BIOC0007: VERIFIED QUESTIONS AND ANSWERS

BIOC0007 What is the difference between a nucleoside and a nucleotide? Nucleoside = sugar and base Nucleotide = sugar, base and phosphate. Which conformation does double stranded DNA most frequently take in cells? B-DNA What is the bond holding the sugar and phosphate in a nucleotide called? Phospho ester bond What type of interaction occurs between stacked bases in the DNA double helix and helps to stabilise the helix? Van der Waals forces The level of DNA packaging brought about by the formation of what looks like beads on a string? Nucleosides with linker DNA between them What are the differences between euchromatin and heterochromatin? Euchromatin is lightly stained, heterochromatin is heavily stained. Euchromatin is transcriptionally active, heterochromatin is usually inactive. Euchromatin mainly found during interphase, when the genome is relatively open and active. Heterochromatin is found towards the periphery of the nucleus during interphase and the centromeres and telomeres of the chromosomes. What is condensed chromatin associated with? Low levels of gene expression. What are histones? Proteins that DNA wraps around approximately 2 times. What is the histone octamer complex consisting of? Eight positively charged histone proteins (two of each H2A, H2B, H3 and H4) that aid in the packaging of DNA. What is the eukaryotic chromosome's level of compression? 1400nm Where does DNA synthesis proceed from? Bidirectionally from the replication origin. Where does DNA replication begin? Replication origins What is the enzyme that breaks DNA, dispels the tension, and reveals the strand ahead of a DNA replication growing fork? Topoisomerase What is the function of the sliding loader? Opens the clamp and loads the clamp onto the ssDNA What is the sliding clamp part of? The replisome How is the energy for DNA synthesis obtained? Through hydrolysis of pyrophosphate Okazaki fragments are small fragments of DNA that eventually are lighted to form what strand of DNA? Lagging What are the initiator proteins responsible for? Recruiting the helicase and stabilise the ssDNA Which strands are replicated when the replication machinery moves along the DNA? Both strands How is torsional stress from supercoiling in replicating DNA relieved ahead of the replication fork? Topoisomerase generates nicks in the DNA strand What enzymes mediate the processing of Okazaki fragments? DNA ligand DNA polymerase I What does DNA ligand do? Forms phosphodiester bonds Which type of DNA damage is considered the most deleterious to the stability of the genome? Double stranded breaks What are the three excision repair systems found in eukaryotes and what do they do? Base excision repair - repairs T-G mismatches and damaged bases. Nucleotide excision repair - repairs chemical adducts and thymine-thymine dimers. Mismatch repair - repairs other base mismatches and small insertions and deletions. Thymine-thymine dimers are chemical adducts that develop in the DNA as a result of damage caused by what? UV radiation. What enzymes play a key role in the base excision repair of nucleotide mismatches and damaged bases? Glycosylases What mechanism can remove a nucleotide that is missing its nitrogenous base with the correct one? Base excision repair In which repair method is photolyses used? Photoreactivation What does homologous recombination do? Repairs DNA double strand breakage During meiosis it generates genetic diversity, Utilised in gene editing systems What is Rad51? Nucleofilament that carries damaged DNA strand to its complementary sister chromatid during homologous recombination repair mechanism. Where does non-homologous end joining occur? In non-dividing cells In what DNA repair mechanism does ATM function? Recombination What does the G2 checkpoint ensure? That DNA replication has been completed correctly Who discovered the double helix structure of DNA and when? Watson and Crick in 1953 What did Rosalind Franklin and Maurice Williams do? X-ray diffraction studies of DNA fibres. What did Edwin Donahue discover? Bases are perpendicular to the sugar backbone, in enol form. What did Erwin Chargaff do? Discovered purines to pyrimidines have a 1:1 ratio What is the composition of a nucleotide? Sugar + phosphate + nitrogenous base Which bases are purines and two-ringed? Adenine and Guanine Which bases are pyrimidines and have one ring? Cytosine and Thymine Where is the phosphate ester bond in DNA? Covalent bond between phosphate and 3' or 5' -OH of nucleoside Where are glycosidic bonds in DNA? Covalent bond between sugar C1 and purine N9 atom or pyrimidine N1. Describe polynucleotides: Linear sequence of nucleotides joined by phosphodiester bonds ( 2 phospho-ester bonds very strong between 3' and 5' -OH on adjacent sugars) Sugar + backbone = backbone repeat unit Bases extend as side groups Double helix = 2 twisted nucleotide sequences Describe 3D DNA conformation: Single strand DNA associates via hydrogen and van der waals bonds to form double stranded DNA A pairs with T with 2 hydrogen bonds C pairs with G with 3 hydrogen bonds These are the Watson-Crick base pairs (weak bonds) Strands are complementary (sequence on one strand dictates sequence on other strand) 5' end of one strand pairs with 3' end of the other (20A/2nm apart) - strands are antiparallel Ratio of purines to pyrimidines is always 1 (Chargraff's rule) What is Chargraff's rule? Ratio of purines to pyrimidines is always 1 How many base pairs is one helical turn? 10.5 base pairs Describe the physical properties of double stranded DNA: Two strands wind around a 360 degree turn in a right handed manner every 10.5 base pairs (most energetically favourable formation) Sugar phosphate backbone is hydrophilic Two grooves (major = 13A and minor = 9A) Grooves allow access of other proteins and molecules to DNA Exposed base edges in grooves aid stability and protein interactions during replication, transcription, recombination and repair. What do exposed edges in DNA grooves do? Aid stability and protein interactions during replication, transcription, recombination and repair. When is A-DNA present? Dehydration When is Z-DNA present? During methylation, high NaCl concentration or during torsional stress. What helix direction is B-DNA? right handed What helix direction is A-DNA? Right handed What helix direction is Z-DNA? left handed/zigzag What is the repeat unit of B-DNA? 10.5 base pairs What is the repeat unit of A-DNA? 11 base pairs What is the repeat unit of Z-DNA? 12 base pairs What is the helix diameter of B-DNA? 20A What is the helix diameter of A-DNA? 26A What is the helix diameter of Z-DNA? 18A Is the central core of B-DNA solid or hollow? solid Is the central core of A-DNA solid or hollow? Hollow Is the central core of Z-DNA solid or hollow? Solid What is the helix rise of B-DNA? 3.4A What is the helix rise of A-DNA? 2.6A What is the helix rise of Z-DNA? 3.7A Describe the major groove dimensions of B-DNA wide/deep Describe the major groove dimensions for A-DNA Narrow/deep Describe the major groove dimensions of Z-DNA Flat Describe the minor groove dimensions for B-DNA Narrow/shallow Describe the minor groove dimensions of A-DNA Wide/shallow Describe the minor groove dimensions of Z-DNA Narrow/deep How many base pairs are there in the human genome? 6.4 x10^9 base pairs organised as 23 chromosomes How many linear double stranded DNA molecules does each chromosome contain? one How much DNA is in the human body (5 x10^12 cells) 100 x10^12m of DNA How much DNA fits into a cell nucleus of 6-15um? 2m DNA What does faithful transmission of genetic information depend on? accurate replication What does successful DNA replication involve? Proper DNA packaging Packaging ensures DNA propagation and protection - how do eukaryotes and prokaryotes do this? Eukaryotes - DNA folds into chromatin Prokaryotes - supercoiling How do eukaryotes organise genome packaging? With the help of histones (main protein) and histone chaperones. What is chromatin composed of? 50% DNA 50% protein What is euchromatin? Decondensed Transcriptionally active Stains light (light as open and not heavily compacted) What is heterochromatin? Compacted Transcriptionally inactive Stains dark What is chromatin remodelling? Structure alteration to allow replication, transcription and repair. What is a histone octamer? Two monomers each of H3 and H4 for a tetramer, association of two H2A-H2B heterodimers follows. What is a nucleosome? 