1. FLS L1. Structure and replication of DNA 2024 - canvas.pptx

Structure and Replication of DNA Dr Laila Kudsiova Acknowledgements: Dr. Laura O’Neill  Components of DNA is a nucleic acid. It is a macromolecule and it is made up of two polynucleotide Each polynucleotide is made up of many small units called NUCLEOTIDE...

Structure and Replication of DNA Dr Laila Kudsiova Acknowledgements: Dr. Laura O’Neill  Components of DNA is a nucleic acid. It is a macromolecule and it is made up of two polynucleotide Each polynucleotide is made up of many small units called NUCLEOTIDES Each NUCLEOTIDE consists of a 5-carbon sugar (deoxyribose), a nitrogen containing base attached to the sugar, and a phosphate group. There are four different types of nucleotides found in DNA, differing only in the nitrogenous base. 5’ A is for adenine 4’ 1’ G is for guanine C is for cytosine 3’ 2’ T is for thymine (2-Deoxyribose)  Nucleoside = sugar + base 5’ P s 3’ Phosphodiester bond s T A P N-Glycosidic bond P Nucleotide = sugar + base + phosphate s s C G P P s s T A P P s s G C P 3’ 5’ ADENINE AND THYMINE BASE PAIRING Hydrogen bonding PURINE PYRIMIDINE GUANINE AND CYTOSINE BASE PAIRING Hydrogen bonding PURINE PYRIMIDINE Remember: A and G are purines, C and T are pyrimidines Purines have short name & long base (2 rings); pyrimidines vice versa purines Adenine Guanine pyrimidines Thymine Cytosine What do I mean by 5’ to 3’? CONDENSATION REACTION H20 3’,5’PHOSPHODIESTER BON 5’ A C T C A A T G C G  DNA REPLICATION When a cell divides, both daughter cells must receive a complete set of genes, so the DNA molecules (chromosomes) must replicate accurately before division.  Asexual Reproduction Prokaryot es REPLICATION PARTITIONING 1. The entire genome is on one circular chromosome = DNA molecule. 2. The chromosome replicates once to produce two chromosomes that are identical (except for rare 3. mutations). The two identical daughter chromosomes move toward opposite end of the cell. 4. When the cell divides the daughter chromosomes are partitioned one to each daughter cell.  Asexual Reproduction Eukaryotes Asexual reproduction by mitosis DNA replicates during S phase Gene expression occurs during G1 and G2 (and S?) Nuclear division (mitosis) occurs during Mitosis Cell division (cytokinesis) occurs at the end of Mitosis  Eukaryotic DNA Replication General feature of DNA replication DNA replication is semi conservative It proceed from a specific point called origin It is bidirectional process It proceed in 5’-3’ direction It occur with high degree of fidelity It is a multi-enzymatic process DNA replication occurs by three steps 1. Initiation: 2. Elongation 3. Termination  DNA Replication is Semi- conservative 1. The strands separate. 2. A new strand is made using each old strand as a template according to the rules of base pairing. Model proposed by Watson and Crick, verified by Matt Meselson and Frank Stahl  DNA Replication: Enzyme Activities Many enzymes are required for DNA 1. Helicase unwinds double-helical DNA. replication. We will only consider enzyme activities, not specific enzymes. Enzymes with these activities are also used for DNA Arthur Kornberg manipulation in the lab. SSBP 2. Single-strand binding protein binds to and stabilizes the single strand to keep DNA unwound. 3. Primase: adds ribonucleoside triphosphates to synthesise an RNA primer Binds at the initiation point of the 3'-5' parent chain Reminder: RNA is like DNA except single-stranded ribose instead of deoxyribose uracil instead of thymine (U pairs with A just as T does) 4. DNA polymerase: proceeds in a 5’ to 3’ direction Adds 1000 bases /second to the growing chain Requires all 4 dNTPs deoxyribonucleotides MUST have a template and a primer Has PROOF READING activity. 5. Exonuclease removes nucleotides from the end of a DNA strand; different enzymes work 5’ to 3’ or 3’ to 5’, and also removes the RNA primer by recognizing the ribose sugar on RNA 6. Ligase joins ends of single DNA strands by making new phosphate bonds There are different types of polymerases  Putting it All Together 1 INITIATION SSB P 3’ 5 Helica se ’ 3’ 5 ’ Primase lays down ’ a primer 3 5’ 5 3’ ’ 5’ 3 ’ ELONGATION 5 3’ ’ 3’ 5 ’ DNA polymerase extends in a 5’ to 3’ direction so it always moves along the direction of the 3’ to 5’ parental strand Putting it All Together Exonuclease removes the RNA primer Polymerase fills the gap Ligase joins the two pieces.....a few more details 3’ Leading strand 5’ 3’ Okazaki fragments Lagging strand 5’  Origin of replication In EUKARYOTES there are multiple origins of replication. In PROKARYOTES there is only ONE origin of replication Bases added at a rate of 1000/second Adding 1000 bases/second So replication of the whole mammalian genome takes approximately 8 hours Origin of replication Parental strand Daughter strand Replication fork Two daughter DNA strands Termination At the end of DNA replication the RNA primer are replaced by DNA by 5’-3’ exonuclease and polymerase activity of ε DNA polymerase. Exonuclease activity of DNA polymerase removes the RNA primer and polymerase activity adds dNTPs at 3’-OH end preceding the primer. In eukaryotic organism with linear DNA, there is a problem. When RNA primer at 5’ end of daughter strand is removed, there is not a preceding 3’-OH such that the DNA polymerase can use it to replace by DNA. So, at 5’ end of each daughter strand there is a gap (missing DNA). This missing DNA causes loss of information contained in that region. This gap must be filled before next round of  Telomeres Found at the linear end of DNA. G:C rich repeats In humans - TTAGGG/AATCCC - highly conserved - NON-CODING but ESSENTIAL to maintain the integrity of DNA  Enzyme Activities to Finish the Job 7. Telomerase uses a short RNA template to add short DNA repeats to the short ends of linear chromosomes when the last primer is removed using RNA template. 8. Gyrase (a topoisomerase) relaxes supercoils produced when the molecule is twisted during replication. Also facilitates unwinding at beginning of replication. Enzyme Activities for Biotechnology These enzyme activities, plus a few others, are also used to manipulate DNA, for example: PCR Making recombinant DNA Detecting mutations at the molecular level Label the structures and enzymes involved in DNA replication 3’ end DNA polymerase SSBPS Leading strand Helicase 5’ end Okazaki fragments Lagging strand Gyrase Ligase Exonuclease Primase 5’ (3) bond N-glycosidic 3’ (1) 3’- (2) 5’phosphodiester bonds (4) 3’ 5’ www.youtube.com/watch?v =TNKWgcFPHqw