Nucleic+DNA+Replication .pdf

Nucleic Acids and DNA Replication bestanimations.com/Science/Biology/DNA/DNA.html Dr. Talat Nasim (School of Pharmacy and Medical Sciences)...

Nucleic Acids and DNA Replication bestanimations.com/Science/Biology/DNA/DNA.html Dr. Talat Nasim (School of Pharmacy and Medical Sciences) [email protected] 1 LEARNT FROM THE LAST LESSION  Discussed the regulation of gene expression in humans (eukaryotes)  Stages where gene expression can be regulated at transcription, splicing and translation  Activities (individual and group) 2  25 September 2024 Intended Learning Outcomes By the end of this lecture, you should be able to: Explain how DNA polymers are formed from nucleotides Describe DNA & RNA structures and the importance of base- pairing Describe the process of DNA replication and understand the basis of some of its applications Techniques that are used in DNA replication and their uses (PCR, DNA sequencing) DNA Replication 3 Biochemistry Mathews/van Holde/Ahren Chapters: 4, 27, 28 Human Molecular Genetics Tom Strachan and Andrew Read Chapters: 1, 3 Molecular Biology of the Cell Alberts/Johnson/Lewis/Raff/Ro berts/Walter Chapters: 4, 5, 6 DNA Replication 4  Resources Medical Sciences Eds. Naish and Syndercombe Court Chapter 2: Biochemistry and Cell Biology Chapter 5: Human Genetics DNA Replication 5  Nucleotides  = Fundamental biochemical building block of DNA (=deoxyribonucleic acid) and RNA (=ribonucleic acid) DNA Replication 6  25 September 2024 Nucleotides C = Cytosine G = Guanine T = Thymine A = Adenine U = Uracil  = Fundamental biochemical building block of DNA (=deoxyribonucleic acid) and RNA (=ribonucleic acid)  Composed of three components:- - Nitrogenous base: Pyrimidine (C, T, U - T in DNA, U in RNA) Purine (G, A) - Pentose sugar: deoxyribose in DNA, ribose in RNA - Phosphate group: acts a bridge between adjacent ribose/deoxyribose groups DNA Replication 7  25 September 2024 Nucleotides C = Cytosine G = Guanine T = Thymine A = Adenine U = Uracil  = Fundamental biochemical building block of DNA (=deoxyribonucleic acid) and RNA (=ribonucleic acid)  Composed of three components:- - Nitrogenous base: Pyrimidine (C, T, U - T in DNA, U in RNA) Purine (G, A) - Pentose sugar: deoxyribose in DNA, ribose in RNA - Phosphate group: acts a bridge between adjacent ribose/deoxyribose groups DNA Replication 8  25 September 2024 Nucleotides C = Cytosine G = Guanine T = Thymine A = Adenine U = Uracil  = Fundamental biochemical building block of DNA (=deoxyribonucleic acid) and RNA (=ribonucleic acid)  Composed of three components:- - Nitrogenous base: Pyrimidine (C, T, U - T in DNA, U in RNA) Purine (G, A) - Pentose sugar: deoxyribose in DNA, ribose in RNA - Phosphate group: acts a bridge between adjacent ribose/deoxyribose groups DNA Replication 9  25 September 2024 Nucleotide: ATP structure    5’ Glycosidic bond DNA Replication 10  25 September 2024 Nucleotide: ATP structure    5’ Glycosidic bond Adenosine Nucleoside (sugar+base) DNA Replication 11  25 September 2024 Nucleotide: ATP structure    5’ Glycosidic bond Adenosine Nucleoside (sugar+base) AMP Nucleoside monophosphate ADP Nucleoside diphosphate DNA Replication 12  25 September 2024 Nucleotide: ATP structure    5’ Glycosidic bond Adenosine Nucleoside (sugar+base) AMP Nucleoside monophosphate ADP Nucleoside diphosphate ATP Nucleoside triphosphate DNA Replication 13  25 September 2024 Ribonucleotides link to form single-stranded RNA DNA Replication 14  25 September 2024 Deoxyribonucleotides link to form DNA double helices DNA Replication 15  DNA helix 25held September 2024 together by base pairing  Core of DNA double helix = bases on complimentary strands held together by H bonds  C (pyrimidine) always pairs with G (purine)  T (pyrimidine) always pairs with A (purine) A-U bonding occurs during gene transcription DNA Replication 16  DNA helix 25held September 2024 together by base pairing  Core of DNA double helix = bases on complimentary strands held together by H bonds  C (pyrimidine) always pairs with G (purine)  T (pyrimidine) always pairs with A (purine) A-U bonding occurs during gene transcription G-C A-T Which interaction is the strongest? DNA Replication 17  DNA helix 25held September 2024 together by base pairing  Core of DNA double helix = bases on complimentary strands held together by H bonds  C (pyrimidine) always pairs with G (purine)  T (pyrimidine) always pairs with A (purine) A-U bonding occurs during gene transcription G-C A-T Which interaction is the strongest? A =T G = C DNA Replication 18 3 Hydrogen