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Dnareplication

This document discusses several key aspects of DNA structure and function: 1) Chromosomes contain DNA, histone proteins, and some RNA. DNA contains a code made up of four nucleotide bases that determines the sequence of amino acids in proteins. 2) DNA replicates semi-conservatively, with each parent strand serving as a template to produce two new DNA double helices. 3) Replication requires DNA polymerase, nucleotides, and energy and proceeds through initiation, elongation, and termination steps at the replication fork.

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Overview A) CHROMOSOME STRUCTURE B) SEMICONSERVATIVE REPLICATION C) THE REPLICATION PROCESS D) THE DNA BLUEPRINT E) THE GENETIC CODE
Chromosomes in Eukaryotic cells consist of: DNA protein some chromosomal RNA
Chromosomes in Prokaryotic cells consist of DNA only :  and so should not be called ‘chromosomes’
DNA has: 2. positively charged (basic) proteins called histones bonded to it 1. negative charges distributed along its length
Chromatin is the : combination of DNA & histones
Functions of the histones: 1. organise the chromosome physically 2. regulate the activities of the DNA histones
Nearly 2m of DNA are crammed into each human cell. What does this mean? A great deal of information can be stored!!
A closer at DNA:
Four Key elements of DNA structure 1) a double-stranded helix 2) of uniform diameter 3) twisting to the right
4) the two strands running in opposite directions
Overview A) CHROMOSOME STRUCTURE B) SEMICONSERVATIVE REPLICATION C) THE REPLICATION PROCESS D) THE DNA BLUEPRINT E) THE GENETIC CODE
1. Semiconservative replication 2. Conservative replication 3. Dispersive replication Three possible replication patterns:
Semiconservative replication Conservative replication Dispersive replication
Semiconservative replication Each parent strand serves as a template for a new strand and the two new DNA strands each have one old and one new strand Parent strands New / daughter strand
Meselson and Stahl experiment [1958] demonstrates semiconservative replication:
Cells broken open to extract DNA E. coli grown in the presence of 15N (a heavy isotope of Nitrogen) for many generations E. coli placed in medium containing only 14N (a light isotope of Nitrogen) • Cells get heavy-labeled DNA Sampled at: 0 min 1 2 3 40 min 20 min Suspended DNA in cesium chloride (CsCl) solution. 4 15N medium
CsCl density gradient centrifugation 5 15N14N DNA Both strands heavy F1 generation DNA (one heavy/one light strand) 0 min 20 min 40 min F2 generation DNA:  Two light strands  (one heavy/one light strand)
Three rounds of replication: Original DNA 1st Round: 2nd Round: 3rd Round: 0 min 20 min 40 min 60 min?
Overview A) CHROMOSOME STRUCTURE B) SEMICONSERVATIVE REPLICATION C) THE REPLICATION PROCESS D) THE DNA BLUEPRINT E) THE GENETIC CODE
FOUR requirements for DNA to replicate 1. DNA to act as a template for complementary base pairing. 2. The four deoxyribonucleoside triphosphates: dATP, dGTP, dCTP & dTTP.
The nucleotides arrive as nucleosides – DNA bases with P–P–P • P-P-P = energy for bonding – DNA bases arrive with their own energy source for bonding dATP dGTP dTTP dCTP 3. A source of chemical energy is needed to drive this highly endergonic reaction.
DNA Polymerase III 4. A DNA polymerase III enzyme brings substrates to the template and catalyses the reactions.
energy ATPGTPTTPCTP Energy of Replication Where does energy for bonding usually come from? ADPAMPGMPTMPCMP modified nucleotide energy We come with our own energy! And we leave behind a nucleotide! You remember ATP! Are there other ways to get energy out of it? Are there other energy nucleotides? You bet!
DNA Template & dATP New strand Template strand 5’ end 3’ end Sugar A T Base C G G C A C OH P P 3’ end 5’ end 5’ end A T C G G C A C T 3’ endPyrophosphate 2 P OH Phosphate 5’ end deoxyribonucleoside triphosphate nucleotide
DNA replication occurs in two steps: 1. DNA is locally denatured (unwound) WHY? To separate the two template strands and make them available for base pairing. Unzipping of DNA
DNA replication occurs in two steps: 2. The new nucleotides are linked by covalent bonding to each growing strand in a sequence determined by complementary base pairing.
REMEMBER: Nucleotides are always added to the growing strand at the 3’ end – the end at which the DNA strand has a free –OH group on the 3’ carbon of its terminal deoxyribose
Three Stages of replication 1) Initiation – occurs at the origin of replication 2) Elongation – involves the addition of new nucleotides based on complementarity of the template strand 3) Termination – occurs at a specific termination site
Origin of replication Site where DNA synthesis starts
A eukaryotic chromosome  May have hundreds or even thousands of replication origins DNA is replicated simultaneously at the origins.
