F: Cell Fundamentals III: DNA Structure, DNA Synthesis And Enzymes Flashcards
What are the main structural features of the DNA helix?
- 2 anti-parallel polynucleotide chains form a RH (right-hand) helix
- Contains minor and major grooves
- Bases on inside of helix, phosphate + sugars on outside
- Nucleotides - repeating base-sugar-phosphate units linked by 3’->5’ phosphodiester bonds
- Bases adenine, guanine, cytosine, thymine
- Sugar = deoxyribose
Why are DNA strands described as anti-parallel?
- Strands run in opposite directions, hence one strand is 5’ -> 3’ and the other is 3’ to 5’
What are grooves in the DNA helix?
- Major groove = backbones far apart (big gap between the backbones)
- Minor groove = backbones close together (small gap between the backbones)
The grooves twist around molecule on opposite sides
What are the base pairs in the DNA helix and how many hydrogen bonds join each base pair?
- A + T = 2 H bonds
- G + C = 3 H bonds
What are purines and what bases are purines?
- Purines are an aromatic heterocyclic composed of carbon and nitrogen. Purines have 2 carbon-nitrogen ring bases
- Guanine + adenine are purine bases
What are pyrimidines and what bases are pyrimidines?
- Pyrimidines are a heterocyclic aromatic compound containing two nitrogen atoms at positions 1 and 3, and contain only one nitrogen-carbon ring
- Thymine, cytosine and uracil are pyrimidine bases
What are the three helical forms of DNA?
- A-DNA - RH helix
- B-DNA - RH helix
- Z- DNA - LH helix
Describe the A form of DNA and how it forms
- RH double helix
- 2 strands of DNA anti-parallel with each other, not symmetrical due to the glycosidic bonds between phosphate and sugar group
- Sugar phosphate chains on outsides, bases on inside
- Has 11 base pairs per helical turn
- Forms when relative humidity of environment is less than 75%, meaning it’s rarely present in normal physiological conditions
Describe the B form of DNA
- RH double helix
- Double strand of B-DNA run in opposite directions
- Structure is asymmetrical with major grooves and minor grooves present alternatively
- Sugar phosphate chains on outside, bases on inside
- In one turn, there are 10 base pairs
What is the difference between A-DNA and B-DNA?
- A-DNA has 11 base pairs per turn, B-DNA has 10 base pairs per turn
Describe the Z form of DNA and how it forms
- LH double helix
- Zigzag appearance of backbone allows it to be distinguished
- Has 12 base pairs per turn
- Occurs when processive enzymes such as polymerase and helicases generate underwound DNA in their wake, causing B-DNA to become Z-DNA
- Formed by 5’ GCGCGCGCGC… or 5’ GTGTGTGTGTG…
What form of DNA is tRNA?
- tRNA is an A-form helix
Describe the levels of DNA structure
- Primary - sequence of bases (DNA sequencing)
- Secondary - Helical structure (X-ray and chemistry)
- Tertiary - DNA supercoiling (Electron microscopy)
- Quaternary - Interlocked chromosomes
How do the sequences of bases in DNA carry the genetic blueprint of life?
- Sequences of bases in DNA ultimately determine the different proteins formed via protein synthesis, and henceforth, regulate protein synthesis
- Proteins are the basic unit of structure and function in organisms’ cells
What is the tetraplex structure of DNA?
What does it require?
- A four stranded DNA helix formed at telomeres
- Requires G-rich DNA sequences
What are telomeres and what do they do?
- A region of repetitive DNA sequences at the end of a chromosome
- Protect end of chromosome from degradation
- Naturally occurring
What is a Holliday junction?
- A 4-way nucleotide strand linking 2 double stranded DNA structures
- Arise naturally in living cells through DNA strand exchange between 2 homologous chromosomes
- Have important role in DNA repair
What is DNA supercoiling?
- Expression of strain on DNA strand, how twisted the double helix is. It’s a higher order DNA structure
- It’s the tertiary structure of DNA
What is Sanger sequencing?
- DNA strand copied with DNA polymerase in presence of inhibitor that arrest (halt) DNA synthesis at specifically A, C, G or T
- DNA strands separated by length on a polyacrylamide gel
- If DNA or incorporated inhibitor is radioactive or fluorescent, DNA bands can be visualised and sequence read
- 700-1000 bases per read
What is higher order structure of DNA?
- The formation of interlocked chromosomes by assemblage of nucleosomes
Describe the features of DNA in bacteria
- Most bacteria have haploid genome, and single chromosome composing of a circular, double stranded DNA molecule
- DNA supercoiled- ribbon is itself twisted in space
What enzymes causes DNA supercoiling?
DNA gyrase
What main protein is DNA complexed with in eukaryotic cells and what does this form?
- Histones (+ other proteins)
- Forms nucleoprotein complex called chromatin
What is a nucleosome?
- Basic building block of chromatin
How do nucleosomes form chromatin?
- They further coil and condense to form chromatin
How are nucleosomes identified in an electron microscope image?