147 base pairs wrapped around a histone complex in left handed manner to form a nucleosome (1.76 turns) What is the first level of DNA compaction? Nucleosomes Describe the histones in a nucleosome: 4 core histones Rich in positively charged amino acid residues, lysine and arginine, counteract negatively charged DNA and stabilises it. Octamer made of tetramer (2 H3 and 2H4) and 2 H2A-H2B heterodimers 147 base pairs wrapped around histone complex in left handed manner to form 1.76 turn nucleosome Describe the histone tails N-terminus tails = H2A, H2B, H3 and H4 C-terminus tails = H2A and H2B 19-39 residues Core = histone protein Tails associate with DNA through major groove Extend from nucleosome complex Required for chromatin condensation (from 10nm to 30nm) Influences chromatin structure List the sections of chromatin packaging from smallest to largest and give their dimensions DNA - 2nm Nucleosome - 11nm 30nm filament - 30nm Extended form of chromosome - 300nm Condensed section of chromosome - 700nm Mitotic chromosome - 1,400nm What do the linker histones bind with? Linker DNA What is the genetic code? Highly orchestrated set of instructions allowing us to live, reproduce and pass information to next generations. Identical in almost all species but number of genes can vary significantly (no clear correlation between genome size, gene number and complexity of an organism). Describe the contributions of Watson and Crick in the discovery of the DNA double helix. How did they utilise the findings of Rosalind Franklin and Maurice Wilkins? Watson and Crick deduced the DNA structure in 1953 by using key information from Rosalind Franklin and Maurice Wilkins' X-ray diffraction studies of DNA fibres. Franklin and Wilkins' work provided crucial data on the helical nature of DNA, which Watson and Crick used to propose the double helix structure. Explain the significance of Edwin Donohue's findings regarding the arrangement of bases in DNA. What did he propose about the orientation of bases in relation to the sugar backbone? Edwin Donohue's findings indicated that the bases in DNA are perpendicular to the sugar backbone, adopting an enol form. This discovery helped in understanding the spatial arrangement of the components within the DNA molecule. What is the 1:1 ratio discussed by Edwin Chargaff in relation to DNA? How does this ratio contribute to our understanding of DNA composition? Edwin Chargaff's 1:1 ratio refers to the observation that the amount of purines (Adenine and Guanine) in DNA is equal to the amount of pyrimidines (Cytosine and Thymine). This discovery was foundational in understanding the base composition of DNA. Define a nucleotide and describe its composition. How do Guanine and Adenine differ from Cytosine and Thymine in terms of structure? A nucleotide is composed of a sugar molecule (such as deoxyribose in DNA), a phosphate group, and a nitrogenous base. Guanine and Adenine are purines characterized by a double-ring structure, while Cytosine and Thymine are pyrimidines with a single-ring structure. Distinguish between a nucleoside and a nucleotide. Provide examples of each and explain their significance in DNA structure. A nucleoside consists of a nitrogenous base attached to a sugar molecule, with or without the phosphate group. Examples include deoxyadenosine (nucleoside with deoxyribose sugar) and adenosine (nucleoside with ribose sugar). A nucleotide, on the other hand, is a nucleoside with one or more phosphate groups attached via phosphoester bonds. Discuss the formation of a polynucleotide chain. What type of bond joins nucleotides in a linear sequence? A polynucleotide chain is formed by the linear sequence of nucleotides joined together by phosphodiester bonds. Each nucleotide is linked to the next one by the phosphate group connecting the 3' carbon of one sugar to the 5' carbon of the next sugar. Describe the structure of the DNA double helix. How are the nucleotides arranged in this structure? The DNA double helix structure consists of two strands of nucleotides twisted around each other. The nucleotides are arranged in a complementary fashion, with Adenine (A) pairing with Thymine (T) via two hydrogen bonds, and Cytosine (C) pairing with Guanine (G) via three hydrogen bonds. Explain the concept of complementary base pairing in DNA. How does it contribute to the stability of the double helix? Complementary base pairing in DNA refers to the specific hydrogen bonding between Adenine and Thymine (A-T) and between Cytosine and Guanine (C-G). This pairing ensures the stability and fidelity of the DNA double helix. Discuss the role of hydrogen bonds in maintaining the structure of DNA. How many hydrogen bonds form between Adenine and Thymine? Between Cytosine and Guanine? Adenine forms two hydrogen bonds with Thymine, while Guanine forms three hydrogen bonds with Cytosine. These hydrogen bonds contribute to the stability of the DNA double helix. Why is the antiparallel nature of DNA significant? How does it relate to the orientation of nucleotide sequences? The antiparallel nature of DNA refers to the arrangement of the two strands running in opposite directions. In a DNA double helix, one strand runs in the 5' to 3' direction, while the complementary strand runs in the 3' to 5' direction. This arrangement is essential for complementary base pairing. Explain Chargraff's rule and its significance in understanding DNA composition. Chargraff's rule states that the ratio of purines (Adenine and Guanine) to pyrimidines (Cytosine and Thymine) is always 1:1 in a DNA molecule. This rule highlights the consistent base pairing in DNA. Describe the physical properties of double-stranded DNA. How do these properties contribute to its function? Double-stranded DNA winds around itself in a right-handed manner, forming a 360° turn every 10.5 base pairs. The sugar-phosphate backbone of DNA is hydrophilic, while the bases are hydrophobic and stack on top of each other. Compare and contrast B DNA, A DNA, and Z DNA. Under what conditions would each of these forms be found? B DNA is the typical Watson-Crick DNA structure found under normal cellular conditions. A DNA is found in environments with low water content, such as in certain microbiomes. Z DNA is a left-handed helical structure formed under conditions of torsional stress, high salt concentrations, or methylation. What are the primary components of human genome packaging? How is the human genome organised within cells? The human genome consists of 6.4 x 10^9 base pairs organized into 23 pairs of chromosomes. Each chromosome contains one linear double-stranded DNA molecule. The human body has approximately 5 x 10^12 cells, with each cell containing around 2 meters of DNA. Discuss the importance of accurate DNA replication. How does DNA packaging contribute to this process? Accurate replication of DNA is essential for faithful transmission of genetic information. Proper DNA packaging ensures that the genetic material is organised and protected, allowing for successful replication. Explain the role of histones in DNA packaging. How do they assist in organizing the genetic material? Histones are proteins that play a key role in DNA packaging. They form complexes called nucleosomes around which DNA wraps. Histones, along with histone chaperones, help in organizing the genetic material within the cell nucleus. Describe the