BIOC0007 L1-L14 Revision Notes Compilation PDF

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BIOC0007 REVISION NOTESLECTURE 1STRUCTURE AND COMPONENT OF DNA, CHARGAFF’S RULEDescribe nucleotides - Nucleotides = cyclic nitrogenous base + pentose sugar + phosphate group Chargaff’s rule states that DNA contains a 1:1 ratio of pyrimidine and purine bases, more specifically, the amount of A equals...

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BIOC0007 REVISION NOTES LECTURE 1 STRUCTURE AND COMPONENT OF DNA, CHARGAFF’S RULE Describe nucleotides - Nucleotides = cyclic nitrogenous base + pentose sugar + phosphate group Chargaff’s rule states that DNA contains a 1:1 ratio of pyrimidine and purine bases, more specifically, the amount of A equals to T, and the amount of C equals to G. This pattern is found in both strands of DNA - In the DNA, nucleotides undergo complementary base pairing (according to chargaff’s rule), more specifically, adenine forms 2 H bonds with thymine, whereas cytosine forms 3 H bonds with guanine Describe DNA strands - DNA is made up of 2 strands of polynucleotide, which is formed by the successive joining of nucleotides by the formation of phosphodiester bond between the 3’-OH group and the 5’-Phosphate group of the next pentose sugar - The pentose sugar is joined to the nitrogenous base by Beta-N-glycosidic linkage - The 2 strands are antiparallel, running in opposite polarity (3’-5’ or 5’-3’) Hydrogen bond formation - Hydrogen bonding between bases are specific that allows A=T and C=G complementary base pairing, making the ratio of purine to pyrimidine of 1:1 Double helix - Stacked base pair interaction leads to formation of double helix - Stacked base pair induced dipole interaction between aromatic bases, contributing to stability - Presence of cations and cationic proteins counteracts electrostatic repulsion of anionic phosphate backbones Characteristics and significance of DNA structure - DNA has a purine-pyrimidine hydrophobic core in the centre, while the phosphate backbone protrudes outwards is hydrophilic and highly negatively charged - The double helix formation ensures the stability of the DNA - Complementary base pair allows the DNA to adapt to the ability to re-anneal after replication or when it is denatured by heat or chemicals for polymerase chain reaction - Stability also ensures that the genetic information are stored in a stable structure for transcriptional purposes

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Every 3 nucleotide reads a codon sequence on the transcribed mRNA and encodes for an amino acid which will form the protein of interest DNA primary sequence determines the protein structure, function and properties

PACKAGING AND ORGANISATION OF DNA (IN EUKARYOTE AND PROKARYOTE) In eukaryotes - At the simplest level, chromatin is a double stranded helical DNA structure - The DNA wraps the histone octamer for 1.6 times - The histone octamer is made up of 2 units of H2A, H2B, H3 and H4 histone monomers; this structure is called the nucleosome - The histone octamer also consists of positively charged arms (arginine and lysine) that contributes in the interaction with negatively charged DNA phosphate backbone - Assembly of the nucleosome and a H1 histone linker protein makes up the structure called chromatosome - The nucleosome folds up to form a 30 nm diameter fibre that forms loops of 300 nm in length - The 300 nm long fibres are compressed and fold into strands of 250 nm in diameter - These 250 nm diameter wide strands compact to form a diameter of 700 nm strand, and tightly coil to produce a single chromatid of a chromosome - Hence, during metaphase, the chromosome is of 1400 nm wide - During interphase, the chromosomes are less condensed, the packaging ratio of the chromosome in comparison to chromosome packaging during metaphase is of 1:10 - Histone proteins are modified by acetylation and methylation for easier access of transcription factors, RNA polymerase for mRNA transcription or DNA polymerases for replication

In prokaryotes - Chromosome is circular - Stored in a structure called nucleoid - The types of proteins associated with the chromatin are known as the nucleoid-associated proteins - These proteins causes the prokaryotic chromosome to form looped structures - Prokaryotes also consist of external, independently replicated plasmid DNA that are useful as a vector in genetic engineering Plasmids - Extrachromosomal circular or linear DNA - Double stranded DNA molecules in bacteria - It is a self replicating genetic unit, known as replicon - Replicon is a DNA or RNA (or a region of DNA or RNA), that replicates from a single origin of replication - For prokaryotic chromosome, the replicon is the entire chromosome

LECTURE 2 DNA REPLICATION Semiconservative - Each of the 2 original strands serve as template for the formation of an entire new strand - Each of the daughter cell inherits a new double helix DNA containing 1 original and 1 new strand REPLICATION PROCESS, ORIGIN OF REPLICATION AND INITIATOR PROTEINS, DNA HELICASES, DNA SYNTHESIS Origin of replication and initiator protein - Process of DNA replication is begun by special initiator proteins that binds to the double-stranded DNA and pry the two strands apart - Hydrogen bonds between the bases at the origin of replication (oriC) has been disrupted - Replication origins are normally rich with A=T base pairing - Initiator protein (DNA A) recognizes a specific DNA sequence within the origin of replication DNA helicase - Recruitment of DNA helicase (DNA B)