Replication fork is the : point at which the two strands of DNA are separated to allow replication of each strand
• Each bacterial DNA has only one Origin of replication
Directionality of the DNA strands at a replication fork Leading strand Lagging strand Fork movement
Directionality of the DNA strands at a replication fork Leading strand Lagging strand Fork movement
Protein Role DNA helicases Unwinds the double helix RNA primase Synthesises RNA primers Single-strand binding proteins Keep the two strands separated DNA polymerase I Erases primer and fills gaps DNA polymerase II [not in syllabus] Proofreading of DNA DNA polymerase III Synthesises DNA; proofreading DNA ligase Joins the ends of DNA segments; DNA repair
Replication: 1st step • Unwind DNA – helicase enzyme • unwinds part of DNA helix • stabilised by single-stranded binding proteins single-stranded binding proteins replication fork helicase
A primer is : - required to start DNA replication—a short single strand of RNA. - synthesised by primase. Then DNA polymerase III begins adding nucleotides to the 3′ end of the primer.
Many Proteins at the Replication Fork
Identical base sequences 5’ 5’ 3’ 3’ 5’ 5’ 3’ 3’
• DNA polymerases: 1. can synthesise DNA only in the 5’ to 3’ direction 2. cannot initiate DNA synthesis Problem at 3’ ends of Eukaryotic Chromosomes
Label structures at the Replication Fork a. Leading strand template b. Leading strand c. Lagging strand d. Lagging strand template e. RNA primer f. Okazaki fragment
The Two New Strands Form in Different Ways
Leading strand (continuous) Lagging strand (discontinuous)
How are Okazaki fragments linked? Each Okazaki fragment requires a primer. The final phosphodiester linkage between fragments is catalyzed by DNA ligase.
The Lagging Strand Story
The Lagging Strand Story
Many Proteins at the Replication Fork
Two dimensional view of a replication fork Direction of synthesis on leading strand 3’ 5’ 3’ 5’ 3’ 5’
Proofreading procedure • DNA replication is not perfect due to: 1) the high speed of replication - (1000 nucleotides per second) 2) spontaneous chemical flip-flops in the bases • occasionally DNA polymerase incorporates incorrectly matched bases
If bases are paired incorrectly, the nucleotide is removed. Proofreading is done by several DNA polymerases including DNA polymerase II
Editing & proofreading DNA • 1000 bases/second = lots of errors! • DNA polymerase I – proofreads & corrects mistakes – repairs mismatched bases – removes abnormal bases • repairs damage throughout life – reduces error rate from 1 in 10,000 to 1 in 100 million bases
Fast & accurate! • It takes E. coli <1 hour to copy 5 million base pairs in its single chromosome – divide to form two identical daughter cells • Human cell copies its 6 billion bases & divide into daughter cells in only few hours – remarkably accurate – only ~1 error per 100 million bases – ~30 errors per cell cycle
What is the advantage of the one-way directionality of the DNA structure? Allows the proofreading enzymes to recognise the parental strand, running in one direction, as the ‘right stuff’.
Overview A) CHROMOSOME STRUCTURE B) SEMICONSERVATIVE REPLICATION C) THE REPLICATION PROCESS D) THE DNA BLUEPRINT E) THE GENETIC CODE BLUEPRINT: a design plan or other technical drawing
DNA ‘Blueprint’ • every cell in the body has the same "blueprint" or the same DNA • blueprint of a house tell the builders how to construct a house
Importance of the DNA ‘Blueprint’ Tells the cell how to build the organism.
How is it possible for cells to have: the SAME DNA different structures & functions? BUT
Proteins are a cell’s “molecular workers” ANSWER: Every cell contains a particular set of proteins Ovum must have receptors to bind the sperm head. Phagocyte must have receptors to engulf the microbe.
If all body cells have the SAME DNA, explain why only the pancreas makes insulin? A cell has the ability to turn off most genes and only work with the genes necessary to do a job.
DNA ‘Blueprint’ • information by itself, does not do anything – e.g. a blueprint may describe the structure of a house in great detail, but unless that information is translated into action, no house will ever be built • likewise, although the base sequence of DNA, the “molecular blueprint” of every cell contains an incredible amount of information, DNA cannot carry out any action on it own
Central dogma: flow of information is from the: DNA of a cell’s genes the proteins that actually carry out the cell’s functions RNADNA Protein to
What is ‘junk DNA’? • 98.5% of human DNA does not code for proteins • Introns (old name: junk DNA) – - the regions of DNA that do not code for proteins • Exons – - the sections of DNA that code for proteins
Split genes: • contain exons and introns • are found only in eukaryotic cells
Exons & Introns: Gene DNA Translation Protein A Protein B Alternative splicing
Evidence for the role of DNA in inheritance: the Hershey and Chase experiment (1952) Martha Chase Alfred Hershey
Hershey and Chase set out to determine whether the: protein or DNA enters the bacterial cells.
• Bacteriophage - a particular type of virus which specifically attacks bacterial cells • bacteriophage T2 : attacks the bacterium Escherichia coli consists of a protein coat and DNA
Which elements to follow? DNA: in nucleotide Protein: BOTH proteins & DNA: C, H, O, N S P in methionine + cysteine
This experiment confirmed that: DNA from bacteriophages infected bacteria Phage head Tail Tail fiber DNA Bacterial cell 100nm
DNA enters bacteria !!