- Look like beads on a string, like a pearl necklace
Outline the features of nucleosomes
- Linker DNA
- Core DNA
- Histone 1
- Histones 2A, 2B, 3, 4
Why is histone 1 important and what does it do?
- This is where linker DNA exits
- Histone 1 can regulate folding of chromatin fibres into more compacted higher-order structure
- Allows for compaction so DNA can fit into the nucleus
What is formed when chromatin undergoes higher order coiling and looping?
- Chromosomes
What are the different stages in the hierarchy of how genetic material is organised?
- 2 chromatids
- One coil
- One rosette
- One loop
- 30 nm fibre
- ‘Beads-on-a-string’ form of chromatin
- DNA
Which anticancer therapies rely on DNA damaging agents?
Radiation:
- UV light produces thymine dimers
- Ionising radiation (x-rays, gamma rays) break DNA chromosomes to cause leukaemia
In what ways can DNA be damaged to cause mutations in DNA?
- Spontaneous damage e.g. loss of bases, hydrolysis of C to U
- Chemicals and radicals generated by oxidative metabolism, causes change in base structure (but also cyclophosphamide) and can cause base insertion (interpolators such as doxorubicin widely used as anticancer drugs)
- Radiation:
- UV light produces thymine dimers (2 thymine molecules join together)
- Ionising radiation (X-rays, gamma rays) break DNA chromosomes to cause leukaemia
Why is the DNA repair system so important?
- Maintains genome stability
- Important as patients with xeroderma pigmentosum have defect in excision repair that deals with UV damage to DNA. Very prone to skin cancer
- Other cancer prone families have DNA repair defects e.g. predisposing to breast cancer and colon cancer
- DNA repair to crucial DNA structure and function
What antibacterial agents inhibit DNA replication and what enzyme do they act on?
- Ciprofloxacin
- Levofloxacin
- Novobiocin
- These are all gyrase inhibitors
What anti tumour agents inhibit DNA replication and what enzyme do they act on?
- Etoposide
- Doxorubicin
- Mitoxantron
- These are all Topo II inhibitors
What antiviral- AIDS agents inhibit DNA replication and what enzyme do they act on?
- AZT and others
- Reverse transcriptase
What inherited defects in mismatch repair genes of bacteria are involved in cancer?
- mutH
- mutL- colon cancer
- mutS - colon cancer
What inherited defects in mismatch repair genes of humans are involved in cancer?
- hMLH - colon cancer
- hMSH - colon cancer
What is the role of DNA helicase?
- Acts at replication fork (the area where the replication of DNA will actually take place)
- Uses ATP to break base pairs between parental DNA, unwinding the double helix
- Opens DNA to allow copying
What is the role of DNA binding protein?
- Prevent the open strand from being digested by nuclear enzymes
What are topoisomerases?
Enzymes that remove the positive supercoils ahead of DNA replication forks- solving the ‘DNA winding problem’
What are the leading and lagging strands of DNA?
- Leading = sense = 5’ to 3’
- Lagging = antisense = 3’ to 5’
How are the leading and lagging strands replicated?
- Replicative DNA polymerases catalyse their synthesis in a multi enzyme process
What are the key properties of DNA polymerase?
- Acts in 5’ to 3’ direction
- Utilises A-T and G-C base pairing to synthesise new DNA strand
- Requires DNA template, a DNA or RNA primer, 4 deoxyribonucleoside triphosphate (dNTP) - the N stands for any nucleotide, so could be replaced by A, C, G or T- building blocks and Mg2+ ions
- Has a proof-reading editing function- able to go back and check right nucleotide has been attached, and if not, it can correct the nucleotide- this function is key to the high fidelity of DNA synthesis
What is dNTP made of and what does dNTP do?
- A phosphate group
- A deoxyribose sugar
- A nitrogenous base
Provides nucleotides to unzipped strand using template strand
What is a primer?
- A short nucleic acid sequence that provides a starting point for DNA synthesis
What are the types of DNA polymerase in bacteria and what do they do?
- DNA polymerase I - Repair
- DNA polymerase II - Repair
- DNA polymerase III - Replication
What are the types of DNA polymerase in eukaryotes and what do they do?
- Alpha - replication
- Beta - repair
- Gama - mitochondrion
- Delta - replication
- Epsilon - replication
Also have repair and translesion polymerase
What is the role of telomerase?
- A reverse transcriptase expressed in stem and cancer cells, makes DNA at telomere ends of chromosomes to offset DNA during normal replication due to ‘end replication problem’
How does DNA polymerase work?
- Add nucleotides to 3’OH end of newly synthesised strand, they elongate in the 5’ to 3’ direction
Describe the structure of dNTP
- 3 phosphate groups attached to 5’ carbon of deoxyribose, which is attached to the 3’OH and one of the 4 bases
What is the difference between dNTPs and nucleotides?
- dNTPs = base + sugar, but these release pyrophosphate (P2O7 4-) to become nucleotides
How does a nucleotide join onto the template strand?