differences between euchromatin and heterochromatin. How are they related to gene activity? Euchromatin is loosely packed, transcriptionally active chromatin that appears light under a microscope. Heterochromatin is densely packed, transcriptionally inactive chromatin that appears dark. These regions play roles in gene regulation and expression. What is a nucleosome? Describe its structure and its role in DNA compaction. A nucleosome consists of DNA wrapped around a core of histone proteins. Approximately 147 base pairs of DNA are wound around the histone octamer (made of two copies each of H2A, H2B, H3, and H4), forming the first level of DNA compaction. Discuss the significance of histone 'tails' in chromatin condensation. How do they influence chromatin structure? Histone 'tails' are extensions of histone proteins that protrude from the nucleosome. They are rich in lysine and arginine residues and play a role in chromatin condensation. These tails interact with neighboring nucleosomes and influence the overall structure of chromatin. Explain the concept of the genetic code. What are its key features, and how does it enable the transmission of genetic information across generations? The genetic code refers to the set of rules by which information encoded in DNA is translated into proteins. It is nearly universal across species, with variations in the number of genes. The genome size and complexity of an organism do not always correlate with each other or with the number of genes. Discuss the importance of accurate DNA replication. How does DNA packaging contribute to this process? What is DNA replication? Semiconservative What must happen before cells divide? DNA must replicate (S phase) What acts as the template for the daughter strand? The parent strand What did the Meselson-Stahl experiment (1958) show? DNA replication is semiconservative In presence of 14N of parent DNA labelled with 15N, daughter strand had one old 15N strand and one new 14N strand. What is the replication origin? Prokaryotes - one origin - OriC - replication takes a few minutes Eukaryotes - many (100,000) origins - ori - replication takes a few hours. Describe what happens at the replication origin DNA unwinds at the origin to form 2 replication forks. Replication moves from ori bidirectionally, forming the replication bubble Two forks (leading and lagging strands) move in opposite direction in 5' to 3' orientation. Bubble grows merging into a large bubble and fork meets. Formation splits into two identical DNA strands Which strand is continuous and which strand is discontinuous? Leading strand = continuous Lagging strand = discontinuous What are the key functions of initiator proteins? Identify Ori-specific sequence elements Bind Ori(s) and open up Ori(s) Recruit and load helicase How are origins of replication in eukaryotes identified? Randomly Have many origins as lots of transcription Describe the role of initiator proteins in prokaryotes DnaA recognises origin of prokaryotes by recognising specific sequences of recognition - ATP breaks hydrogen bonds at AT rich region where initiator protein binds to origin DNA strands are separated at AT-rich regions after initiator protein binding Initiator protein recruits replication proteins Describe the role of helicase in DNA replication: Helicase is a hexameric ring shaped ATPase Binds DNA backbone Unwinds by ATP hydrolysis Initiates formation of replication bubble Travels along replication fork, continuously unwinding it by ATP hydrolysis and initiates formation of replication bubble Dissociates when it reaches the end of the DNA strand Describe the role of ssDNA binding proteins in DNA replication SSB-single stranded binding protein (prokaryotes) RPA Replication factor A (eukaryotes) Bind to backbone of DNA and bonds with each other (adjacent) creating long strands Stabilises and protects ssDNA Prevents helix re-association Interact with other proteins Describe the role of topoisomerase in DNA replication Gyrase in prokaryotes and Topo I, II in eukaryotes Acts on dsDNA ahead of helicase Transiently breaks DNA to alleviate torsional stress Describe the role of ligase in DNA replication Seals nicks in Okazaki fragments (lagging strand) during DNA synthesis Describe the role of primase in DNA replication Primase = RNA polymerase Adds a 10 base pair RNA primer in 5' to 3' direction for DNA polymerase to bind Part of the primosome complex (primase + associated proteins) Later, RNA primer is degraded by RnaseH/Pol and substituted by DNA What is the function of DNA polymerase? Catalyses the polymerisation of deoxyribonucleotides into a DNA strand Describe the palm of DNA polymerase Most highly conserved Composed of a beta sheet Catalytic site - links incoming dNTPs to template Proofreading activity Describe the finger of DNA polymerase Composed of alpha helices Binds and enclose incoming dNTPs Move dNTPs in contact with the palm's catalytic site Describe the thumb of DNA polymerase Composed of alpha helices Interacts with newly synthesised DNA Helps maintain primer/template position Helps maintain polymerase/template association Describe the sequence of events of DNA polymerase ssDNA winds through the fingers Growing dsDNA fits into palm Fingers help position incoming dNTP and dNTP pairs with the next available template base Fingers close around the base-paired dNTP Palm catalyses the base paired dNTP phosphodiester bond Thumb holds elongated dsDNA Attachment of paired dNTP to primer leads to opening of fingers Primer - template junction moves by one base pair Next dNTP addition cycle begins How many DNA polymerases does E. coli have and which is most important? At least 5 - Pol I-V Pol III is most important What is the function of Pol I in E. coli and how many subunits does it have? RNA primer removal DNA repair 1 subunit What is the function of Pol III core in E. coli and how many subunits does it have? Chromosome replication 3 subunits What is the function of Pol III holoenzyme in E.coli and how many subunits does it have? Chromosome replication 9 subunits How many DNA polymerases do eukaryotes typically have? Over 15 What is the function of Pol alpha in eukaryotes and how many subunits does it have? Primer synthesis during DNA replication 4 subunits What is the function of Pol delta in eukaryotes and how many subunits does it have? Lagging strand DNA synthesis Nucleotide and base excision repair 2-3 subunits What is the function of Pol epsilon in eukaryotes and how many subunits does it have? Leading strand DNA synthesis Nucleotide and base excision repair 4 subunits What is the sliding clamp and what is its role in DNA synthesis? Highly stable ring-shaped complex Highly conserved Binds and keeps polymerase tethered to DNA and in place Remains associated until replication ends What is the clamp loader and what is its role in DNA synthesis? 