Overview A) CHROMOSOME STRUCTURE B) SEMICONSERVATIVE REPLICATION C) THE REPLICATION PROCESS D) THE DNA BLUEPRINT E) THE GENETIC CODE
What does DNA code for? DNA specifies only the production of protein synthesis
DNA nucleotide base sequence: determines the amino acid sequence of protein molecules
GENETIC CODE is the relationship between the: bases and amino acids
The code • DNA nucleotide bases:- adenine, guanine, cytosine and thymine • RNA has four nucleotide bases:- adenine, guanine, cytosine and uracil • this ‘alphabet’ of 4 letters is responsible for carrying the code that results in the synthesis of a potentially infinite number of protein molecules
How many bases code for one amino acid? Recall that there are 20 different amino acids in proteins. Only 4 amino acids would be possible. A, T, C, G1? 2? 3? 16 amino acids would be possible: still not large enough. e.g. AU, CU, or CC. 42 = 16 64 amino acids would be possible: e.g. AUU, GCG, or UGC. This vocabulary provides more than enough words to describe the amino acids. 43 = 64
Conclusion: The code is a triplet code i.e. three bases code for one amino acid.
Codon: a set of three adjacent nucleotides, also called triplet, in DNA or mRNA that designates a specific amino acid to be incorporated into a polypeptide
Six features of the genetic code 1. Triplet code 2. Specificity 3. Degeneracy 4. Universality 5. Non-overlapping 6. Punctuated
1) The code is a triplet code • the DNA code for a protein is first copied into messenger RNA (mRNA) before a protein is made • mRNA is complementary to the DNA DNA mRNA
RNA base sequence DNA RNA DNA A – T C – G RNA A – U C – G
One mRNA molecule may contain hundreds or even thousands of bases  the cell recognises where the code for a protein starts and stops as the mRNA has: START CODON STOP CODON start and stop codons
64 codons in all 61 for amino acids 3 ‘stop codons’ (UAA, UAG, UGA) 1 ‘start codon’ (AUG – codes for methionine)
Codons in RNA
 Methionine is specified by the codon AUG - known as the start codon  Note: it may be removed after the protein is synthesised All proteins originally begin with the amino acid methionine. Why?
When the ribosome encounters a stop codon, it releases the : 1. newly synthesised protein 2. mRNA
2) The code is specific (non ambiguous) • each triplet code specifies only one amino acid • e.g. UUU = phenylalanine
3) The code is degenerate Valine GUU GUC GUA GUG  a given amino acid may be coded for by more than one codon 64 codons and only 20 amino acids: so some amino acids are coded for by several codons – exceptions [next slide]: Tyrosine UAU UAC Lysine AAA AAG
Tryptophan UGG Methionine AUG
First TWO bases determine the amino acid • Third Base is usually less specific than the first two. • This is also known as the "Wobble Hypothesis" because often the: Valine GUU GUC GUA GUG third base can change BUT the amino acid remains the same. Wobble position of a codon refers to the 3rd nucleotide in a codon
What is the advantage of a degenerate code? This allows for possible mutations to be less damaging.
No change in polypeptide:
Polypeptide structure is changed • deletion or addition of one or two bases, leads to a change in reading frame (reading sequence) THE FAT CAT ATE THE BIG RAT Delete C: THE FAT ATA TET HEB IGR AT Insert A: THE FAT ATA ATE THE BIG RAT
Six features of the genetic code 1. Triplet code 2. Specificity 3. Degeneracy 4. Universality 5. Non-overlapping 6. Punctuated
4) The code is nearly universal • the genetic code is the same in all organisms, except in: e.g. AGA = arginine in: all organisms whose genetic code has been studied mitochondria protozoan nuclear DNAand
The universality of the genetic code is among the strongest evidence that all living things share a common evolutionary heritage
What is the importance of the universality of the code? GENETIC ENGINEERING IS POSSIBLE
Aim: to map out the entire genetic code of a human -2.1 million base pairs -(30,000 – 40,000 protein coding genes) The Human Genome Project (1990 – 2003)
The Human Genome Project (1990 – 2003)
What is the ‘Genome’? The total DNA in an organism
The human genome = 46 chromosomes The total DNA in an organism
What is the size of a gene? • average gene in humans: 3000 bases • but sizes vary greatly • the largest known human gene: - 2.4 million bases
Six features of the genetic code 1. Triplet code 2. Specificity 3. Degeneracy 4. Universality 5. Non-overlapping 6. Punctuated
5) The code is non-overlapping non-overlapping: - no base of a given triplet contributes to part of the code of the adjacent triplet non-overlappingoverlapping
• the genetic code is read in groups (or “words”) of three nucleotides • after reading one triplet, the “reading frame” shifts over the next three letters, not just one or two
Six features of the genetic code 1. Triplet code 2. Specificity 3. Degeneracy 4. Universality 5. Non-overlapping 6. Punctuated
6. The code is punctuated: REMEMBER: Excluding the start & stop codons, the actual code determining the sequence of amino acids is UNPUNCTUATED NOTE: according to the syllabus, the code is punctuated due to start and stop codons however the majority of text books consider the code as being unpunctuated i.e. comma less
MUTATIONS
A mutation is a change in the • amount, arrangement or structure of the DNA of an organism
A mutation produces a change in the genotype & is passed on when a cell nucleus divides by:  mitosis or  meiosis from the mutant cell Mutant daughter cells Mutant daughter cells Mutant cell Mutant cell
Which type of mutation can be inherited by the offspring? germinal somatic Occur in somatic cells: are NOT passed on the offspring Occur in gamete cells: are passed on to the offspring
A mutation may result in the change in appearance of a characteristic of a population e.g. red eyes in Drosophila appeared in 1909
e.g. dark-coloured moth appeared in 1848 The "typica" form of the moth. The "carbonaria" form.
