DNA Structure and Replication PDF

Nucleic acids: Structure and Function Lynn OConnor Nov,2024  DNA Structure & Replication Structure of nucleic acids DNA replication and repair Transcription Translation, synthesis of proteins Structure of DNA https://www.youtube.com/watch?v=o_-6JXLYS-k DNA packaging and r...

Nucleic acids: Structure and Function Lynn OConnor Nov,2024  DNA Structure & Replication Structure of nucleic acids DNA replication and repair Transcription Translation, synthesis of proteins Structure of DNA https://www.youtube.com/watch?v=o_-6JXLYS-k DNA packaging and replication https://www.youtube.com/watch?v=dKubyIRiN84 Replication https://www.youtube.com/watch?v=TNKWgcFPHqw Replication (HHMI) 2:19 https://www.youtube.com/watch?v=gJzcYbt7_E4  What is the eurkaryotic genome? A set of genetic information What is the genome for? Some parts contain instructions used daily by the cell Some parts contain instructions when the cell is stressed Some parts are not used (as far as we know)  Physical Organisation 2 distinct compartments i) nucleus ii) mitochondria Most of the genome (about 30,000 genes) – contained in chromosomes within the nucleus – info from both maternal and paternal origins  Data Storage Analogy  Why are ‘nucleic acids’ important? DNA is the information store of the cell It determines what proteins are made, when and how Proteins = major players in the cell – Enzymes influence rate of reactions N.B. synthesis and degradation of lipids and carbohydrates – Structural proteins determine cell shape – Cell-cell communication; peptide hormones, cell adhesion Central dogma of molecular biology Unidirectional flow of genetic information DNA replication = DNA synthesis Transcription = RNA synthesis Translation = protein synthesis Structure of nucleic acids Composed of nucleotide building blocks (polynucleotides) 2 types: – ribonucleic acid (RNA) – deoxyribonucleic acid (DNA) Structure of nucleotides Each nucleotide a base a sugar and one or more phosphate groups  Bases are Nitrogen-containing molecules 5 different bases – Adenine (A) – Cytosine (C) – Guanine (G) – Thymine (T) – Uracil (U) A, C, G and U are found in RNA A, C, G and T are found in DNA purines: A and G pyrimidines: C, T and U  Sugars (monosaccharides) containing 5 Carbon atoms (pentoses) ribose in ribonucleic acid (RNA) deoxyribose in deoxyribonucleic acid (DNA) deoxyribose has a H instead of an OH group at the 2’ position  Phosphates Contain 1 mono- 2 di- or 3 tri- phosphate groups e.g. adenosine monophosphate (AMP) adenosine diphosphate (ADP) adenosine triphosphate (ATP) A nucleotide  Nucleic acid structure Nucleotides are joined to each other through their phosphate groups = 3’-5’ phosphodiester linkages (Covalent bonds) Nucleic acids are directional i.e. have 5’ and 3’ ends Sequence of bases along nucleic acids is not restricted stores genetic information  Base pairing Bases interact with each other via Hydrogen bonds in a process called base-pairing H bonds are individually weak – Any advantage of weak bonds here? Base-pairing is specific A:T in DNA or A:U in RNA 2 H bonds G:C in DNA and RNA 3 H bonds Base-pairing occurs between complementary bases  DNA is a ‘double helix’ 2 strands of DNA are coiled around the same axis to form a right-handed double helix approx. 10 base pairs per turn of the double helix Length of DNA Base pair = bp 1,000 base pairs = 1 kilobase pair = 1kb Human genome – 3.2 x 109 base pairs Chemical Structure of dsDNA N.B. Strands run in opposite directions - They are anti- parallel - A in one strand with T in the opposite - G in one strand with C in the opposite  DNA vs. RNA Double-stranded Single-stranded A, C, G, T A, C, G, U Deoxyribose Ribose (H at 2’C position) (OH at 2’C position)  What is a genome? the complete genetic code of an organism Human genome 3 x 109 bp A gene is a part of the genome that encodes a specific RNA and/or protein Human genome has 20,000-30,000 genes  DNA packaging A full copy of the human genome is found in the nucleus of each cell if unravelled and straightened would be 1.8m long DNA in the cell is tightly packed as chromosomes in the nucleus Human cells contain 23 pairs of chromosomes Higher order structures formed during progressive compaction of chromatin  Chromatin = DNA and various proteins that package it in eukaryotic cells Histones Major proteins of chromatin H1, H2A, H2B, H3 and H4 Small proteins Rich in basic amino acids, e.g. Lys, Arg N.B. + a.a.s -DNA Opposite charges attract  Nucleosomes 146 bps of DNA are associated with nucleosome particle 50-70 bp span of linker DNA bound by a linker histone H1 1 x H1 2 x (H2A, H2B, H3, H4)= octamer Nucleosome