5-subunit ring structure Opens clamp and loads it onto DNA ATP hydrolysis binds clamp on DNA and releases loader Loader removes clamp when replication finishes What is the role of the replication fork in DNA synthesis? Both DNA strands are synthesised together at the replication fork DNA polymerase elongates only in 5' to 3' direction as 3' -OH group required Leading strand = replicated continuously from a single RNA primer Lagging strand = replicated in short discontinuous fragments from many RNA primers - Okazaki fragments Describe the role of the okazaki fragments in the lagging strand Primase synthesises RNA primer DNA polymerase extends RNA primer into Okazaki fragment but can not attach the end of the synthesised DNA to the next DNA fragment, leaving a 'nick' between the two Okazaki fragments. New Okazaki fragment is synthesised A DNA polymerase with exonuclease activity removes rNTPs. Same polymerase then fills the gap with dNTPs DNA ligase binds and seals the DNA 'nick' to form a long continuous DNA strand Describe the structure and function of the DNA polymerase holoenzyme It is crucial to leave DNA single stranded as little as possible and to couple leading and lagging strand synthesis. Therefore, multiple polymerases can function at the same time Holoenzyme = 3 core enzymes, 2 clamps and 1 loader Holoenzyme speeds up process compared to 1 DNA polymerase, 1 pol can act as backup or aid in lagging strand synthesis Describe the 'trombone model' of coupling of polymerases: Lagging strand loop formation Loop growth Loop disassembly and clamp loading Reformation of the loop Label the replisome Describe initiation of DNA Replication: Ori(s) recognised by initiator proteins that upon up helix Initiator proteins recruit DNA helicase DNA helicase unwinds the helix to expose ssDNA SSBs coat ssDNA to stabilise and protect Primase synthesises a 10 base pair RNA primer Describe elongation in DNA replication: Sliding clamp is recruited Clamp loader is associated with the sliding clamp Replication machinery moves along the DNA simultaneously Describe termination of DNA replication Two replication forks meet where they reach the end Replication complexes are disassembled and two daughter DNA strands are formed In what direction are new DNA strands formed? 5' to 3' Where does DNA replication begin? The origin What is DNA replication machinery called? replisome What does the replisome include? Helicase Primase Polymerase Sliding clamp Clamp loader ss binding protein Other than the replisome, what other proteins participate in DNA replication? Initiator protein Topoisomerase Ligase In what direction does polymerase act? 5' to 3' What does polymerase need to initiate synthesis? RNA primer What was demonstrated by the Meselson-Stahl experiment in 1958? DNA replication is semiconservative. In DNA replication, each parent strand acts as a template for the synthesis of what? One new parent strand and one new daughter strand. Which direction does DNA replication proceed along the leading strand? 5' to 3' What is the function of the helicase enzyme in DNA replication? Unwinds the DNA double helix Which enzyme is responsible for synthesizing short RNA primers during DNA replication? Primase What is the role of single-stranded binding proteins (SSBs) during DNA replication? Protect and stabilise single-stranded DNA Which enzyme removes RNA primers and fills in the gaps with DNA nucleotides during DNA replication? Polymerase Okazaki fragments are found in which strand of DNA during replication? Lagging strand How are adjacent Okazaki fragments on the lagging strand joined together? Ligase seals the gaps between fragments. Which enzyme is responsible for sealing the nicks between adjacent DNA fragments during DNA replication? Ligase What is the function of the sliding clamp in DNA replication? Holds the DNA polymerase in place during replication The DNA polymerase holoenzyme consists of: Three core enzymes, two clamps, and one loader. Which direction does DNA polymerase add nucleotides to the growing DNA strand? 5' to 3' What is the primary function of the clamp loader in DNA replication? Opens the sliding clamp and loads it onto DNA During DNA replication, the leading strand is synthesized how? Continuously from a single RNA primer. How does DNA polymerase proofread its work during replication? Backtracks and removes the incorrect nucleotide. What initiates the formation of the replication bubble during DNA replication? Helicase How does DNA replication terminate? When the two replication forks meet. Explain the significance of the Meselson-Stahl experiment in 1958 in understanding DNA replication. The Meselson-Stahl experiment provided evidence for the semiconservative nature of DNA replication. They demonstrated that when DNA replicates, each new double helix contains one parental (old) strand and one newly synthesized (daughter) strand. This was shown by labelling the parental DNA with a heavy isotope of nitrogen (15N) and then allowing replication to occur in the presence of a lighter isotope (14N). The resulting DNA molecules showed a hybrid band, confirming the presence of one old and one new strand in each newly replicated DNA molecule. Describe the process of DNA replication initiation, including the roles of initiator proteins, helicase, and primase. Recognition of origin of replication (ori) by initiator proteins that bind ori and open up DNA helix. Helicase then unwinds double helix by breaking hydrogen bonds between the base pairs, creating a replication bubble. Primase is then recruited to synthesise a short RNA primer complementary to DNA template. Primer provides starting point for DNA polymerase to begin synthesising new DNA strand. Compare and contrast the replication processes in prokaryotes and eukaryotes, focusing on the number of origins of replication and the time taken for replication. In prokaryotes, such as bacteria, there is typically one origin of replication (oriC) where replication begins. Replication in prokaryotes is relatively rapid, taking a few minutes to complete. In contrast, eukaryotic cells have multiple origins of replication (approximately 100,000) scattered throughout the genome. This allows for the simultaneous replication of the large eukaryotic genome, but it also takes longer, with replication generally taking a few hours to complete. Explain the roles of the following enzymes in DNA replication: DNA polymerase, helicase, ligase, and primase: DNA polymerase catalyses polymerisation of deoxyribonucleotides into new DNA strand and has proofreading activity to correct errors. Helicase unwinds DNA double helix by breaking hydrogen bonds between base pairs, creating replication fork. Ligase seals nicks or gaps between Okazaki fragments on lagging strand by catalysing formation of phosphodiester bonds. Primase synthesises short RNA primers complementary to DNA template, providing starting point for DNA polymerase. Describe the process of lagging strand synthesis during DNA replication, including the formation of Okazaki fragments and their subsequent joining. Primase synthesises short RNA primer on lagging strand template. DNA polymerase elongates RNA primer into an Okazaki fragment. When polymerase reaches the next RNA primer, it detaches, leaving a gap between fragments. Another RNA primer is synthesised, and process repeats to form multiple Okazaki fragments. Finally, DNA ligase seals nicks between adjacent Okazaki fragments by catalysing formation of phosphodiester bonds. Discuss the significance of the sliding clamp and clamp loader in DNA replication. The sliding clamp, a ring-shaped protein complex, plays crucial role in DNA replication by tethering DNA polymerase to DNA strand. This ensures processivity, allowing polymerase to move along template without falling off. The clamp loader is responsible for opening the sliding clamp, loading it onto the DNA, and then releasing it once replication is complete. This coordinated action of the sliding clamp and clamp loader ensures efficient and accurate DNA replication. Explain how DNA replication ensures the fidelity of the genetic code, including the role of DNA polymerase in proofreading. DNA polymerase has proofreading activity, meaning it can detect and correct errors in base pairing during replication. If an incorrect nucleotide is added to the growing DNA strand, the polymerase can remove the nucleotide and replace it with the correct one. This proofreading function helps to minimise occurrence of mutations and ensures accurate transmission of genetic information from one generation to the next. Describe the events that occur during the termination phase of DNA replication. Two replication forks, moving in opposite directions, eventually meet at the termination site. The replication complexes are disassembled, and the replication machinery dissociates from the DNA. Two identical daughter DNA molecules are formed, each consisting of one parental strand and one newly synthesized daughter