occur in: any gene at any time be: Mutations can Spontaneous Induced
Spontaneous Mutations:  are permanent changes in the genome that occur without any outside influence  occur because the machinery of the cell is imperfect Both chromatids are sent to one daughter cell, the other gets none. One chromatid goes to each daughter cell.
Induced Mutations:  occur when some outside agent causes a permanent change in DNA  mutagens:  anything that causes a mutation  examples: • Asbestos • Tar from tobacco • Ionising radiation e.g. UV • Pesticides • Caffeine
Mutation rates vary between organisms In general, the mutation rate in: unicellular eukaryotes bacteria Chernobyl disaster was a catastrophic nuclear accident that occurred on 26 April 1986 is roughly 0.003 mutations per genome per generation. Chernobyl: mutant dog
Ionising radiation is radiation that: carries enough energy to liberate electrons from atoms or molecules, thereby ionizing them.
Ionising radiation e.g. UV, X-rays, -rays
Ionising radiation damages the DNA UV light causes adjacent thymines to cross link
Mutations can be: Chromosomal [covered in 2nd year] Gene mutations or point mutations: INSERTION INVERSION DELETION SUBSTITUTION describe a change in the structure of DNA at a single locus 1 2
Fig. 12 Gene or point mutation 1) INSERTION: the addition of an extra nucleotide A GT G C A T A TT G A C A G 2) DELETION: involves the loss of a nucleotide A GT G C A T A TT C A G
Fig. 12 Gene or point mutation 4) SUBSTITUTION: a particular base is substituted by another (e.g. sickle-cell anaemia) A GT G C A T A TT G T A G 3) INVERSION: two nucleotides become arranged in the wrong order A GT G C A T T TA G C A G
Sickle Cell Anaemia in humans is an example of base substitution • a base in one of the genes involved in producing haemoglobin is substituted • at position 14 in the DNA: thymine is replaced by adenine
Sickle Cell Anaemia:  at low oxygen tensions, haemoglobin S crystallises in the red cells distorting them into a sickle shape
Point mutations No mutation DNA level TTC TTT ATC TCC mRNA level AAG AAA UAG AGG Protein level Lys Lys STOP Arg Silent Nonsense Missense Missense mutation Nonsense mutation is a point mutation in a sequence of DNA that results in a premature stop codon is a point mutation that results in the substitution of one amino acid in protein for another
Frameshift mutations The addition or deletion of a single base has much more profound consequences than does the substitution of one base for another THE CAT SAW THE DOG
A frameshift mutation: alters the reading frame in the mRNA downstream of the mutation TA deleted
Changing the reading frame early in a gene, and thus in its mRNA transcript, means that the majority of the protein will be altered. Amino acid Deletion of a single nucleotide DNA bases Original DNA code for an amino acid sequence. Incorrect amino acid sequence, which may produce a malfunctioning protein.
End-Of-Year SEP 2013 Use your knowledge of the genetic code to explain statements (a) and (b) below. Use your knowledge of genetic mutations to answer statements (c), (d) and (e). [5 marks each] i) Distinguish between a base substitution and an inversion. i) Distinguish between a deletion and an insertion. ii) Explain how deletions and insertions lead to frameshift mutations
Use your knowledge of biology to explain the following. The structure of the DNA molecule permits vast amounts of information to be stored. (5 marks) Question: [SEP, 2007] 1. Information on the DNA molecule is in the form of a sequence of bases, where three consecutive bases specify an amino acid. Thus a small number of bases are needed to code for an amino acid. Considering that DNA within a eukaryotic cell is 2m long, it allows for a large amount of information to be stored. 2. In many eukaryotic cells, split genes occur. These contain regions which code for the protein called exons and introns which do not code. The way in which the exons are linked together determines the type of polypeptide to be formed. Thus one gene can form a number of closely related polypeptides.
THE END

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Dnareplication

  • 2.
    Overview A) CHROMOSOME STRUCTURE B)SEMICONSERVATIVE REPLICATION C) THE REPLICATION PROCESS D) THE DNA BLUEPRINT E) THE GENETIC CODE
  • 3.
    Chromosomes in Eukaryoticcells consist of: DNA protein some chromosomal RNA
  • 4.
    Chromosomes in Prokaryoticcells consist of DNA only :  and so should not be called ‘chromosomes’
  • 5.
    DNA has: 2. positivelycharged (basic) proteins called histones bonded to it 1. negative charges distributed along its length
  • 6.
    Chromatin is the: combination of DNA & histones
  • 7.
    Functions of thehistones: 1. organise the chromosome physically 2. regulate the activities of the DNA histones
  • 8.
