Genetics and Genetic Engineering - Second Lecture PDF

# Genetics and Genetic Engineering - Second Lecture ## Faculty of Veterinary Medicine - Department of Animal Husbandry and Development of Animal Wealth - General Program - Genetics and Genetic Engineering - First Year Students ## Dr/ Ahmed Ibrahim Ateya ## The Genetic Material ### DNA replicatio...

# Genetics and Genetic Engineering - Second Lecture ## Faculty of Veterinary Medicine - Department of Animal Husbandry and Development of Animal Wealth - General Program - Genetics and Genetic Engineering - First Year Students ## Dr/ Ahmed Ibrahim Ateya ## The Genetic Material ### DNA replication ### Table: DNA vs RNA | Feature | DNA | RNA | |---|---|---| | Sugar | Deoxyribose | Ribose | | Pyrimidine bases | Thymine | Uracil | | Strands | Double strand | Single strand | | As a genetic material | In most living organisms | Only in some viruses | ### Table: Types of RNA | Criteria | rRNA | mRNA | tRNA | |---|---|---|---| | Size | Largest | Differ according to gene that transcribes it or number of amino acid coded by certain gene | Smallest | | Genetic function | Synthesis of ribosomes | Carry genetic information from nucleus to cytoplasm | Carry amino acid during translation | | S value (Svedberg unit) | 5 S, 16 S, 23 S in *E. coli* 5 S, 18 S, 28 S in mammals | 6-25 S | 4 S | ## The DNA replication - DNA replication is the process of producing two identical replicas from one original DNA molecule. ### Possible Models of DNA Replication - **Conservative replication**: The parental double helix should remain intact and the second DNA molecule should be constructed as entirely new DNA. - **Semi-conservative replication**: Each of the two resulting DNA molecules should be composed of one original or conserved strand and one new DNA strand. - **Dispersive replication**: Both strands of the two newly produced DNA molecules should contain a mixture of old and new DNA. ## The DNA synthesis - The enzyme responsible for The DNA synthesis is mainly **DNA polymerase** which has three types. ### DNA polymerase I - DNA Polymerase I (or Pol I) is an enzyme that participates in the process of DNA replication. Discovered by Kornberg in 1956, it was the first known DNA polymerase (and, indeed, the first known of any kind of polymerase). - It was initially characterized in *E. coli*. - It is able to synthesize DNA in cell-free system or *in vitro*. #### The requirement of this enzyme - Four types of deoxynucleotides in triphosphate form. - Mg++ ions - DNA template. #### Direction of building: - it adds C-5 deoxynucleotides to the C-3end of the growing chain by phosphodiester bond. #### Function *in vivo*: - only repair function due to - It can synthesize only short pieces of DNA. - It has slow rate of building, 650 nucleotide per minute, which 100 times less than in vivo rate. ### DNA polymerase II: - Need the same requirement. - Only used in repair function *in vivo* as DNA polymerase I. - Also discovered in *E. coli*. - Direction of building C-5 to C-3 ### DNA polymerase III (DNA polymerase alpha in eukaryotes) - Instable outside the cell. It work at 37°C - **Requirements:** as DNA polymerase I and II in addition to free 3- OH end of polynucleotide chain to be able to add new nucleotides and it is ATP dependent. - Has high rate of building 700 nucleotide in second so it is the main enzyme in DNA building inside the cell. - Direction of building as DNA polymerase I and II. - It can proof-read its own product (repair function), remove mistakes and then correct the error. ## Steps of DNA replication ### Unwinding the DNA helix - **Point of origin:** the point at which the unwinding of helix and replication start and continue in both directions around it. - In prokaryotes there is only one point of origin to whole DNA while in eukaryotes where is DNA is huge there are many point of origin and each segment of chromosome whose replication is under the control of one point of origin is called **replicon**. ### Enzymes involved in unwinding process - **Helicase**: unwind the double helix through breaking of hydrogen bonds which require ATP as a source of energy. - **Single strand binding protein (SSB):** bind to the single strands to stabilize the unwind strands until be replicated. It is called HSP (Helix Stabilizing Protein) - **DNA Gyrase or Topoisomerase I:** reduce the tension produced by supercoiling at the ends of each replicon as a result of unwinding. This is through cutting in two strands (nicking) and when tension released it seals the nick again. ## Initiation of DNA Synthesis - The **DNA polymerase III** is the main enzyme in replication of DNA. However, this enzyme requires free 3-OH group to synthesize the polynucleotide chain. - **RNA polymerase (Primase)** synthesize short RNA primer to provide 3'- OH end for DNA polymerase. - The **DNA polymerase III** adds deoxyribonucleotides to the RNA primer. - The primer is cleaved by exonuclease activity of DNA polymerase I which also replace the primer with a short DNA linked to the DNA synthesized by DNA polymerase III. ## DNA replication fork - A diagram of a DNA replication fork is presented. ## Continuous, discontinuous bidirectionals - Due to the fact that the 2 strands of DNA are antiparallel, DNA synthesis becomes bidirectional and at one side of point of origin DNA synthesis is continuous, other side is discontinuous. - Continuous strand called leading strand while the discontinuous one called lagging strand. - **Okazaki Fragments** are the discontinuous fragments formed during DNA synthesis. Okazaki Fragments are sealed by **DNA ligase**. ## Semi-conservative Replication - A diagram of the semi-conservative model of DNA synthesis is presented, with Okazaki fragments.