strand. The process is complete, and the two daughter DNA molecules are ready for further cellular processes. Discuss the significance of the multiple DNA polymerases present in eukaryotic cells during DNA replication. DNA polymerase δ and ε are mainly involved in synthesising leading and lagging strands, respectively. DNA polymerase γ is found in mitochondria and is responsible for replicating mitochondrial DNA. The presence of multiple polymerases allows for coordination of various aspects of DNA replication, repair, and maintenance of genomic integrity. Explain the importance of DNA replication in the faithful transmission of genetic information from one generation to the next. DNA replication is essential for the faithful transmission of genetic information during cell division. It ensures that each daughter cell receives an exact copy of the genetic material present in the parent cell. Through the semiconservative process of DNA replication, genetic mutations are minimized, and the integrity of the genetic code is maintained. This fidelity is crucial for the proper functioning and survival of organisms, as errors in DNA replication can lead to genetic diseases, developmental abnormalities, and other detrimental effects. What is the role of initiator proteins in DNA replication? Identifying and binding replication origins What is the function of DNA ligase in DNA replication? Joining Okazaki fragments on the lagging strand Which enzyme synthesizes RNA primers during DNA replication? Primase What is the purpose of SSBs (Single-Stranded Binding Proteins) during replication? Stabilising and protecting single-stranded DNA What is the main function of DNA polymerase during replication? Adding nucleotides to the growing DNA strand Which enzyme alleviates torsional stress ahead of the replication fork? Topoisomerase What is the purpose of the sliding clamp in DNA replication? Keeping DNA polymerase tethered to DNA Which protein recognizes and binds to the replication origin in prokaryotes? Initiator protein (DNAa/DnaA) Which phase of DNA replication involves the recruitment of the sliding clamp and clamp loader? Elongation What enzyme adds deoxyribonucleotides to the growing DNA strand during replication? DNA polymerase Why are RNA primers necessary for DNA replication? They provide a template for DNA polymerase to initiate synthesis. How does the replisome speed up the process of DNA replication? By coordinating the activities of multiple enzymes Describe the process of DNA replication, including the roles of initiator proteins, helicase, DNA polymerase, and DNA ligase. How does the leading strand differ from the lagging strand in terms of synthesis? Initiation - initiation proteins recognise and bind to origins of replication (Ori), these proteins open up the double helix structure of DNA to expose the single-stranded DNA (ssDNA). Helicase unwinds DNA strands by breaking the hydrogen bonds between the base pairs. Elongation - DNA polymerase, the main enzyme responsible for DNA synthesis, adds nucleotides to the exposed single-stranded DNA template. Leading strand synthesised continuously in the 5' to 3' direction, following the unwinding of the DNA. Lagging strand synthesised discontinuously in Okazaki fragments, as it moves away from the replication fork. Leading strand synthesised continuously by DNA polymerase in the direction of the replication fork. A primase enzyme creates an RNA primer, which DNA polymerase extends along the template DNA strand. The sliding clamp helps to keep DNA polymerase firmly attached to the template strand, allowing for continuous synthesis. Lagging strand synthesised in the opposite direction of the replication fork. Primase creates RNA primers at intervals along the lagging strand. DNA polymerase then synthesizes short Okazaki fragments from each RNA primer. DNA ligase seals the nicks between the Okazaki fragments, creating a continuous strand. Termination - replication continues bidirectionally until the entire DNA molecule is replicated. At termination, two daughter DNA molecules are formed, each containing one original parental strand and one newly synthesized daughter strand. Roles of Key Enzymes and Proteins: Initiator Proteins recognise and bind to origins of replication, initiating the replication process. Help in opening up the DNA helix and recruiting other replication proteins. Helicase - unwind DNA double helix by breaking hydrogen bonds between base pairs, creates the replication fork by separating the two DNA strands. What is the role of initiator proteins in DNA replication? Identifying and binding replication origins Explain the significance of the Meselson-Stahl experiment in understanding the mechanism of DNA replication. What were the key observations made during this experiment? Observations: They used isotopes of nitrogen (15N and 14N) to label DNA. DNA was initially grown in a medium with heavy nitrogen (15N). Then, the DNA was transferred to a medium with light nitrogen (14N) and allowed to replicate. DNA samples were taken at different time points and analyzed using density gradient centrifugation. Key Findings: After one round of replication in the 14N medium, a hybrid DNA band was observed, indicating a mix of old (15N) and new (14N) DNA. After two rounds of replication, two distinct DNA bands were observed: one with hybrid DNA and one with completely new (14N) DNA. This supported the semiconservative model of DNA replication, where each new DNA molecule consists of one old parental strand and one newly synthesized daughter strand. Significance: Confirmed the semiconservative model proposed by Watson and Crick. Provided strong evidence for the mechanism of DNA replication where each parent strand acts as a template for the synthesis of a new complementary strand. Demonstrated the accuracy and fidelity of the DNA replication process. During DNA replication, the leading strand is synthesized how? Discuss the functions of topoisomerase and single-stranded binding proteins (SSBs) during DNA replication. How do they contribute to the overall process? Alleviates torsional stress ahead of the replication fork caused by unwinding of the DNA. Prevents the DNA from becoming overly twisted or tangled. Creates temporary breaks in the DNA strands to relieve strain, then reseals the breaks. SSBs (Single-Stranded Binding Proteins): Bind to and stabilize single-stranded DNA (ssDNA) exposed during replication. Prevents the ssDNA from re-annealing or forming secondary structures. Facilitates the processivity of DNA polymerase by keeping the template strand accessible. Compare and contrast the roles of DNA polymerase and primase in DNA replication. How do they work together to synthesize new DNA strands? DNA Polymerase: Adds nucleotides to the growing DNA strand during replication. Requires a primer (usually RNA) to initiate synthesis. Has proofreading activity to ensure accuracy by removing incorrectly paired nucleotides. Synthesizes DNA in the 5' to 3' direction. Primase: Synthesizes short RNA primers complementary to the DNA template. Provides the starting point for DNA polymerase to begin synthesizing new DNA strands. Collaborates with DNA polymerase to initiate DNA synthesis at the replication fork What happens at a replication fork? Leading strand is elongated continuously Lagging strand formed as a series of discontinuous Okazaki fragments from primers synthesised every 100-200 nucleotides Adjacent Okazaki fragments are joined by DNA ligase What causes spontaneous DNA damage? Normal cellular process e.g., DNA replication (polymerase errors, topoisomerase nick not sealed properly) Cellular metabolic by-products (ROS, RNS, formaldehyde, uric acid Spontaneous damage (hydrolytic bond severing, deamination, depurination) Repeat slippage (expansion or decrease) rNTPs instead of dNTPs Describe the formation and significance of Okazaki fragments during DNA replication: Okazaki