    Nearly 2m ofDNA are crammed into each human cell. What does this mean? A great deal of information can be stored!!
  • 9.
  • 10.
    Four Key elementsof DNA structure 1) a double-stranded helix 2) of uniform diameter 3) twisting to the right
  • 11.
    4) the twostrands running in opposite directions
  • 12.
    Overview A) CHROMOSOME STRUCTURE B)SEMICONSERVATIVE REPLICATION C) THE REPLICATION PROCESS D) THE DNA BLUEPRINT E) THE GENETIC CODE
  • 13.
    1. Semiconservative replication 2.Conservative replication 3. Dispersive replication Three possible replication patterns:
  • 14.
  • 15.
    Semiconservative replication Each parentstrand serves as a template for a new strand and the two new DNA strands each have one old and one new strand Parent strands New / daughter strand
  • 16.
    Meselson and Stahlexperiment [1958] demonstrates semiconservative replication:
  • 17.
    Cells broken open toextract DNA E. coli grown in the presence of 15N (a heavy isotope of Nitrogen) for many generations E. coli placed in medium containing only 14N (a light isotope of Nitrogen) • Cells get heavy-labeled DNA Sampled at: 0 min 1 2 3 40 min 20 min Suspended DNA in cesium chloride (CsCl) solution. 4 15N medium
  • 18.
    CsCl density gradient centrifugation 5 15N14N DNA Both strands heavy F1 generation DNA(one heavy/one light strand) 0 min 20 min 40 min F2 generation DNA:  Two light strands  (one heavy/one light strand)
  • 21.
    Three rounds of replication: Original DNA 1st Round: 2ndRound: 3rd Round: 0 min 20 min 40 min 60 min?
  • 23.
    Overview A) CHROMOSOME STRUCTURE B)SEMICONSERVATIVE REPLICATION C) THE REPLICATION PROCESS D) THE DNA BLUEPRINT E) THE GENETIC CODE
  • 24.
    FOUR requirements forDNA to replicate 1. DNA to act as a template for complementary base pairing. 2. The four deoxyribonucleoside triphosphates: dATP, dGTP, dCTP & dTTP.
  • 25.
    The nucleotides arriveas nucleosides – DNA bases with P–P–P • P-P-P = energy for bonding – DNA bases arrive with their own energy source for bonding dATP dGTP dTTP dCTP 3. A source of chemical energy is needed to drive this highly endergonic reaction.
  • 26.
    DNA Polymerase III 4. ADNA polymerase III enzyme brings substrates to the template and catalyses the reactions.
  • 27.
    energy ATPGTPTTPCTP Energy of Replication Wheredoes energy for bonding usually come from? ADPAMPGMPTMPCMP modified nucleotide energy We come with our own energy! And we leave behind a nucleotide! You remember ATP! Are there other ways to get energy out of it? Are there other energy nucleotides? You bet!
  • 28.
    DNA Template &dATP New strand Template strand 5’ end 3’ end Sugar A T Base C G G C A C OH P P 3’ end 5’ end 5’ end A T C G G C A C T 3’ endPyrophosphate 2 P OH Phosphate 5’ end deoxyribonucleoside triphosphate nucleotide
  • 30.
    DNA replication occursin two steps: 1. DNA is locally denatured (unwound) WHY? To separate the two template strands and make them available for base pairing. Unzipping of DNA
  • 31.
    DNA replication occursin two steps: 2. The new nucleotides are linked by covalent bonding to each growing strand in a sequence determined by complementary base pairing.
  • 32.
    REMEMBER: Nucleotides are alwaysadded to the growing strand at the 3’ end – the end at which the DNA strand has a free –OH group on the 3’ carbon of its terminal deoxyribose
  • 34.
    Three Stages ofreplication 1) Initiation – occurs at the origin of replication 2) Elongation – involves the addition of new nucleotides based on complementarity of the template strand 3) Termination – occurs at a specific termination site
  • 35.
    Origin of replication Sitewhere DNA synthesis starts
  • 36.
    A eukaryotic chromosome May have hundreds or even thousands of replication origins DNA is replicated simultaneously at the origins.
  • 37.
    Replication fork isthe : point at which the two strands of DNA are separated to allow replication of each strand
  • 38.
    • Each bacterialDNA has only one Origin of replication
  • 39.
    Directionality of theDNA strands at a replication fork Leading strand Lagging strand Fork movement
  • 40.
    Directionality of theDNA strands at a replication fork Leading strand Lagging strand Fork movement
  • 41.
    Protein Role DNA helicasesUnwinds the double helix RNA primase Synthesises RNA primers Single-strand binding proteins Keep the two strands separated DNA polymerase I Erases primer and fills gaps DNA polymerase II [not in syllabus] Proofreading of DNA DNA polymerase III Synthesises DNA; proofreading DNA ligase Joins the ends of DNA segments; DNA repair
  • 42.
    Replication: 1st step •Unwind DNA – helicase enzyme • unwinds part of DNA helix • stabilised by single-stranded binding proteins single-stranded binding proteins replication fork helicase
  • 43.
    A primer is: - required to start DNA replication—a short single strand of RNA. - synthesised by primase. Then DNA polymerase III begins adding nucleotides to the 3′ end of the primer.
