DNA and Genetics Learning Outcomes

DNA & Genetics Dr Katherine Karakoula A.Karakoula@wlv.ac.uk 4PY019 – CPP1 Learning Outcomes ➢ Understand differences between DNA & RNA ➢ Transcription, translation and replication ➢ Post-translational modifications ➢ DNA damage - mutations in DNA ➢ Gene expression and eukaryotic control 2 Deoxyribonucleic acid (DNA) ➢ DNA is a polymer composed of two polynucleotide chains that coil around each other to form a double helix. ➢ Carries the genetic instructions for the development, functioning, growth and reproduction of all known organisms and many viruses. 3 ➢ The two DNA strands are known as polynucleotides as they are composed of simpler monomeric units called nucleotides. ➢ Each nucleotide is composed of one of four nitrogen-containing nucleobases (cytosine [C], guanine [G], adenine [A] or thymine [T]), a sugar called deoxyribose, and a phosphate group. 4 Nucleotide     A = Adenine T = Thymine G = Guanine C = Cytosine RNA only: U = Uracil 5 DNA strands ➢ Two strands held together by strong covalent bonds formed between the deoxyribose sugar of one nucleotide and the phosphate of the next nucleotide (phosphate linkage). ➢ The bases are lined up along the sugar-phosphate backbone held together by hydrogen bonds 6 Antiparallel strands ➢ The strands are anti-parellel; run in opposite directions. One runs 3' to 5', the other run 5' to 3'. ➢ This is done by the sugar phosphate backbone twisting around itself in a coil. ➢ The purpose of this twisting is to protect the bases inside it and prevent them from being damaged by the environment. 7 ds DNA ➔ ss DNA  Since CG pairs have 3 H-bonds and AT pairs have 2 Hbonds, the more CG bonds there are in a piece of DNA, the more energy is required to denature it Rosalind Franklin 1953 ➢ Crystallised DNA and X-ray diffraction ➢ It was clear that DNA was in a helix with symmetrically organised bases in centre 9 DNA Is a Double Helix ➢ James Watson and Francis Crick (1953) suggested double-helix model of DNA structure. ➢ Their molecular model of DNA was based on (a) X-ray diffraction image taken by Rosalind Franklin, Maurice Wilkins and Raymond Gosling and (b) the complementary base pairing in DNA. Adapted from dataphys.org 10 Complementary base pairs ➢ The bases of one strand pair up with the bases of another strand by hydrogen bonds. ➢ A purine must always be base paired to a pyrimidine: ❖ A pairs with T (2 hydrogen bonds ❖ G pairs with C (3 hydrogen bonds Complementary: when you read the message on one strand you automatically know the message on the other strand. NOT IDENTICAL but exact same message on both strands. 11 Complementary base pairing 1950 – Erwin Chargaff 12  Adenine = Thymine  Cytosine = Guanine A T G C 30% 30% 20% 20% Total A+T+G+C = 100% 13 Chromosomes and Chromatin Chromatin is the complex of genomic DNA with proteins called histones that support its structure Chromosomes are thread-like structures located inside the nucleus of eukaryotic cells. Each chromosome is made of protein and a single molecule of DNA. 14 Chromosome structure  Chromosomes are the means of packing huge amounts of DNA into the restricted space of the nucleus In most cells, humans have 22 pairs of these chromosomes and 1 pair of the sex chromosomes (XX in females and XY in males) for a total of 46 per cell. Each chromosome has a constriction point called the centromere, which divides the chromosome into two sections, or “arms.” 16 Centromeres Structure: Regions of repetitive DNA that give chromosomes their constricted appearance  Functions: Holds sister chromatids together in mitosis and meiosis and point of attachment of the spindle  Telomeres Structure:  Regions of short repetitive DNA  Humans (TTAGGG) 250 - 1000 repeats  Functions:  Protect chromosomal ends from shortening  Linked with ageing and cancer protection  GENE Genes are section of DNA that provide the instructions for one PROTEIN molecule Chromosomes are made up of GENES. 19 DNA replication 20 DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule. 21 Why is it important? ➢ To make new (identical) copies of chromosomes for cell division (Growth) ➢ To help maintain continuity of genetic information (Reproduction) ➢ Continuous surveillance of DNA and its repair 22 Requirements for DNA replication? ➢ Original DNA template - DNA is a double helix made of two complementary strands. Each strand can be used as a template to create a new DNA molecule. ➢ Free DNA nucleotides – needed to form the new strands. ➢ Replication enzymes ➢ Primers – needed to start the process because DNA polymerase can only add nucleotides to an existing strand of DNA. 23 Three Theories: 1. Semi-conservative  The one Watson and Crick suggested where the two strands of a DNA molecule separate during replication. Each strand then acts as a template for synthesis of a new strand. The conservative hypothesis proposed that the entire DNA molecule acted as a template for the synthesis of an entirely new one. 24 2. Conservative Makes copy of double strand from double strand (without splitting) 3. Dispersive Production of two copies of the DNA, both containing distinct regions of DNA composed of either both original strands or both new strands. 