fragments are short, newly synthesized DNA fragments that are formed on the lagging strand during DNA replication. They are a result of the discontinuous synthesis of DNA on the strand that runs in the opposite direction of the replication fork. Initiation of Lagging Strand Synthesis - as replication fork opens up and helicase unwinds the DNA, lagging strand template is exposed. Primase recognises exposed single-stranded DNA and synthesises a short RNA primer complementary to the template DNA which provides the starting point for DNA synthesis on the lagging strand. Synthesis of Okazaki Fragments - DNA polymerase III, the main polymerase enzyme in prokaryotes, starts adding deoxyribonucleotides to the 3' end of the RNA primer, synthesises a short DNA fragment (Okazaki fragment) in the 5' to 3' direction, moving away from the replication fork. However, since the DNA polymerase can only synthesize DNA in the 5' to 3' direction, it moves in the opposite direction of the replication fork on the lagging strand. Multiple Okazaki Fragments - as the replication fork continues to unwind and progress, primase generates more RNA primers along the lagging strand. Each RNA primer initiates the synthesis of a new Okazaki fragment, results in the lagging strand being synthesized in a series of short, discontinuous segments. RNA Primer Removal and Gap Filling - after the completion of each Okazaki fragment, DNA polymerase I in prokaryotes (or a similar enzyme in eukaryotes) removes the RNA primer. Gap left behind by the removed RNA primer is filled in by DNA polymerase I with deoxyribonucleotides. This process creates a continuous DNA strand on the lagging strand, complementary to the leading strand. Sealing of Okazaki Fragments - even after the gap is fille d there remains a nick between the adjacent Okazaki fragments. DNA l What causes induced DNA damage? External factors e.g., Alkylating agents disrupt base pairing and backbone (nitrosamines) Intercalating agents - insert between stacked bases and distort helix (EtBr) Base analogs - substitutional bases (thymine by 5-bromouracil) Environment (ionising, UV radiation - pyrimidine dimers) Genotoxic agents (phthalates). ssDNA damage is frequent and common. If untreated, what can it lead to? Mutations Distortions (lesions) Can be tolerated but usually leads to disease Name the types of ssDNA repair? Mismatch repair (MMR( during replication)) Mismatch repair immediately after replication Nucleotide excision repair (NER) Base excision repair (BER) Direct reversal All have same principal but different enzymes What is post replication mismatch repair (MMR)? Repairs damage generated during DNA replication DNA polymerase proofreading 3' to 5' exonuclease activity. Identified mismatch is immediately restored DNA fidelity improves 100-fold What are the key steps in post replication mismatch repair (MMR)? Mismatch recognition DNA replication stops - polymerase pauses Polymerase transfers the 3' end of the growing strand to its exonuclease site Removes incorrect base 3' end is transferred back to the polymerase site Replication resumes Describe mismatch repair immediately after replication Mismatch repair escaping Pol III proofreading In E. coli, 3x mutator proteins Identify mismatched and indels immediately after replication Parent strand transiently tagged by Dam methylase - hemi methylation - methylates parent strand transiently As DNA synthesis proceeds, parental strand is methylated while other strand is unmethylated What are the key steps in mismatch repair immediately after replication? DNA polymerase misincorporates a nucleotide, creating a mismatch - the newly synthesised GATC site is hemimethylated. MutS binds the mismatch and forms a complex with MutL MutS-MutL scans DNA bidirectionally, forming a loop. MutS-MutL finds nearest GATC site and recruits MutH, which cleaves the newly synthesised unmethylated GATC sequence Helicase II and Pol I exonuclease unwind and degrade the newly replicated DNA strand past the mismatch. Pol III fills the gap Ligase seals the DNA. Describe the key steps in base excision repair (BER): DNA glycosylase recognises and binds damaged bases It flips out exposing it It cleaves bond between sugar + phosphate and base AP endonuclease recognises AP site and cleaves phosphodiester bond Short patch BER - nucleotide synthesised by Pol I Long patch BER - nucleotide stretch synthesised by Pol I. Ligase seals the gap Describe the key steps of nucleotide excision repair of bulky lesions (NER): 4 UVR proteins - UvrA to D. UvrA + UvrB scan genome UvrA leaves UvrB recruits UvrC - local helix unwinding UvrC - exonuclease 2x precise incisions UvrD (helicase) removes 12-13 base pairs Pol I fills gap Ligase seals What are the two types of translesion DNA synthesis (TLS) Enables replication to proceed across DNA damage Two types are the polymerase switching model and the gap filling model Describe the polymerase switching model: High fidelity pol stalls and switched with TLS pol TLS pol replicates across damage High fidelity pol switches back Describe the gap filling model High fidelity pol skips ahead of lesion Gap is filled by TLS pol dsDNA damage is lethal if left unrepaired. What are the dsDNA damage response systems? Homology directed repair Non-homologous end joining Homologous recombination Describe non homologous end joining: Always available Occurs predominantly in non-dividing cells (G0, G1, early S) No need for template DNA Error prone What are the key steps in non homologous end joining? Recognition and binding of DSB by Ku70/80 Ku70/80 recruits DNA-PKcs - formation of DNA-PK complex Phosphorylation and end trimming by the nuclease artemis Nucleases / polymerases / phosphates trim, fill / modify DNA ends Blunt ends are formed Ligase seals the break What is homology directed repair? Occurs only during S/G2 phase Needs template DNA Highly accurate Describe pre-synapsis in homology directed repair DSB recognised by MRN complex. MRN recruits and activates ATM which activates variety of target proteins End resection - exonucleases remove nucleotides creating ss 3' overhangs (large - around 50bp) ssDNA is coated with RPA (binding protein) BRCA2 and RAD54 aid exchange of RPA with RAD51 so RAD51 nucleofilament is formed Describe synapsis in homology directed repair. Rad51 nucleofilament searches for and invades homologous DNA Temporary D-loop (displacement) formation Describe the post-synapsis stage in homology directed repair RAD51 dissociates and allows for DNA synthesis to proceed DNA polymerase elongates the 3' end of the damaged DNA Repaired fragment pairs with the ssDNA of the damaged strand Remaining gaps filled by DNA polymerase and sealed with ligase What are the cell cycle damage checkpoints? G1-DNA integrity, cell size signalling G2-DNA damage, chromosome duplication Spindle-attachment of kinetochore and spindle fibre S- DNA fidelity, genome stability What is involved in cell cycle surveillance? Cyclins - associated with specific checkpoints, drive cell division events and activate CDKs Cyclin dependent kinases (CDKs) - master cell cycle positive regulators Cyclin dependent kinase inhibitors - cell cycle negative regulators, inhibit CDK and involved in cell cycle arrest. What is the role of cyclins in cell cycle surveillance? Associated with specific checkpoints Drive cell division events Activate CDKs What is the role of cyclin dependent kinases (CDKs) in cell cycle surveillance? Master cell cycle positive regulators What is a common cause of spontaneous DNA damage? Hydrolytic bond severing Which enzyme is responsible for synthesizing RNA primers during DNA replication? Primase Which repair mechanism is responsible for correcting base-pair mismatches during DNA replication? Mismatch repair (MMR) What is the function of DNA polymerase I in prokaryotic cells during DNA repair? Removing RNA primers Which repair mechanism is responsible for repairing bulky DNA