  • 44.
    Many Proteins atthe Replication Fork
  • 45.
  • 46.
    • DNA polymerases: 1.can synthesise DNA only in the 5’ to 3’ direction 2. cannot initiate DNA synthesis Problem at 3’ ends of Eukaryotic Chromosomes
  • 47.
    Label structures atthe Replication Fork a. Leading strand template b. Leading strand c. Lagging strand d. Lagging strand template e. RNA primer f. Okazaki fragment
  • 48.
    The Two New StrandsForm in Different Ways
  • 49.
  • 50.
    How are Okazakifragments linked? Each Okazaki fragment requires a primer. The final phosphodiester linkage between fragments is catalyzed by DNA ligase.
  • 51.
  • 52.
  • 53.
    Many Proteins atthe Replication Fork
  • 54.
    Two dimensional viewof a replication fork Direction of synthesis on leading strand 3’ 5’ 3’ 5’ 3’ 5’
  • 55.
    Proofreading procedure • DNAreplication is not perfect due to: 1) the high speed of replication - (1000 nucleotides per second) 2) spontaneous chemical flip-flops in the bases • occasionally DNA polymerase incorporates incorrectly matched bases
  • 56.
    If bases arepaired incorrectly, the nucleotide is removed. Proofreading is done by several DNA polymerases including DNA polymerase II
  • 57.
    Editing & proofreadingDNA • 1000 bases/second = lots of errors! • DNA polymerase I – proofreads & corrects mistakes – repairs mismatched bases – removes abnormal bases • repairs damage throughout life – reduces error rate from 1 in 10,000 to 1 in 100 million bases
  • 58.
    Fast & accurate! •It takes E. coli <1 hour to copy 5 million base pairs in its single chromosome – divide to form two identical daughter cells • Human cell copies its 6 billion bases & divide into daughter cells in only few hours – remarkably accurate – only ~1 error per 100 million bases – ~30 errors per cell cycle
  • 59.
    What is theadvantage of the one-way directionality of the DNA structure? Allows the proofreading enzymes to recognise the parental strand, running in one direction, as the ‘right stuff’.
  • 60.
    Overview A) CHROMOSOME STRUCTURE B)SEMICONSERVATIVE REPLICATION C) THE REPLICATION PROCESS D) THE DNA BLUEPRINT E) THE GENETIC CODE BLUEPRINT: a design plan or other technical drawing
  • 61.
    DNA ‘Blueprint’ • everycell in the body has the same "blueprint" or the same DNA • blueprint of a house tell the builders how to construct a house
  • 62.
    Importance of theDNA ‘Blueprint’ Tells the cell how to build the organism.
  • 63.
    How is itpossible for cells to have: the SAME DNA different structures & functions? BUT
  • 64.
    Proteins are acell’s “molecular workers” ANSWER: Every cell contains a particular set of proteins Ovum must have receptors to bind the sperm head. Phagocyte must have receptors to engulf the microbe.
  • 65.
    If all bodycells have the SAME DNA, explain why only the pancreas makes insulin? A cell has the ability to turn off most genes and only work with the genes necessary to do a job.
  • 66.
    DNA ‘Blueprint’ • informationby itself, does not do anything – e.g. a blueprint may describe the structure of a house in great detail, but unless that information is translated into action, no house will ever be built • likewise, although the base sequence of DNA, the “molecular blueprint” of every cell contains an incredible amount of information, DNA cannot carry out any action on it own
  • 67.
    Central dogma: flowof information is from the: DNA of a cell’s genes the proteins that actually carry out the cell’s functions RNADNA Protein to
  • 68.
    What is ‘junkDNA’? • 98.5% of human DNA does not code for proteins • Introns (old name: junk DNA) – - the regions of DNA that do not code for proteins • Exons – - the sections of DNA that code for proteins
  • 69.
    Split genes: • containexons and introns • are found only in eukaryotic cells
  • 70.
    Exons & Introns: Gene DNA Translation ProteinA Protein B Alternative splicing
  • 71.
    Evidence for therole of DNA in inheritance: the Hershey and Chase experiment (1952) Martha Chase Alfred Hershey
  • 72.
    Hershey and Chaseset out to determine whether the: protein or DNA enters the bacterial cells.
  • 73.
    • Bacteriophage -a particular type of virus which specifically attacks bacterial cells • bacteriophage T2 : attacks the bacterium Escherichia coli consists of a protein coat and DNA
  • 74.
    Which elements tofollow? DNA: in nucleotide Protein: BOTH proteins & DNA: C, H, O, N S P in methionine + cysteine
  • 78.
    This experiment confirmedthat: DNA from bacteriophages infected bacteria Phage head Tail Tail fiber DNA Bacterial cell 100nm
  • 79.
  • 80.
    Overview A) CHROMOSOME STRUCTURE B)SEMICONSERVATIVE REPLICATION C) THE REPLICATION PROCESS D) THE DNA BLUEPRINT E) THE GENETIC CODE
  • 81.
    What does DNAcode for? DNA specifies only the production of protein synthesis
  • 82.
    DNA nucleotide basesequence: determines the amino acid sequence of protein molecules
  • 83.