bonds 2 Hydrogen bonds  25 September 2024 DNA double helix – structural features  Hydrophobic bases are in the centre of the duplex away from water, and stabilised by H-bonding between bases on complimentary strands  Hydrophilic sugar-phosphate backbone stabilised by electrostatic and H-bonding interactions with water DNA Replication 19  25 September 2024 DNA double helix – structural features  Stacked bases have weak transient electrostatic interactions van der Waal’s forces (pi-pi interactions) DNA Replication 20  25 September 2024 RNA – structural features base pairing no base ↑ pairing · gives RNA stability  Single-stranded RNA molecules adopt secondary structures through base-pairing for similar reasons DNA Replication 21  25 September 2024 Nucleic acids defined by size and direction  The only difference between nucleotides in a DNA/RNA polymer is the base on individual monomeric units  Therefore the polymer is described by the sequence and number of bases  Thus size is expressed in the number of monomeric units e.g. X bases long X base pairs long DNA Replication 22  25 September 2024 Nucleic acids defined by size and direction  Nucleotides always added to the 3’- end of a polynucleotide chain  The -phosphate of the new nucleotide reacts with the 3’-OH group of the polymer to form a 3’-5’ phosphodiester bond  The nucleotide being added must base pair with the base on the template strand before linkage to chain Chain grows in a 5’ to 3’ direction DNA Replication 23  25 September 2024 Nucleic acids defined by size and direction  A nucleic acid chain of any length has a 5’ phosphate at the start and terminates with a 3’ OH  By convention, the base sequence of a DNA chain is written in the 5' to 3' direction e.g 5’-AGTCT- 3’ Complement is 3’-TCAGA-5’ Written as 5’-AGACT- 3’ DNA Replication 24  DNA25replication September 2024 Replication follows several steps that involve multiple proteins called replication enzymes and RNA DNA replication occurs in the S phase of interphase during the cell cycle DNA replication is vital for cell growth, repair, and reproduction DNA Replication 25 DNA replication 25 September 2024 is semi-conservative DNA Replication 26  25 September 2024 DNA replication - mechanism Step 1: Replication Fork Formation 5’ 3’ DNA Replication 27  25 September 2024 DNA replication - mechanism Step 1: Replication Fork Formation DNA helicase Double stranded DNA “unzipped” into two single strands To do this, base pairs broken by DNA helicase Strands form a Y shaped replication fork which is the template for replication to begin DNA Replication 28  25 September 2024 DNA replication - mechanism Step 1: Replication Fork Formation DNA helicase Double stranded DNA “unzipped” into two single strands To do this, base pairs broken by DNA helicase Strands form a Y shaped replication fork which is the template for replication to begin Proteins bind and stabilise unwound single stranded DNA DNA can only replicate from the 5’ to 3’ direction DNA Replication 29  25 September 2024 DNA replication - mechanism Replication fork is bi-directional One strand oriented 3' to 5' direction (=leading strand) Other oriented 5' to 3’ (=lagging strand) Each side replicated by different processes to accommodate the directional difference DNA Replication 30  25 September 2024 DNA replication - mechanism Replication fork is bi-directional One strand oriented 3' to 5' direction (=leading strand) Other oriented 5' to 3’ (=lagging strand) Each side replicated by different processes to accommodate the directional difference DNA Replication 31  DNA25replication September 2024 – leading strand Step 2: RNA primer binding A short piece of RNA (=primer) binds to the 3' end of the strand. The primer is the replication starting point Primers generated by the enzyme DNA primase DNA Replication 32  DNA25replication September 2024 – leading strand Step 3: Elongation DNA pol  In humans, DNA polymerase  binds to the strand at the site of the primer and adds new complementary base pairs to the strand during replication After extending approx 20 base pairs, elongation taken over by DNA pol  DNA pol  and  have proofreading 3'->5' exonuclease activity Prevents incorporation of incorrect nucleotides DNA Replication 33  DNA25replication September 2024 – leading strand Step 3: Elongation DNA pol  Results in the synthesis of one continuous replicated DNA strand in the 5’-3’ direction DNA Replication 34  25 September 2024 DNA replication - mechanism Replication fork is bi-directional One strand oriented 3' to 5' direction (=leading strand) Other