structure Histone Modification: By acetylation, methylation or phosphorylation – changes charge and shape – prepare the chromatin for DNA replication and transcription Modifications are reversible Modification by acetylation decreases the affinity for DNA and causes decompaction of the chromatin, allowing gene transcription to take place  Structure of nucleic acids DNA replication and repair Transcription Translation, synthesis of protein  Main Features of eukaryotic DNA synthesis 1. Semiconservative with respect to parental strand 2. Bidirectional with multiple origins of replication 3. Primed by short stretches of RNA 4. Semidiscontinuous with respect to the synthesis of new DNA DNA replication is semi-conservative Each strand of DNA serves as a template for the synthesis of a new strand Each daughter molecule of DNA contains one parental strand and one newly synthesized strand N.B. DNA sequence is conserved in this way A. Small prokaryotic circular DNA B: Very long eukaryotic DNA Replication is bidirectional with multiple origins of replication Advantage of multiple origins of replication for eukaryotic DNA?  DNA synthesis Successive addition of nucleotides to the 3’ end of a growing DNA chain Incoming nucleotide is complementary DNA polymerases catalyse formation of phosphodiester bond between incoming nucleotide and the growing chain. They require Template DNA strand dNTPs Primer with free 3’ end Replication fork Leading strand is synthesized continuously Lagging strand - discontinuous  A short piece of RNA serves as a primer for the synthesis of each Okazaki fragment RNA primer Primase = DNA-directed RNA polymerase  Mechanism of action of HELICASE 1. Maintains the separation of the parental strands 2. Unwinds the double helix ahead of the advancing replication fork Supercoils As DNA unwinds supercoils are formed DNA topoisomerases remove supercoils Antimicrobial agents Target bacterial DNA gyrase e.g. Quinolones, such as ciprofloxacin Anticancer agents Target human topo II e.g. etoposide Mechanism of action of DNA ligase Seals the nicks in the DNA after DNA pol fills the gaps left by RNA primers HELICASE: – motor proteins – strand separation and formation of replication fork TOPOISOMERASES: - relieve torsional stress in DNA DNA POLYMERASES: - several with distinct activities- some have proofreading ability PRIMASE: initiate synthesis of RNA molecule as primer SINGLE-STRANDED DNA BINDING PROTEINS: prevent premature annealing of ss DNA to ds DNA LIGASE: seals the nicks Eukaryotic cell cycle S phase (DNA synthesis) DNA replication G phases (gap) Cell growth M phase (mitosis) Chromosomes segregate Eukaryotic DNA polymerases Note * denotes 3’-5’ exonuclease activity  Telomeres What is it? A protective repetitive stretch of DNA complexed with protein at the end of a chromosome, shortens with every cell division Structure At ends of eukaryotic chromosomes = DNA-protein complex In humans it is (TTAGGG)n n=1,000s ssDNA at 3’ end Function Protect ends of chromosomes Telomerase Telomer maintenance enzyme - Maintains telomere length  Telomere Telomers: Complexes of non-coding DNA plus proteins located at the ends of linear chromosomes FUNCTIONS: 1. To maintain the structural integrity of the chromosome – preventing attack by nucleases 2. Allow repair systems to distinguish the “end” of the chromosome from a double strand break  Dolly the sheep First animal to be cloned in 1997  Normal at birth  Normal development up to age 3  Chromosomal examination – her telomers were shorter than would be expected for a sheep of her chronological age  Dolly died of lung disease at 6 years of age Biological age much older than her chronological age - suggestion  Medical relevance In normal human somatic cells, telomeres shorten with each successive cell division. Once too short, cells cannot divide anymore. In cancer and stem cells, telomeres do not shorten and cells can continue to divide Study of telomeres - understand aging Related pharmacology DNA synthesis can be inhibited by nucleoside analogues – modified in sugar component e.g. AZT – Anti-retroviral drug – 3’ C on deoxyribose has N3 – Terminates DNA chain elongation – (used to treat HIV) Cytosine arabinoside – anticancer – arabinose replaces deoxyribose All halt DNA chain elongation  Causes of DNA damage Environmental agents – Chemicals – Radiation, e.g. UV radiation causes adjacent T’s to dimerise Errors in normal cellular processes – e.g. incorporation of wrong nucleotide during DNA replication Normal chemical changes – e.g. oxidation Mutation: A heritable change in the sequence of nucleotides in DNA that causes a permanent alteration of genetic information