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The helicase unwind the DNA double helix by moving in the 5’ to 3’ direction along one strand, or in the 3’ to 5’ direction along the other - Both types of DNA helicase exist - Initiator proteins (DNA A) and the recruitment of DNA helicases (DNA B) allowed the establishment of the replication fork At the replication fork - To begin chain growth, DNA polymerases require short, preexisting RNA or DNA strand called primer - The primer complementary base pair to the template strand, a DNA polymerase adds deoxynucleotides to the free hydroxyl group at the 3’ end of the primer as directed by the sequence of the template strand - Multienzyme complex containing the DNA polymerase synthesize the DNA of both new daughter strands - Antiparallel orientation of the 2 DNA strands in the DNA double helix, this mechanism would require 1 daughter strand to be synthesised in the 5’ to 3’ direction and the other strand in the 3’ to 5’ direction - Such a distinct fork would require 2 distinct types of DNA polymerase - Most of the DNA polymerase can only synthesize the DNA strand in the 5’-3’ direction - DNA polymerase catalyses the stepwise addition of nucleotides to the 3’-OH end of a polynucleotide chain, the primer strand which is paired to a template strand - Process is driven by large, favourable free-energy change, caused by the release of pyrophosphate and its subsequent hydrolysis into 2 inorganic phosphate The multienzyme complex → THE REPLISOME - Contains several enzymatic activities and protein activity - Helicase, primase, SSB proteins and DNA polymerase - At the front of the fork, DNA helicase opens the DNA helix - 2 DNA polymerase works at the fork; one on the leading strand and one on the lagging strand - DNA polymerase are held to the DNA strands by a sliding clamp and a clamp loader; sliding clamp keeps DNA polymerase firmly on the DNA - DNA polymerase on the lagging strand must start at short intervals, using a short RNA primer made by DNA primase molecule - The lagging strand is being fold back to increase the efficiency of replication, so that the leading and lagging strands are synthesised in the same direction - Single-stranded DNA binding (SSB) proteins, are also called helix-destabilizing proteins, binds tightly and cooperatively to exposed single-stranded DNA without covering the bases - This helps helicases by stabilizing the unwound, single stranded conformation

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This also prevents the formation of hairpin helices that readily form in single stranded DNA

SYNTHESIS AND FEATURES OF LEADING AND LAGGING STRANDS OF DNA REPLICATION Leading strand - Synthesis of one daughter strand, called the leading strand, can proceed from a single RNA primer continuously in the 5’ to 3’ direction, the same direction as the movement of the replication fork Lagging strand - Synthesis of the other daughter strand, called the lagging strand, occurs in the opposite direction from the movement of the replication fork - A cell accomplishes this by synthesizing a new primer by DNA primase every few hundred bases on the second parental strand, as more of the strand is exposed by unwinding - The primers are properly base-paired and has a 3’ -OH group at the end - Each of these primers, base-paired to their template strand is elongated in the 5’ to 3’ direction, forming discontinuous segments called Okazaki fragments - Synthesis of each Okazaki fragments ends when the DNA polymerase I runs into the RNA primer attached to the 5’ end of the previous fragment - DNA polymerase ‘dock’ at the primer site and finishes the elongation of the DNA fragment - The old RNA primer is erased, by 5’-3’ exonuclease, and replaced by DNA - An enzyme called DNA ligase carries out nick sealing

STRUCTURAL AND FUNCTIONAL FEATURES OF DNA POLYMERASES DNA polymerase - Enzyme that catalyses the polymerisation of deoxyribonucleotides into DNA strand - Catalyse the addition of deoxynucleotides to the 3’-end of the growing replicated DNA strand, complementary to the template strand - Via nucleophilic attack and phosphodiester bond formation - E. coli has at least 5 DNA polymerases, Pol I-V - DNA Pol III is the subunit with the highest processivity (consecutive reactions without releasing its substrate), and exists as part of the Pol III holoenzyme - DNA Pol III is responsible for chromosome replication Structure - Resembles a right hand in which the palm, fingers and thumb grasp the DNA and form the active site - Incoming nucleotide causes the fingers to tighten, initiating the nucleotide addition reaction - Dissociation of the pyrophosphate causes the release of the fingers and causes the translocation of DNA by 1 nucleotide - Active site of the polymerase is now ready to receive the next nucleotide Function - Replicate the DNA leading strand using its template strand - Structurally, it contains the trombone model replisome machinery that contains DNA B Helicase that unwinds the DNA double helix - The primosome in which RNA primers are synthesised by enzyme primase - Beta-sliding clamp hold DNA double strands tightly - Alpha-polymerase core complexes carry out polymerase activity