    GENETIC CODE isthe relationship between the: bases and amino acids
  • 84.
    The code • DNAnucleotide bases:- adenine, guanine, cytosine and thymine • RNA has four nucleotide bases:- adenine, guanine, cytosine and uracil • this ‘alphabet’ of 4 letters is responsible for carrying the code that results in the synthesis of a potentially infinite number of protein molecules
  • 85.
    How many basescode for one amino acid? Recall that there are 20 different amino acids in proteins. Only 4 amino acids would be possible. A, T, C, G1? 2? 3? 16 amino acids would be possible: still not large enough. e.g. AU, CU, or CC. 42 = 16 64 amino acids would be possible: e.g. AUU, GCG, or UGC. This vocabulary provides more than enough words to describe the amino acids. 43 = 64
  • 86.
    Conclusion: The code isa triplet code i.e. three bases code for one amino acid.
  • 87.
    Codon: a set ofthree adjacent nucleotides, also called triplet, in DNA or mRNA that designates a specific amino acid to be incorporated into a polypeptide
  • 88.
    Six features ofthe genetic code 1. Triplet code 2. Specificity 3. Degeneracy 4. Universality 5. Non-overlapping 6. Punctuated
  • 89.
    1) The codeis a triplet code • the DNA code for a protein is first copied into messenger RNA (mRNA) before a protein is made • mRNA is complementary to the DNA DNA mRNA
  • 90.
    RNA base sequence DNA RNA DNA A– T C – G RNA A – U C – G
  • 91.
    One mRNA moleculemay contain hundreds or even thousands of bases  the cell recognises where the code for a protein starts and stops as the mRNA has: START CODON STOP CODON start and stop codons
  • 92.
    64 codons inall 61 for amino acids 3 ‘stop codons’ (UAA, UAG, UGA) 1 ‘start codon’ (AUG – codes for methionine)
  • 93.
  • 94.
     Methionine isspecified by the codon AUG - known as the start codon  Note: it may be removed after the protein is synthesised All proteins originally begin with the amino acid methionine. Why?
  • 95.
    When the ribosomeencounters a stop codon, it releases the : 1. newly synthesised protein 2. mRNA
  • 96.
    2) The codeis specific (non ambiguous) • each triplet code specifies only one amino acid • e.g. UUU = phenylalanine
  • 97.
    3) The codeis degenerate Valine GUU GUC GUA GUG  a given amino acid may be coded for by more than one codon 64 codons and only 20 amino acids: so some amino acids are coded for by several codons – exceptions [next slide]: Tyrosine UAU UAC Lysine AAA AAG
  • 98.
  • 99.
    First TWO basesdetermine the amino acid • Third Base is usually less specific than the first two. • This is also known as the "Wobble Hypothesis" because often the: Valine GUU GUC GUA GUG third base can change BUT the amino acid remains the same. Wobble position of a codon refers to the 3rd nucleotide in a codon
  • 100.
    What is theadvantage of a degenerate code? This allows for possible mutations to be less damaging.
  • 101.
    No change inpolypeptide:
  • 102.
    Polypeptide structure ischanged • deletion or addition of one or two bases, leads to a change in reading frame (reading sequence) THE FAT CAT ATE THE BIG RAT Delete C: THE FAT ATA TET HEB IGR AT Insert A: THE FAT ATA ATE THE BIG RAT
  • 103.
    Six features ofthe genetic code 1. Triplet code 2. Specificity 3. Degeneracy 4. Universality 5. Non-overlapping 6. Punctuated
  • 104.
    4) The codeis nearly universal • the genetic code is the same in all organisms, except in: e.g. AGA = arginine in: all organisms whose genetic code has been studied mitochondria protozoan nuclear DNAand
  • 105.
    The universality ofthe genetic code is among the strongest evidence that all living things share a common evolutionary heritage
  • 106.
    What is theimportance of the universality of the code? GENETIC ENGINEERING IS POSSIBLE
  • 107.
    Aim: to map outthe entire genetic code of a human -2.1 million base pairs -(30,000 – 40,000 protein coding genes) The Human Genome Project (1990 – 2003)
  • 108.
    The Human GenomeProject (1990 – 2003)
  • 109.
    What is the‘Genome’? The total DNA in an organism
  • 110.
    The human genome= 46 chromosomes The total DNA in an organism
  • 111.
    What is thesize of a gene? • average gene in humans: 3000 bases • but sizes vary greatly • the largest known human gene: - 2.4 million bases
  • 112.
    Six features ofthe genetic code 1. Triplet code 2. Specificity 3. Degeneracy 4. Universality 5. Non-overlapping 6. Punctuated
  • 113.
    5) The codeis non-overlapping non-overlapping: - no base of a given triplet contributes to part of the code of the adjacent triplet non-overlappingoverlapping
  • 114.
    • the geneticcode is read in groups (or “words”) of three nucleotides • after reading one triplet, the “reading frame” shifts over the next three letters, not just one or two
  • 115.
    Six features ofthe genetic code 1. Triplet code 2. Specificity 3. Degeneracy 4. Universality 5. Non-overlapping 6. Punctuated
  • 116.