lesions caused by UV radiation? Nucleotide Excision Repair (NER) What is the primary function of the protein Ku70/80 in DNA repair? Binding to DNA double-strand breaks Which repair mechanism is known for its error-prone nature but is always available in cells? Non-Homologous End Joining (NHEJ) Which phase of the cell cycle is primarily responsible for checking DNA integrity before entering mitosis? G1 phase What is the significance of DNA damage checkpoints in the cell cycle? They ensure accurate DNA replication and repair Describe the process of DNA repair through Base Excision Repair (BER). Include the key steps involved and the significance of this repair mechanism. Base Excision Repair (BER) used by cells to repair damaged DNA bases. First, DNA glycosylase enzyme recognizes and binds to the damaged base, flipping it out of the DNA helix. The DNA glycosylase then cleaves the bond between the damaged base and the sugar-phosphate backbone, removing the damaged base, creates an apurinic/apyrimidinic (AP) site. An AP endonuclease enzyme recognises AP site and cleaves DNA backbone at this location. In the short patch BER pathway, a single nucleotide is then synthesized by DNA polymerase I to fill the gap. In the long patch BER pathway, a stretch of nucleotides is synthesized to replace the damaged segment. Finally, DNA ligase seals the nick in the DNA backbone. Explain the process of Nucleotide Excision Repair (NER) and its role in repairing DNA damage. Provide examples of the types of lesions repaired by NER. Nucleotide Excision Repair (NER) is a versatile DNA repair mechanism that corrects a variety of DNA lesions, particularly bulky adducts caused by UV radiation. Recognition of damaged DNA segment by a protein complex consisting of UvrA, UvrB, and UvrC. UvrA and UvrB scan the DNA until they detect a lesion, at which point UvrA dissociates. UvrB then recruits UvrC, which makes two incisions around the damaged site. This creates a small oligonucleotide containing the lesion, which is removed by the UvrD helicase. DNA polymerase I fills in the gap with new nucleotides, and DNA ligase seals the nick. NER is crucial for repairing lesions such as pyrimidine dimers caused by UV radiation, as well as chemical adducts from compounds like polycyclic aromatic hydrocarbons. Discuss the importance of the cell cycle checkpoints in maintaining genomic stability. Explain how these checkpoints function to prevent the proliferation of cells with damaged DNA. Cell cycle checkpoints ensure accurate replication and division of cells and serve to prevent cells with damaged DNA from undergoing further proliferation, which could lead to the propagation of mutations. G1 checkpoint = occurs before entering S phase, checks for DNA integrity and cell size signalling. If DNA damage detected, cell cycle is arrested to allow for repair or, if the damage is irreparable, to trigger apoptosis. G2 checkpoint = occurs before entering the M phase, verifies DNA replication is complete and accurate, any remaining DNA damage is repaired before cell division proceeds. Spindle checkpoint = ensures proper attachment of kinetochores to spindle fibres during mitosis, preventing chromosome mis-segregation. These checkpoints function by employing cyclins, cyclin-dependent kinases (CDKs), and cyclin-dependent kinase inhibitors (CDKIs). Cyclins are associated with specific checkpoints and activate CDKs, which drive cell division events. CDKIs inhibit CDK activity. What is the role of cyclin-dependent kinase inhibitors in cell cycle surveillance? Cell cycle negative regulators Inhibit CDK Involved in cell cycle arrest What can DNA damage be caused by? Single stranded mutations - insertions, deletions, substitutions or slippage. Lesions - structural distortions Double stranded breaks - lethal What do defects in DNA repair mechanisms result in? Susceptibility Mutation Disease Eventually evolution After 3 cycles of PCR, how many copies of the double stranded DNA sequence exist? 8 What is required for PCR to occur? Nucleotides Target DNA Taq polymerase In Sanger sequencing, what is the difference between dNTPs and ddNTPs? ddNTPs lack the 3'OH group of their sugar What are the benefits of real-time PCR over standard PCR? Can be used downstream for cloning Easily quantifiable Does the CRISPR-Cas9 system involve DNA polymerase? No What can genome editing via the CRISPR-Cas9 system generate? Gene silencing Short sequence deletions Single nucleotide mutations Short sequence insertions What is not found in a plasmid vector? A replication origin A multiple cloning site A drug resistant gene Enhancer elements Enhancer elements In next generation sequencing by synthesis (NGS), what is a characteristic feature of the sequencing process? Incorporation of fluorescently labelled dideoxynucleotides What is the purpose of the gRNA? To navigate the Cas9 protein to the correct target sequence What do cloning vectors include? Yeast artificial chromosomes What is PCR? Polymerase chain reaction Faithful amplification of a specific DNA sequence in a short time What is standard PCR? Developed in 1980s Used for DNA detection and downstream DNA processing However is low resolution and non quantitative, rather than qualitative method What is real-time qPCR? Developed in 1990s and used for DNA detection, high resolution and continuous monitoring, tag fluorescently DNA of interest, interacts and inhibits downstream modification. However, can't be used for post-PCR DNA manipulation What are the reaction components for PCR chain reaction? DNA DNA polymerase (thermostable polymerase - taq polymerase) 2 primers - 10bp used as starting points as 3'OH group needed to start replication) dNTPs (A, C, G, T) Polymerase buffer - mix of reagents that stabilise environment for polymerase and give suitable pH for polymerase to act. H2O - adapt water into mix for further downstream applications. What are the 3 main steps in PCR? Denaturation of DNA (95oC) - breaks hydrogen bonds between bases and opens helix to become ssDNA. Annealing of primers (50-70oC) - temperature drops due to GC content of primers, depends on if more 2H or 3H bonds. Elongation (70oC) - DNA polymerase elongates strands - 30-45 seconds - longer if more DNA. Many repeated cycles (number of molecules doubles with each cycle). Time for each step and cycles depends on sequence length/structure + primers. In what direction do primers always go? 3' to 5' Describe the process of Base Excision Repair (BER) and explain its significance in the context of DNA damage repair. Provide examples of the types of lesions repaired by BER: Base Excision Repair (BER) is a critical mechanism for repairing damaged DNA bases. The process involves several steps: Recognition and removal of the damaged base by a DNA glycosylase enzyme. Creation of an abasic site (AP site). Cleavage of the sugar-phosphate backbone at the AP site by an AP endonuclease. Removal of the damaged region and replacement with the correct nucleotide by DNA polymerase. Sealing of the nick in the DNA backbone by DNA ligase. BER plays a key role in repairing lesions such as deaminated bases, oxidative damage, and alkylated bases. For example, it can repair DNA damage caused by reactive oxygen species (ROS) generated during cellular metabolism. Discuss the importance of DNA damage checkpoints in maintaining genomic stability. Explain how these checkpoints function to prevent the proliferation of cells with damaged DNA: DNA damage checkpoints are crucial for ensuring the integrity of the genome and preventing the propagation of mutations. The main checkpoints, such as the G1, G2, and spindle checkpoints, function as follows: G1 Checkpoint: This checkpoint occurs before the cell enters the synthesis (S) phase. It checks for DNA integrity and cell size signalling. If DNA damage is detected, the cell cycle is arrested to allow for repair or apoptosis if the damage is irreparable. G2 Checkpoint: This