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It also has a 3’-5’ exonuclease activity that enables the cleavage of incorrectly added deoxynucleotide detected by its proof reading component; clipping off any unpaired residues at the primer terminus. - Hence, DNA polymerase functions as a “self-correcting” enzyme End product - 2 semi conserved DNA double helix LECTURE 3 DNA DAMAGE AND MUTATIONS - ~1 incorrect nucleotide per 10,000 nucleotides - Mutation rate is around ~1 in 1,000,000,000 nucleotides - Due to proof-reading by all 3 E. coli DNA polymerases; 3’-5’ exonuclease activity - Most frequent mutation is the deamination of cytosine to thymine REPAIR OF SINGLE MUTATION: BASE EXCISION Enzymes involved - GT mismatch - Involve DNA glycosylase which recognizes a specific altered base a catalyses the removal of the base by hydrolysing the glycosidic bond - Involve APEI endonuclease which cuts the baseless phosphodiester backbone - AP lyase removes the deoxyribose phosphate (baseless nucleotide) that is left after APEI endonuclease cuts the phosphodiester backbone - TT dimer - Helicase TFIIH and RPA which partially unwinds the DNA double strand at the damage site - Endonuclease XP-F and XP-G cut the damaged DNA (24-32 bases apart) around the lesion GT mismatch - DNA glycosylase specific for GT mismatch cuts the thymine base - APEI endonuclease specific for baseless deoxyribose cuts the DNA backbone - Deoxyribose phosphate is removed by an endonuclease AP lyase - The gap is filled by DNA polymerase and sealed by DNA ligase

Thymine-Thymine dimers - TT dimers arise from damage by UV irradiation - UV causes dimerisation - It causes the distortion of the double helix - RECOGNITION OF DAMAGE SITE. The damage site is recognised by a nucleotide excision complex - UNWINDING THE DAMAGE SITE. The opening of the DNA double helix, by partially unwinding, by helicase activity - Helicase factors: TFIIH and RPA - REMOVING THE DAMAGE SITE. Endonucleases XP-F and XP-G cut the damaged DNA around the lesion - REFILLING AND RESEALING THE GAP. The gap is filled by DNA polymerase and sealed by DNA ligase

REPAIR OF DOUBLE-STRAND BREAKS Enzymes involved - Homologous recombinant - ATM kinase activates and recruits a set of exonuclease that removes nucleotides at the break, first from the 3’ end and then from the 5’ end, creating single stranded 3’ ends - Non-homologous end joining - Ku and DNA dependent protein kinase binds to the ends of the double strand break

Homologous recombination - ACTIVATION OF ENZYME. Double strand break activated the ATM kinase - ENDONUCLEASE CLEAVE. ATM activates and recruits a set of exonucleases that removed nucleotides from the 3’ end, and then from the 5’ end - FORMATION OF OVERHANGS. This forms 2 3’ end overhangs - HOMOLOGY DUPLEX SEQUENCE SEARCH. RAD51 nucleoprotein filament searches for the homologous duplex DNA sequence on the sister chromatid - COMPLEMENTARY BASE PAIRING OF THE OVERHANG TO THE HOMOLOGOUS DUPLEX SEQUENCE. The 3’ overhang invade the duplex to form a joint molecule in which the single stranded 3’ end is paired to the complementary strand - ELONGATION OF THE 3’ SINGLE STRAND. DNA polymerase elongate the 3’ end of the damaged DNA, using the complementary undamaged DNA strand as a template - BASE PAIRING WITH THE OTHER 3’ END SINGLE STRAND. The repaired 3’ end of the damaged DNA pairs with the single stranded 3’ end of the other damage strand - GAP FILLING AND LIGATION. The remaining gaps are then filled in by DNA polymerase and ligase.

Non-homologous end joining (NHEJ) - Occurs during G1 when there is no sister chromatids

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Ku and DNA-dependent protein kinase binds to the ends of the DSB Formation of a synapse, the ends are processed by nucleases (exonucleases), resulting in the removal of a few bases The 2 double strands are ligated together by DNA ligase However, several base pairs are lost which can introduce mutations Occasionally, ends from different chromosomes are accidentally joined together

THE CELL DIVISION CYCLE - Events of the cell cycle are controlled by checkpoints - Checkpoints are action of specific biochemical pathways that causes delay in the cell cycle progression if a previous cell cycle event is not completed - Cyclin-dependent kinases (CDKs) are regulators of the cell cycle - The cell cycle is an ordered set of events that results in the generation of two copies of a preexisting cell - CDKs are activated when cyclin protein joins and forms a complex Example of CDK and Cyclin complexes - During G1, CDK2 and cyclin E complex initiates replication - During G1, CDK4 and cyclin D complex promotes progression from G1 to S - During S phase, CDK2 and cyclin A complex initiates replication

