according to the base complementarities to the template DNA strand and then connects this new deoxynucleotide to the RNA primer by a phosphodiester bond.  Steps of DNA Replication in Prokaryotes (Cont.,) Synthesis of DNA proceeds only in one direction the 5' -----> 3' direction (in the newly growing strand) ✓One strand is synthesized continuously needing one RNA primer and grows in 5’ → 3’ direction. This strand is called leading strand. ✓The other strand must grow discontinuously in small pieces; each piece has its RNA primer and grows in 5’→ 3’ direction but opposite the direction of replication fork. This is called lagging strand and the small fragments forming the lagging strand are called Okazaki fragments.  Steps of DNA Replication in Prokaryotes (Cont.,) 3) Excision of RNA primers and their replacement by DNA RNA primer of each Okazaki fragment is removed by 5’→3’ exonuclease activity of DNA polymerase I enzyme. DNA pol I replaces the removed RNA nucleotides with deoxynucleotides, by its 5’→3’ polymerase activity. 4- DNA ligase enzyme, legate the DNA fragments together.  Medical applications 1- Quinolones antimicrobial drugs e.g. nalidixic acid which act by inhibiting bacterial gyrase preventing bacterial replication and transcription. 2- Bloom disease: characterized by short stature and predisposition to cancer due to genetic defect in DNA helicase.  Post replication modifications Proofreading or editing of the newly synthesized DNA (replication fidelity) Proofreading activity is a function of 3’→5’exonuclease activity (proofreading activity) of DNA pol III and DNA pol. I. Misreading of the template sequence could result in dangerous mutations. To ensure replication fidelity, DNA pol III and DNA pol. I have in addition to their 5’→3’ polymerase activity, a 3’→5’ exonuclease activity (proofreading activity).  Post replication modifications DNA polymerase III and I always check the newly added nucleotide, and if it is incorrect (not complementary to template), they remove the incorrect base by its exonuclease activity from 3’ to 5’ and replace it with the correct base this called proofreading.  Differences between 5′→3′ and 3′→5′ exonucleases The 5′→3′ exonuclease activity of DNA polymerase I differs from the 3′→5′ exonuclease used by both DNA polymerases I and III in: ✓5′→3′ exonuclease can remove one nucleotide at a time from a RNA primer that is properly base-paired to the template (excision of RNA primers). ✓So, 3′→5′ exonuclease can remove incorrect deoxynucleotides in the 3′→5′ direction. This ability is important in proofreading.  Note DNA Pol I has three activities: 1- Polymerase activity from 5′→3′for the synthesis of DNA segments replacing RNA primers. 2- Exonuclease activity from 5′→3′ for excision of RNA primers and repair 3-Exonuclease activity from 3′→ 5′ for proofreading activity during the synthesis of DNA segments. Comparison of prokaryotic DNA polymerases  Eukaryotic DNA Replication 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 origins of replication in prokaryotes, have already been noted. In eukaryotes, RNA primers are removed by RNase H and FEN1 rather than by a DNA polymerase.  Eukaryotic DNA Replication (Cont.,) Eukaryotic DNA polymerase: - At least five key eukaryotic DNA polymerases have been identified and categorized on the basis of: molecular weight, cellular location, sensitivity to inhibitors, and the templates or substrates on which they act. - They are designated by Greek letters  Post replication modifications Packing of Eukaryotic DNA THANK YOU