25 Meselson and Stahl 1957 Density Shift Experiments  Labeled DNA with “heavy” Nitrogen  Follow label:  Does it split equally = Semi-conservative Does it stay together = Conservative Does it split randomly = Dispersive 26 Meselson and Stahl 1954 Hybrid of light and heavy DNA ▪ Growth of E. coli in medium, or nutrient broth, containing a "heavy" and light isotope of nitrogen, 15N, 14N ▪ Measurement of DNA’s density using density gradient centrifugation (cesium chloride CsCl). 27 DNA replication is semi-conservative, meaning that each strand in a DNA molecule serves as a template for synthesis of a new, complementary strand. 28 Replication Three main steps in the replication process: 1. Initiation 2. Elongation 3. Termination 29 Replication fork formation ➢ DNA replication starts with a single molecule of DNA made up of two complementary strands. ➢ Before replication begins, ds DNA must be unzipped into two single strands. ➢ The helix structure is unwound by breakage of the weak hydrogen bonds between bases, which are holding the two strands together. This process occurs at several locations on a DNA molecule. 30 ➢ DNA helicase disrupts the hydrogen bonding between base pairs separating thus, the two strands into a Y shape known as replication fork. https://bam.files.bbci.co.uk/bam/live/content/zvvjtfr/large ➢ This area will now be the template for replication to begin. 31 ➢ DNA is directional in both strands 5’ to 3’ and only progresses in the 5’ to 3’ direction ➢ The 5’end has a phosphate group attached while the 3’end has a hydroxyl group attached. ➢ Leading strand 5’ to 3’ - CONTINUOUS ➢ Lagging strand 3’ to 5’ - DISCONTINUOUS 32 https://cdn.serc.carleton.edu/images/microbelife/microbservatories/methods/replication.v3.jpg 33 Enzymes required for DNA replication DNA helicase – unwinds and separate double stranded DNA as it moves along the DNA. Itforms the replication fork by breaking the hydrogen bonds between nucleotide pairs in DNA. DNA Primase – synthesises RNA primers that act as templates for the starting point of DNA replication. DNA Polymerase – synthesises new DNA molecules by adding nucleotides to leading and lagging strands of DNA. Topoisomerase- unwinds and rewinds DNA strands to prevent the DNA from becoming supercoiled. Exonuclease – group of enzymes that remove nucleotide bases from the end of a DNA chain. DNA Ligase – facilitates joining of DNA strands by formation of phosphodiester bonds 34 The parent strand and its complementary DNA strand coils into double helix shape. 35 DNA Replication ➢ DNA from cell to cell, has to be replicated with fidelity ➢ Mistakes – mutations, cancer, etc. ➢ Only one mistake in a billion nucleotides is made by the replication machinery ➢ DNA replication is a result of the coordination of > 10 protein complexes and enzymes 36 Error Correction DNA polymerase proofreads the newly formed strand: 1. Excise bases that are “mismatched” 2. Replace with the correct base 3. Then move forward to next base 37 Transcription & Translation Central dogma of molecular biology The central dogma of molecular biology is the concept that genetic information flows from DNA to mRNA and then to protein. This concept is the basic principle common to all organisms. 3 Eukaryotic structure All eukaryotic cells bacteria. have a membrane enclosed nucleus compared to 4 Eukaryotic cell ➢ In eukaryotes genetic material (DNA) is found in the cell’s nucleus. ➢ Transcription and translation are spatially and temporally separated in eukaryotic cells. ➢ Transcription occurs in the nucleus to produce a pre-mRNA molecule. ➢ The pre-mRNA is typically processed to produce the mature mRNA, which exits the nucleus and is translated in the cytoplasm. Copyright © Pearson Education, Inc. 4 In Transcription: the DNA sequence of a gene is "rewritten" using RNA nucleotides. In eukaryotes, the RNA must go through additional processing steps to become a messenger RNA, or mRNA. In Translation: the sequence of nucleotides in the mRNA is "translated" into a sequence of amino acids in a polypeptide (protein or protein subunit). 42 Cracked the genetic code Made artificial sequences then determined which AAs were joined up together Nirenberg (right) and Matthaei at the National Institutes of Health in 1961 Lysine ACACACACACACAC Threonine Histidine ➢ The genetic code is the set of rules used by living cells to translate information encoded within genetic material (DNA or RNA sequences of nucleotide triplets, or codons) into proteins. ➢ The genetic code is highly similar among all organisms and can be expressed in a simple table with 64 entries. Amino acids are written in the 5’ to 3’ direction. 2 amino acids -selenocysteine and pyrrolysine- use tRNAs that are able to base pair with stop codons in the mRNA during translation. 