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During G2, CDK1 and cyclin B complex promotes mitosis

Mutation in this gene abolished the DNA-damage checkpoint Is found in 50% of human cancers

LECTURE 4 GENETIC ENGINEERING - Direct manipulation of the genome of an organism using biotechnology Example of enzymes used in genetic engineering - Polymerases - DNA polymerases, reverse transcriptase - Nucleases (restriction endonucleases, RNAse) - DNA ligase - Alkaline phosphatase PCR - A widely used technique to amplify DNA in vitro - The principle is a cyclic thermal process, to be repeated n times - Total product count after n cycles: 2^n Usage of PCR - Sequence-specific detection and amplification of DNA - Generation of DNA for specific applications - Engineering of DNA - Quantitation of DNA There are 3 formal steps - Strand separation of dsDNA (denaturation/melting) - 94-96 degree celsius - Denaturation is close to boiling temperature - Melting occurs first at A=T base pairing - Annealing of primers - 55-70 degree celsius - Extension of primers - 66-72 degree celsius - By using DNA polymerase, it has 3 main enzymatic activities, - 5’-3’ DNA dependent DNA polymerase, which is primer and template dependent - 3’-5’ exonuclease, relevant for proofreading

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5’-3’ for DNA reparation

DNA SEQUENCING The Sanger method - Also known as the chain termination sequencing - To start the sequencing experiment, a short oligonucleotide is annealed to the template DNA - This oligonucleotide acts as a primer for synthesis of a new DNA strand that is complementary to the template - Other than the recruitment of the 4 deoxyribonucleotide triphosphates (dATP, dTTP, dCTP and dGTP), dideoxynucleotides are also added to the reaction. - Each of these dideoxynucleotide is labelled with a different fluorescent marker - The polymerase enzyme does not differentiate the difference between dNTP and ddNTPs - Once incorporated, there will not be further elongation of the strand as there is a lack of 3’-OH group that normally forms the phosphodiester bond with the next dNTP - To work out the sequence, we have to identify the ddNTP at the end of each chain terminated molecules - The mixture is loaded into the capillary gel and electrophoresis carried out to separate the molecules according to their lengths - After seperation, the molecules are run past a fluorescence detector capable of discriminating the labels attached to the ddNTP - The sequence can show which base the chain terminated molecules ends in Next generation DNA sequencing - Breakage of the starting DNA into fragment of sizes that are suitable for the sequencing method being used - Immobilization of the fragments onto the solid support - Amplification of the immobilized fragments using PCR RECOMBINANT DNA cDNA library and genomic library - A genomic library keeps amplified copies of multiple fragments from complete genome of organisms - It includes both introns and exons in its collection - It is meant for DNA sequencing rather than protein synthesis - Genomic library are prepared by restriction nuclease digestion and PCR - A cDNA library contain cloned, reverse-transcribed mRNA

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cDNA libraries are used for functional protein synthesis Therefore it lacks DNA sequences corresponding to genomic regions that are not expressed due to post-transcriptional modification and alternative splicing Hence in a cDNA library, there is no introns (noncoding regions) After reverse transcription of mRNA into cDNA, it is amplified multiple rounds via PCR for collection cDNA libraries contain smaller fragments than genomic DNA libraries, and are usually cloned into plasmid vectors

Creating recombinant cDNA plasmid - DNA is digested using type II restriction endonucleases to obtain gene of interest - Type II restriction endonucleases cleaves the DNA at a specific restriction site - The sequence at the restriction site is palindromic - Plasmid, which is used as a vector, is also digested with the same restriction enzyme to create the same sticky ends (overhangs) - The gene of interest (restriction enzyme cut DNA) is ligated to the plasmid vector using DNA ligase, forming recombinant plasmid - Recombinant plasmid is taken up by E.coli in the presence of CaCl2 + heat pulse - The culture is spread on nutrient agar plates containing ampicillin - The transformed cell survives, as it possess ampicillin resistant genes - The transformed cells are grown to allow cell multiplication - The colony of cells formed contain copies of the same recombinant plasmid When and why would you use a plasmid cloning vector and what are its main features - Plasmid cloning vector is used when the DNA that has to be cloned is up to ~15kb in size - It is used to shuttle genes within a colony via transformation, to allow bacteria to express gene of interest, and also to monitor protein synthesis via tracking with a tagged reporter gene - It contain genes for resistance to antibiotics or production of toxins - Hence this is often used as a selectable marker to ensure that the bacteria in a culture contain a particular plasmid - It contains at least 1 DNA sequence that act as the origin of replication - They are able to multiply independently, within the cell, of the main bacteria ...