    6. The codeis punctuated: REMEMBER: Excluding the start & stop codons, the actual code determining the sequence of amino acids is UNPUNCTUATED NOTE: according to the syllabus, the code is punctuated due to start and stop codons however the majority of text books consider the code as being unpunctuated i.e. comma less
  • 117.
  • 118.
    A mutation isa change in the • amount, arrangement or structure of the DNA of an organism
  • 119.
    A mutation producesa change in the genotype & is passed on when a cell nucleus divides by:  mitosis or  meiosis from the mutant cell Mutant daughter cells Mutant daughter cells Mutant cell Mutant cell
  • 120.
    Which type ofmutation can be inherited by the offspring? germinal somatic Occur in somatic cells: are NOT passed on the offspring Occur in gamete cells: are passed on to the offspring
  • 121.
    A mutation mayresult in the change in appearance of a characteristic of a population e.g. red eyes in Drosophila appeared in 1909
  • 122.
    e.g. dark-coloured mothappeared in 1848 The "typica" form of the moth. The "carbonaria" form.
  • 123.
    occur in: anygene at any time be: Mutations can Spontaneous Induced
  • 124.
    Spontaneous Mutations:  arepermanent changes in the genome that occur without any outside influence  occur because the machinery of the cell is imperfect Both chromatids are sent to one daughter cell, the other gets none. One chromatid goes to each daughter cell.
  • 125.
    Induced Mutations:  occurwhen some outside agent causes a permanent change in DNA  mutagens:  anything that causes a mutation  examples: • Asbestos • Tar from tobacco • Ionising radiation e.g. UV • Pesticides • Caffeine
  • 126.
    Mutation rates varybetween organisms In general, the mutation rate in: unicellular eukaryotes bacteria Chernobyl disaster was a catastrophic nuclear accident that occurred on 26 April 1986 is roughly 0.003 mutations per genome per generation. Chernobyl: mutant dog
  • 127.
    Ionising radiation isradiation that: carries enough energy to liberate electrons from atoms or molecules, thereby ionizing them.
  • 128.
    Ionising radiation e.g.UV, X-rays, -rays
  • 129.
    Ionising radiation damages theDNA UV light causes adjacent thymines to cross link
  • 130.
    Mutations can be: Chromosomal [covered in2nd year] Gene mutations or point mutations: INSERTION INVERSION DELETION SUBSTITUTION describe a change in the structure of DNA at a single locus 1 2
  • 131.
    Fig. 12 Geneor point mutation 1) INSERTION: the addition of an extra nucleotide A GT G C A T A TT G A C A G 2) DELETION: involves the loss of a nucleotide A GT G C A T A TT C A G
  • 132.
    Fig. 12 Geneor point mutation 4) SUBSTITUTION: a particular base is substituted by another (e.g. sickle-cell anaemia) A GT G C A T A TT G T A G 3) INVERSION: two nucleotides become arranged in the wrong order A GT G C A T T TA G C A G
  • 133.
    Sickle Cell Anaemiain humans is an example of base substitution • a base in one of the genes involved in producing haemoglobin is substituted • at position 14 in the DNA: thymine is replaced by adenine
  • 134.
    Sickle Cell Anaemia: at low oxygen tensions, haemoglobin S crystallises in the red cells distorting them into a sickle shape
  • 135.
    Point mutations No mutation DNAlevel TTC TTT ATC TCC mRNA level AAG AAA UAG AGG Protein level Lys Lys STOP Arg Silent Nonsense Missense Missense mutation Nonsense mutation is a point mutation in a sequence of DNA that results in a premature stop codon is a point mutation that results in the substitution of one amino acid in protein for another
  • 136.
    Frameshift mutations The additionor deletion of a single base has much more profound consequences than does the substitution of one base for another THE CAT SAW THE DOG
  • 137.
    A frameshift mutation: altersthe reading frame in the mRNA downstream of the mutation TA deleted
  • 138.
    Changing the readingframe early in a gene, and thus in its mRNA transcript, means that the majority of the protein will be altered. Amino acid Deletion of a single nucleotide DNA bases Original DNA code for an amino acid sequence. Incorrect amino acid sequence, which may produce a malfunctioning protein.
  • 139.
    End-Of-Year SEP 2013 Useyour knowledge of the genetic code to explain statements (a) and (b) below. Use your knowledge of genetic mutations to answer statements (c), (d) and (e). [5 marks each] i) Distinguish between a base substitution and an inversion. i) Distinguish between a deletion and an insertion. ii) Explain how deletions and insertions lead to frameshift mutations
  • 140.
    Use your knowledgeof biology to explain the following. The structure of the DNA molecule permits vast amounts of information to be stored. (5 marks) Question: [SEP, 2007] 1. Information on the DNA molecule is in the form of a sequence of bases, where three consecutive bases specify an amino acid. Thus a small number of bases are needed to code for an amino acid. Considering that DNA within a eukaryotic cell is 2m long, it allows for a large amount of information to be stored. 2. In many eukaryotic cells, split genes occur. These contain regions which code for the protein called exons and introns which do not code. The way in which the exons are linked together determines the type of polypeptide to be formed. Thus one gene can form a number of closely related polypeptides.
  • 141.