Lecture 5: Central Dogma of Molecular Biology, DNA, & RNA Replication (PDF)

CENTRAL DOGMA OF MOLECULAR BIOLOGY, DNA, RNA DNA replication  CENTRAL DOGMA OF MOLECULAR BIOLOGY Replication Transcription Translation DNA RNA protein Reverse transcription ✓DNA is the c...

CENTRAL DOGMA OF MOLECULAR BIOLOGY, DNA, RNA DNA replication  CENTRAL DOGMA OF MOLECULAR BIOLOGY Replication Transcription Translation DNA RNA protein Reverse transcription ✓DNA is the carrier of genetic information; ✓DNA consists of two strands that are linked together, and can be split similarly to a zip. ✓It has coded instructions, genes, that are necessary for the construction and functioning of cells. ✓The DNA molecule can make an exact copy of itself by accurately passing instructions on cell division.. DNA encodes genetic information thanks to four "building blocks" called bases: adenine, thymine, guanine, and cytosine. They are abbreviated as A, T, G and C and have the property of each "connecting" with only one of the other three bases: A + T, T + A, G + C, C + G; so that "A" on one strand of DNA will successfully "bind" only to "T" on the other strand THE ORDER IS OF GREAT IMPORTANCE: A + T IS NOT EQUIVALENT TO T + A, JUST AS C + G IS NOT THE SAME AS G + C. ⦿ During cell division, the bases bond to form two identical new strands, which begin to wrap up. DNA replication or synthesis is the process of copying the double strand of DNA before cell division. The obtained two double chains (threads) are the same, but sometimes replication errors occur, which can lead to the production of slightly modified copies, and each of the fields consists of one original and one newly synthesized strand. This is called semi-conservative replication. The replication process consists of three cases: initiation, replication and termination. Nucleus functions. Storage and reproduction of genetic information - DNA replication and reparation. Enzymes and factors that provide the mechanism of replication Replication 1. Basic features DNA Matrix principle (complementarity A-T, G-C) Semi-conservative mechanism - the new molecule contains: - old (mother) strand - new strand Replication speed: - prokaryotes - 500 bp./sec - eukaryotes - 50 bp./sec 2. Mechanism - Opening the double helix - it is necessary to make them the bases - accessible Helicase - destroys hydrogen and "stacking" account interactions of energy from ATP / without enzyme it can be achieved at heating up 90 ° C Prevent pairing of single-stranded parts of the DNA template SSB (single-stranded binding) proteins single chain part of DNA template with short, Internal chain connected areas monomeric SSB proteins cooperative binding of SSB proteins stretches single-stranded DNA -Enzymes that release pressure in unfolding DNA -and which protect against interlacing DNA topoisomerase І and ІІ For the release of pressure, from the unwinding of the helix turning is necessary When opening every 10 bp. DNA should be rotate 180 ° DNA polymerase topoisomerase І - relieves tension, Which arises from the entanglement of DNA so that Breaks one strand. The torn strand can now turn around the other and release the pressure who was created DNA topoisomerase І with tyrosine in the active center the energy of the torn phosphodiesterase bond is preserved allowing it to resume after rotation DNA topoisomerase І at no new cost to binds with phosphate and energy breaks down the phosphodiesterase connection in one strand the spontaneous reconstruction of the phosphodiester bond Reconstruction of DNA integrity and topoisomerase I topoisomerase ІІ - releases entanglements by tearing the both strands of the DNA molecule. double ATP domain helix 1 of topoisomerase II double helix 2 ATP binding, crossing of sewing on dimerization of helix 1 through helix 2 and ATPase domains, The torn part in Release of scattering of helix 2 helix 1 helix 2  265 Synthesis of a new DNA molecule 3’ end 5’ end matrix 5’ strand growing strand 3’ край 3’ pyrophosphate new deoxyribonucleoside triphosphate 5’ end The addition of new nucleotides to the growth strand is performed by an enzyme DNA polymerase (DNA polymerase III and prokaryotes and DNA polymerase δ - eukaryotes) new 5’ triphosphate deoxyribonucleoside triphosphate new deoxyribonucleoside growing triphosphate strand “thumb” growing matrix strand gap pyrophosphate 