checkpoint occurs before the cell enters the mitotic (M) phase. It ensures that DNA replication is complete and accurate. Any remaining DNA damage is repaired before cell division proceeds. Spindle Checkpoint: This checkpoint ensures proper attachment of kinetochores to spindle fibres during mitosis, preventing chromosome mis-segregation. By halting the cell cycle progression, these checkpoints provide time for DNA repair mechanisms to fix any damage. This prevents the proliferation of cells with mutations, reducing the risk of cancer and maintaining genomic stability. Describe the process of Nucleotide Excision Repair (NER) and its role in repairing DNA damage. Provide examples of the types of lesions repaired by NER: Nucleotide Excision Repair (NER) is a versatile DNA repair mechanism that corrects various types of DNA lesions, particularly bulky adducts caused by UV radiation. The process involves the following steps: Recognition of the damaged DNA segment by a protein complex consisting of UvrA, UvrB, and UvrC. UvrA and UvrB scan the DNA until they detect a lesion, at which point UvrA dissociates. UvrB then recruits UvrC, which makes two incisions around the damaged site. This creates a small oligonucleotide containing the lesion, which is removed by the UvrD helicase. Describe the 3 phases of the PCR reaction: Exponential - reagents in excess, efficient reaction, high fidelity. Linear - reagents consumed, reaction slows, may become unstable (varying products). Plateau - reaction complete, no more product generation, if left long enough degradation. What is gel electrophoresis? Size dependent DNA separation Contains agarose (white powder added to gel - higher the agarose content, less DNA) DNA migration towards positive pole. Upon completion, DNA can be visualised by staining gel with intercalating agents. Marker ran alongside fragments (marker contains known fragments). What is in the reaction mix for Sanger low throughput DNA sequencing? 1 primer annealed to DNA of interest DNA polymerase All 4 dNTPs Small amounts of 4 ddNTPs, each labelled with different colour fluorescent dye, no 3'OH group on sugar, added to lower dNTPs Describe Sanger low throughput DNA sequencing: Polymerase will stop if ddNTP is added (chain terminator) Generation of multisized sequences Electropherogram generated - peaks can read sequence - can read up to 800bp but usually reads at 600bp What are the features of high throughput DNA sequencing - NGS: Flexible - genome wide or sequence specific Cost effective - human genome 700 (depends on sequencing depth) Accurate - low error rate Sensitive - can detect low frequency variants What are the uses of high throughput DNA sequencing? Research Ancestry Health issues prognostic, diagnostic Prenatal Give some examples of sequencing platforms in high throughput DNA sequencing? Pyrosequencing (biotinylation) Single cell sequencing (fluorescent tagging) Illumina sequencing (sequencing by synthesis (SBS) Nanopore - sequencing electric current density charges. Ion torrent - detects proton release from NTP incorporation Describe the process of high throughput DNA sequencing Library preparation - prepare with DNA fragments you're interested in synthesising, use of restriction enzymes to create ds fragments. Adapter ligation - adapters composed of 3 elements - sequence homologous to sequence on flow cell, fragments must attach for sequence to happen, adapter has sequence corresponding to primer used to amplify sequencing primer and unique barcodes to identify different DNA fragments. Library hybridisation - occurs after denaturation - ss hybridised to flow cell (millions of sequences at the same time). Cluster formation - DNA fragments stick. Sequencing - DNA polymerase synthesises complementary strand, original strand is denatured so left with ssDNA stuck to flow cell, identifies homologous sequence in flow cell, creates ss bridge. Data analysis - DNA polymerase amplifies and creates dsDNA and then it opens, linker sequences are removed, overlapping regions determine fragment order, use of computational analysis What can genetic engineering be used for? Livestock Crops Drugs Vaccines Diagnostics Industry Waste management What is recombinant DNA? Composed of DNA sequences derived from different sources. What are restriction endonucleases? One of the earliest tools developed for DNA manipulation Nuclease activity that breaks bonds between adjacent nucleotides Hundreds available from different bacteria with different mechanisms and different DNA recognition sites. However, always same outcome - DNA degradation. How many types of restriction endonucleases are there? 3 types Which restriction endonuclease is most common? Type II Describe restriction endonucleases: All 3 types have same recognition and cleavage site Recognise specific short palindromic sequences (4-8bp) Blunt cut - does not leave overhangs Methylated DNA cut Types I and III recognition site and cleavage site are different and usually not palindromes Describe DNA cloning: Cloning vector cleaved with restriction endonucleases DNA fragments of interest obtained by cleaving chromosome with restriction endonuclease. Fragments are ligated to prepared cloning vector DNA is introduced into host cell Propagation (cloning) of transformed cell produces many copies of recombinant DNA. Name and describe the type of cloning vectors: Plasmid - circular, with MCS, selection marker, oris, bacterial host, insert size _15Kb Viral - part or whole inactivated virus, any cell host, insert size _25Kb Cosmid - hybrid between plasmid and phage vectors - bacterial host, insert size _50kb BAC - artificial - derived from E. coli F-plasmid, bacterial host, base _300kb YAC - artificial chromosomes with ori, centromere, telomeres, selection marker, yeast cell, _1Mb insert size HAC - artificial chromosome, mimics cell, episome host, no limit on insert size What are cloning vectors selected and engineered for? Safety Selection ability High growth rate What is genome editing? Process of precisely, permanently and heritably changing the genetic code of an organism by inserting, deleting or modifying or replacing specific sites of DNA. What does CRISPR stand for? Clustered Regularly Interspaced Palindromic Repeats Describe the CRISPR timeline: 1987 - repetitive palindromic DNA sequences in E. coli Mid 2000s - identified as a prokaryote adaptive defence mechanism against viruses 2012 - all CRISPR components were discovered and characterised 2019 - first clinical trials What is the CRISPR locus composed of? tracrRNA Cas genes Leader (AT rich) Repeat (20-50bp) Spacer (30bp) Repeat Spacer Repeat Spacer What are spacers in CRISPR? Segments of unique sequence, captured and integrated into CRISPR locus through a cas1-cas2 protein dimer. Describe the process of adaptation / spacer acquisition in CRISPR: Leader - sequence differs between species - may carry promoter for CRISPR transcription and may contain signals for CRISPR-Cas adaptions. Several CRISPR associated or cas genes. Trans-activating crRNA aids maturation of crRNA precursor and with crRNA forms the guide RNA (gRNA) CRISPR locus is transcribed as long precursor RNA from upstream promoter. Precursor RNA is processed by cas proteins to give rise to short crRNAs Describe CRISPR prime editing: ssDNA cut PE:pegRNA complex binds to target DNA Cas9 Nickase cuts one strand of DNA Reverse transcription of RNA incorporates desired sequence into target DNA. Edited strand is incorporated - original DNA is cleaved by cellular endonucleases Unedited strand is repaired to match newly edited sequences There are two main approached to deliver the CRISPR complex - what are these? Ex vivo gene editing In vivo gene editing (lipofection, microinjection, electroporation, viral vectors, nanoparticles) What are the applications of CRISPR? Disease modelling Basic research Diagnostics Gene therapy Agriculture Bioenergy Epigenome editing Precisio