44 ➢ Amino acids are molecules that combine to form proteins. ➢ Amino acids and proteins are the building blocks of life. When proteins are digested or broken down, amino acids are left. The human body uses amino acids to make proteins to help the body to bread down food. 45 The genetic information for a protein contained in DNA is transcribed into mRNA (messenger RNA), which is synthesized using DNA as a template. This process is called transcription. Transcription takes place in the nucleus. Triplets are groups of three successive nucleotide bases in DNA. Codons Cells decode mRNAs by reading their nucleotides in groups of three, called codons. Here are some features of codons: •Most codons specify an amino acid •Three "stop" codons mark the end of a protein •One "start" codon, AUG, marks the beginning of a protein and also encodes the amino acid methionine Codons in an mRNA are read during translation, beginning with a start codon and continuing until a stop codon is reached. mRNA codons are read from 5' to 3' , and they specify the order of amino acids in a protein from N-terminus (methionine) to Cterminus. 4 bases = 64 possible combinatio ns taken 3 at a time © 2014 Nature Education AUG is an initiation codon; UAA, UAG, and UGA are termination (stop) codons. Suzanne Clancy, Ph.D. & William Brown, Ph.D. (Write Science Right) © 2008 Nature Education Citation: Clancy, S. & Brown, W. (2008) Translation: DNA to mRNA to Protein. Nature Education 1(1):101 Transcription Three main steps in the transcription process: 1. Initiation 2. Elongation 3. Termination 49 Transcription Initiation ➢ Initiation is the beginning of transcription. ➢ TFIID binds to the TATA box via the TBP (TATAbinding protein). ➢ Five more transcription factors and RNA polymerase combine around the TATA box in a series of stages to form a transcription-initiation complex. 50 Elongation ➢ Elongation is the addition of nucleotides to the mRNA strand. ➢ RNA polymerase moves along the DNA template from 3’ to 5’ building the mRNA molecule using complementary base pairing with nucleotides being added to the 3’ end of the growing RNA molecule. 51 Termination The mechanisms of termination are different in prokaryotes and eukaryotes. ➢In bacteria, the polymerase stops transcription at the end of the terminator and mRNA can be translated without further modification. In eukaryotes, the termination of transcription is different for the different RNA polymerases and mRNA needs to be modified before translation takes place. ➢ 52 TRANSLATION 53 During translation process, a protein is synthesised from mRNA by ribosomes. In prokaryotes transcription and translation are coupled; that is, translation begins while the mRNA is still being synthesized while in eukaryotes, transcription occurs in the nucleus and translation occurs in the cytoplasm. Translation is accomplished by the ribosome, which links proteinogenic amino acids in an order specified by messenger RNA (mRNA), using transfer RNA (tRNA) molecules to carry amino acids and to read the mRNA three nucleotides at a time. Consists of 3 phases: Initiation, Elongation, Termination 54 Translation always begins at a start codon, which has the sequence AUG and encodes the amino acid methionine (Met) in most organisms. Thus, every polypeptide typically starts with methionine, although the initial methionine may be snipped off in later processing steps. A start codon is required to begin translation, but the codon AUG can also appear later in the coding sequence of an mRNA, where it simply specifies the amino acid methionine. 55 Translation Initiation Initiation of translation begins with the assembly of the components of the translation system: ❖2 ribosomal subunits (60S and 40S) ❖mRNA template to be translated ❖Aminoacyl tRNA synthetase catalyzes the bonding between specific tRNAs and the amino acids. ❖Peptidyl transferase that connects A site and P site by forming a peptide bond [the nitrogen carbon bond] during elongation phase. ❖Initiation factors eIFs. ❖The codon AUG initiates the process of translation and one of three stop codons i.e. UAA, UAG, or UGA for chain termination. In eukaryotes and archaea, the amino acid encoded by the start codon is methionine. ❖GTP – energy source 56 Ribosomes are complexes of rRNA molecules and proteins and are the cell's protein synthesis factory. Mainly found bound to the endoplasmic reticulum and the nuclear envelope, as well as freely scattered throughout the cytoplasm. Ribosome biogenesis is facilitated by the nucleolus, through the processing and assembly of rRNA into preribosomal particles. 57 Transfer RNA 3 loops , one of which contains the anti codon Anti codon CUU CCG Codon GAA GGC An amino acid residue is attached to the 3’ end Each tRNA has an anticodon for the amino acid codon it carries which are complementary to each other. 