- The 3’OH transfers a pair of electrons to the phosphate group attached to the 5’ CH2 which then transfers the electron pair to the O on the pyrophosphate, causing the pyrophosphate to split from the dNTP
- This forms a phosphodiester bond with the dNTP, which is now a nucleotide as the pyrophosphate has split off
- The chain extends in the 5’ to 3’ direction
What are the problems associated with DNA synthesis always proceeding in the 5’ to 3’ direction?
- 5’ to 3’ activity of DNA polymerase means 1 DNA strand must be discontinuously made, causing the formation of Okazaki fragments
- It’s discontinuously made due to anti-parallel nature, meaning replication has to be constantly reinitiated, and Okazaki fragments then need to be processed, so the lagging strand is synthesised discontinuously
What are Okazaki fragments?
Short sections of DNA formed at time of discontinuous synthesis of the lagging strand during DNA replication
How does DNA being a multi-enzyme process fix the problem of the discontinuous synthesis of the lagging strand?
Multi priming steps on the lagging DNA and processing of Okazaki fragments
How does DNA polymerase ensure DNA synthesis occurs with high fidelity?
- Complementary base pairing with proofreading by DNA polymerase, and mismatch repair system ensure DNA replication proceeds with high fidelity
- Mismatch repair system - corrects most of the polymerase errors. Multienzyme system highly conserved across species
What are the features of the DNA helix that are important for DNA replication?
- 2 anti-parallel polynucleotide chains form a RH helix, bases inside, phosphate + sugars on outside. One strand 5’ to 3’, other 3’ to 5’
- Polynucleotide chains linked together by H bonds between A+T and G+C
- One strand is complementary to the other via complementary base pairing
- DNA synthesis requires unwinding and opening of helix, followed by copying of each DNA strand
Why is DNA replication semiconservative?
1 strand of the replicated DNA is the same as one of the strands of the original DNA, and the other is a newly synthesised complementary strand
Where is DNA replication initiated?
At specific sites on DNA called replication origins
What are replication origins recognised by?
An initiation complex- a sequence of proteins required to begin DNA replication
What happens to DNA at replication origins?
DNA unwinds to form a replication bubble and allow access to the replication machinery
What are replication forks?
On either side of the replicator bubble, replication forks are the points at which the parental DNA duplex (double helix) is being unwound
What phase does DNA synthesis occur in?
S phase of the cell cycle, it involves complete unwinding of the parental DNA
Define enzymes
Proteins that speed up (catalyse) specific chemical reactions
What are the key properties of enzymes?
- Increase reaction rate
- Show specificity
- Unchanged at end of reaction
- Do no alter reaction equilibrium
- Facilitate reaction by decreasing the free energy of activation of the reaction
How do enzymes increase reaction rate?
- Decrease activation energy required to initiate reaction
What are some types of enzymes?
- Proteases
- Nucleases
- Polymerases
- Kinases
Describe the active site of enzymes
3D cavity of cleft that binds substrates with specificity through electrostatic, hydrophobic, hydrogen bonding and van der Waals interactions
Form ES complex at active site
What is the lock and key mechanism?
Active site complementary to substrate, substrate binds perfectly, like a key in a lock
What is the induced fit hypothesis?
Active site of enzyme changes shape as it binds to substrate, making binding easier and more effective
What is Vmax?
Maximum rate of reaction of enzyme at a specific concentration of substrate
What is Km?
Concentration of substrate at which half of active sites of enzymes are occupied by the substrate
Describe competitive inhibition and what happens to Km and Vmax?
Inhibitor competes with substrate for binding to enzyme active site, forming an inactive enzyme-inhibitor complex
Km is increased as more substrate is needed to achieve Vmax/2
Vmax is unchanged as the effects of the inhibitor can be outcompeted by higher substrate concentrations
Describe non-competitive inhibition and what happens to Km and Vmax?
Inhibitor binds at different site and does not compete with substrate for binding at active site, but causes change in shape of active site
Km unaltered
Vmax reduced because active site no longer binds as well with substrate, therefore, reaction takes longer to complete
What is feedback inhibition
A cellular control mechanism, in which the activity of an enzyme is inhibited by the end product of a biochemical pathway
What is allosteric regulation?
- A regulatory molecule (acting at pocket distinct from active site)
- Changes the enzyme conformation to influence the active site
- Can cause decrease or increase in enzyme activity
- Allosteric enzymes possess sites for allosteric binding
- Controls the flux of material through a metabolic pathway
Describe compartmentation
- Sequences in enzyme polypeptide chain target enzyme to ER, mitochondrion, nucleus etc
Describe covalent modification of enzyme
Change enzyme shape and activity -e.g. phosphorylation
What are allosteric enzymes?
Enzymes that have an additional binding site for effector molecules other than the active site
What are the properties of allosteric enzymes?
- Multisubunit complexes
- Regulatory sites and catalytic sites on different subunits
- Regulation occurs via conformational changes
- Exhibit non- Michaelis- Menten kinetics:
- V & S plots sigmoidal
- Involved in feedback inhibition of metabolic pathways