oriented 5' to 3’ (=lagging strand) Each side replicated by different processes to accommodate the directional difference DNA Replication 35  DNA25replication September 2024 – lagging strand Steps 1 = same as leading strand Step 2: RNA primer binding Step 3: Elongation DNA pol  Direction DNA pol  of replication DNA pol  Multiple RNA primers needed, each only several bases apart DNA pol  generates complimentary DNA (Okazaki fragments) to the strand between the RNA primers Replication discontinuous as the new fragments are not joined DNA Replication 36  DNA25replication September 2024 – both strands Step 4: Termination RNA primers degraded and filled by action of RNAse H and DNA pol  DNA ligase joins any breaks in the leading and lagging strands to generate continuous double stranded DNA DNA Replication 37  DNA replication occurs 25 September 2024 at multiple sites Replication cannot just originate from 1 site per chromosome (chromosome = 1 dsDNA strand) – would take too long! Genome (=total DNA in a cell) replicates in 8 hrs Multiple origins of replication with replication forks proceeding in opposite directions necessary DNA Replication 38  DNA replication occurs 25 September 2024 at multiple sites 2 double stranded sister chromatids Replication cannot just originate from 1 site per chromosome (chromosome = 1 dsDNA strand) – would take too long! Genome (=total DNA in a cell) replicates in 8 hrs Multiple origins of replication with replication forks proceeding in opposite directions necessary “Replication bubbles” DNA Replication 39 Inheritance of histones 25 September 2024 after replication Histones removed in front of the replication bubble H3/H4 tetramers remain intact adjacent to the DNA after synthesis New H3/H4 tetramer cores bind followed by H2A/H2B. DNA Replication 40 Inheritance of histones 25 September 2024 after replication Histones removed in front of the replication bubble H3/H4 tetramers remain intact adjacent to the DNA after synthesis New H3/H4 tetramer cores bind followed by H2A/H2B. DNA Replication 41 Inheritance of histones 25 September 2024 after replication Histones removed in front of the replication bubble H3/H4 tetramers remain intact adjacent to the DNA after synthesis New H3/H4 tetramer cores bind followed by H2A/H2B. DNA Replication 42  Histones involved 25 September 2024 in epigenetic inheritance Epigenetics = heritable changes in phenotype/cell behaviour or gene expression in cells caused by changes other than in the DNA base sequence that control the activity of genes Examples of changes: Histone modifications E.g. acetylation of Lys, methylation of Lys and Arg) DNA modifications E.g. methylation of cytosine DNA Replication 43  Histones involved 25 September 2024 in epigenetic inheritance Epigenetic modifications alter chromatin structure to control accessibility of transcription factors and co-activators necessary for gene transcription Epigenetic marks can be altered by environmental stimuli E.g. smoking, nutritional status Provides a mechanism for environmental factors to be imprinted genetically DNA Replication 44  Histones involved 25 September 2024 in epigenetic inheritance Epigenetic modifications alter chromatin structure to control accessibility of transcription factors and co-activators necessary for gene transcription Epigenetic marks can be altered by environmental stimuli E.g. smoking, nutritional status Provides a mechanism for environmental factors to be imprinted genetically DNA Replication 45 DNA POLYMERASES  Eukaryotes have 15 or more  Humans  DNA Polymerase α, δ, ε - nuclear DNA  DNA Polymerase γ, θ - mitochondrial DNA  Lagging strand  Pol α (primase) - makes the RNA primer and adds nucleotides (replicative primer)  Pol δ – synthesises – displaces the RNA primer/DNA to create a flap  Nucleases remove the flap  Okazaki fragments are joined by DNA ligase  Leading strand  Pol ε - synthesises 46 POLYMERASE CHAIN REACTION (PCR)  We can make DNA in vitro  Mimic the natural process of DNA replication  DNA primers can be designed to amplify specific parts of the genome 47  Summary Double stranded DNA versus single stranded RNA structure Strands are directional (from 5 ’phosphate – 3’ hydroxyl) Base pairing critical for structural integrity of DNA and important for stability of RNA DNA/RNA defined by sequence and length (in bases or base pairs [bp]) DNA replication proceeds through several steps each requiring specific enzymes Several important applications of DNA replication processes PCR DNA sequencing Precision medicine 48 25 September 2024 DNA Replication