59 ➢ Once translation has begun at the start codon, the following codons of the mRNA will be read one by one, in the 5' to 3' direction. ➢ As each codon is read, the matching amino acid is added to the Cterminus of the polypeptide. Most of the codons in the genetic code specify amino acids and are read during this phase of translation. ➢ Translation continues until a stop codon (UAA, UAG, and UGA) is reached. ➢ Unlike start codons, stop codons don't correspond to an amino acid. Instead, they act as "stop" signals, indicating that the polypeptide is complete and causing it to be released from the ribosome. ➢ More nucleotides may appear after the stop codon in the mRNA, but will not be translated as part of the polypeptide. 60 ❖ DNA sequence start codon : ATG; T sequence U mRNA ❖ Three stop codons for chain termination. DNA RNA TAG TAA TGA UAG UAA UGA 61 The process of mRNA translation differs between prokaryotes and eukaryotes. Prokaryotic mRNA has a Shine-Dalgarno sequence for ribosome binding Eukaryotic mRNA has a 5' cap and poly-A tail for protection and ribosome binding. The first amino acid also differs: formylmethionine in prokaryotes and methionine in eukaryotes. The Shine-Dalgarno sequence helps recruit the ribosome to the mRNA to initiate protein synthesis by aligning the ribosome with the start codon. It's not used in eukaryotes because the initiation of translation is far more complicated in eukaryotes than prokaryotes. The Shine-Dalgarno sequence is typically found around position -7 to -4 of the translational Start codon and has the sequence AGGAGG. This sequence is complementary to part of the 3′ end of 16S rRNA: GAUCACCUCCUUA-3 Genetics 63 Genetics is the study of genes. Genes carry information that gets passed from one generation to the next eg parents to offspring and determine who we are. Genetics research studies how individual genes or groups of genes are involved in health and disease. Understanding genetic factors and genetic disorders is important in learning more about promoting health and preventing disease. 64 https://www.luriechildrens.org/ 65 DNA DAMAGE 66 DNA DAMAGE ➢ DNA is a relatively stable molecule however damage to DNA is inevitable. ➢ DNA is subjected to many alterations i.e. chemical, environment etc. ➢ DNA can also be altered by mistakes made during its own replication or recombination. ➢ The genetic information encoded in the DNA has to remain uncorrupted. ➢ Any chemical changes must be corrected. ➢ Damage and sequence alterations to DNA are often quickly repaired, but when they are not, the DNA becomes permanently altered resulting in the production of mutation (s). 67 ➢ Mutations are permanent alterations/changes in a DNA sequence that are propagated through cellular generations. ➢ A cancer cell has mutations that prevent cell death, resulting in loss of cell cycle control and unregulated cell division, which leads to malignant tumours. ➢ The cell has a limited amount of time to fix the initial alteration and restore the DNA to its normal sequence, before replication converts the alteration into a mutation that will be passed on to the next generation. ➢ Mutations can be: ▪ Somatic mutations : occur in somatic cells and only affect the individual in which the mutation arises. ▪ Germ-line mutations: alter gametes and passed to the next generation. 68 69 Types of mutation Missense mutation: a change in one DNA base pair that results in the substitution of one amino acid for another in the protein. This amino acid substitution may have no effect, or it may render the protein nonfunctional. 70 Nonsense mutation: is a point mutation in a sequence of DNA that results in a premature stop codon, or a nonsense codon in the transcribed mRNA, and in a truncated, incomplete, and usually nonfunctional protein product. The functional effect of a nonsense mutation depends on the location of the stop codon within the coding DNA. 71 Insertion mutation: An insertion changes the number of DNA bases in a gene by adding a piece of DNA. As a result, the protein made by the gene may not function properly. 72 Deletion mutation: A deletion changes the number of DNA bases by removing a piece of DNA. Small deletions may remove one or a few base pairs within a gene, while larger deletions can remove an entire gene or several neighbouring genes. The deleted DNA may alter the function of the resulting protein (s). 73 Duplication mutation: A duplication consists of a piece of DNA that is abnormally copied one or more times. This type of mutation may alter the function of the resulting protein. 74 Frameshift mutation: This type of mutation occurs when the addition or loss of DNA bases changes a gene's reading frame. A reading frame consists of groups of 3 bases that each code for one amino acid. A frameshift mutation shifts the grouping of these bases and changes the code for amino acids. The resulting protein is usually nonfunctional. Insertions, deletions, and duplications can all be frameshift mutations. 75 Agents that damage DNA Radiations ➢ Highly reactive oxygen radicals produced during normal cellular respiration and other biochemical pathways. ➢ Ionizing radiation (gamma and x-rays). ➢ Ultraviolet rays (UV-C and –B) Chemicals in the environment ➢ Aromatic hydrocarbons, plant and microbial products, chemotherapy agents 76 Types of DNA damage 77 DNA repair mechanisms 78 79