5’-3’ direction of “fingers” matrix growing on the new strand “palm” Characteristics of DNA polymerase III: - attaches deoxyribonucleoside triphosphates to the 3'OH group - the presence of even a short strand of nucleic acid is required - It is catalyzed by the growth of the strand in the direction 5‘ 3' - has 3‘ 5' corrective exonuclease activity DNA polymerase III has 3‘ 5' corrective exonuclease activity growing strand 3’-5 ’corrective activity of DNA polymerase allows it to remove the wrong matrix wrong nucleotide paring С-А After removing the wrong nucleotide normal extension of the strand can continue further extension of the strand is blocked Accuracy - 1 error per 109 nucleotides Only growth in the 5’ 3' direction allows correction of possible errors growing strand hypothetical 5’- 3’ 3’- 5’ growing growing check 5’-end, created after 3’-end, created after the removal of one the removal of one nucleotide after screening nucleotide after screening new, correct new, correct deoxyribonucleoside deoxyribonucleoside triphosphate triphosphate high energy high energy connection can not be connection can be demolished and upgraded demolished and upgraded can not continue can continue The properties of DNA polymerase ІІІ and the antiparallelity of DNA determine the differences in the growth of the two new strands 5’ 3’ 5’ Replication fork 3’ Leading strand The backlog with Okazaki fragments / 100-200 bp. in eukaryotes 1000-2000 bp. in prokaryotes /  DNA primer synthesizes RNA primers necessary for the synthesis of Okazaki fragments RNA new RNA primer primer matrix DNA polymerase builds on the RNA primer a new fragment of Okazaki DNA polymerase it the fragment ends of Okazaki previous RNA primer is degraded by (RNAase H) and replaced by DNA DNA ligase builds phosphodiesterase RNA primers are about 10 bp long. Missing connection and are synthesized at distances of about 200 bp.  DNA replication in prokaryotes 5’3’ 3’ 5’ Replication complex in prokaryotes Sliding pliers  Replication fork in eukaryotes Differences from prokaryotes - Two enzymes work on the backlog strand polymerases - α and δ. DNA polymerase α / primer complex forms RNA primers and short pieces of DNA, which are then further elongated by DNA polymerase δ. Helicase is made up of 6 different subunits. Telomerase lengthens the telomere parts of the maternal strand DNA to complete replication of the newly constructed strand Telomeres - tandem repeated parental strand short G rich sequences незавршена, новосинтезирана (in man - telomerase заостаната верига 10 000 connects direction of nucleotides telomerase from synthesis repetitive теломеразата GGGTTA Го удолжува 3’крајот на telomerase with linked RNA matrix родителската верига sequences) telomerase extends the 3 'end of parent strand DNA polymerase Replication bubble (eye) replication start replication start АТ-rich binding of initiating sequence protein starting at replication local opening DNA helicase, of the DNA strand bound to an inhibitor DNA binding helicase with Initiating proteins synthesis of DNA binding RNA primer helicases helicase with inhibitor initiating proteins the helix opens the helix, synthesis of connects the primacy and leading DNA strand starts forms a primosome RNA primer synthesis DNA primase it allows DNA polymerase to start DNA synthesis RNA primers initiate the synthesis of and begins the synthesis of and other DNA strands DNA RNA the remaining strand polymerase primer formation of two replicators forks moving in opposite directions replication fork 1 replication With one start of replication at a speed of 50 bp secondary fork 2 a large human chromosome / 150x106 bp / will replicate in 800 hours. In reality, many beginnings of replication are used. DNA repair - a mechanism for repairing: 1) Mistakes in one of the strands of DNA - the injured part is cuttings and then reconstructed according to the matrix principle, so that the new strand is synthesized using the normal strand, like a matrix 2) Mistakes in both strands are corrected by: a) the free ends are sewn to each other (usually there is a loss of genetic material) b) the lost piece is reconstructed in a way that the homologous chromosome is used as a matrix (there is a complex mechanism for recognizing and pairing the corresponding parts of the mistake and healthy homologous chromosome