Molecular B1 Flashcards
What is a genome,gene,genotype,phenotype,nucleic acid
All life depends on three critical
Molecules name them and their functions
Genome: an organism’s genetic material
Gene: a discrete units of hereditary information located on the chromosomes and consisting of DNA.
Genotype: The genetic makeup of an organism
Phenotype: the physical expressed traits of an
organism
Nucleic acid: Biological molecules (RNA and DNA) that allow organisms to reproduce; Nucleic acids are polynucleotides—that is, long chainlike molecules composed of a series of nearly identical building blocks called nucleotides. Each nucleotide consists of a nitrogen-containing aromatic base attached to a pentose (five-carbon) sugar, which is in turn attached to a phosphate group.
All life depends on 3 critical molecules
DNAs:
• Stores and transmit information on how cell works and divide.
RNAs:
• Act to transfer short pieces of information to different parts of cell
• Provide templates to synthesize into protein
Proteins:
• Form enzymes that send signals to other cells and regulate gene activity
• Form body’s major components (e.g. hair, skin, etc.)
What is denaturation
What is the structure of nucleic acids
What is the difference between a nucleoside and a nucleotide
The presence of the 2’OH (the OH is on the second carbon ) confers special chemical and structural properties to RNA compared to DNA true or false(DNA has 2’H (H is on the second carbon) not 2’OH)
While DNA contains deoxyribose, RNA contains ribose, characterised by the presence of the 2′-hydroxyl group on the pentose ring (Figure 5). This hydroxyl group make RNA less stable than DNA because it is more susceptible to hydrolysis true or fakse
The process of separating two DNA strands into two single strands is called denaturation.
Nucleic Acids Structure 1.Nucleic acid bases and nucleotides 2-Double Helix Structures of DNA B-DNA A-DNA and A-dsRNA Z-DNA 3-Transition DS(double stranded) SStranded (single stranded) DNA 4- Structure of tRNA(transfer RNA)
Nucleoside: composed of sugar(ribose or deoxyribose) and the base (purine or pyrimidine)
Nucleotide:composed of phosphate 1,2,3 and sugar(ribose or deoxyribose) and the base (purine or pyrimidine)
Or A nucleoside consists of a nitrogenous base covalently attached to a sugar (ribose or deoxyribose) but without the phosphate group. A nucleotide consists of a nitrogenous base, a sugar (ribose or deoxyribose) and one to three phosphate groups
A nucleotide: Pentose+phosphate+base
What is the consequence of Aromaticity of bases (purines and pyrimidines)
Bases are planar true or false
Why are purines and pyrimidines) called bases?
Ring nitrogens of bases are normally not protonated at physiological pH
True or false
Pyrimidines:
-Large number of electrons -
in the pi orbital system
Protonation of ring nitrogens:
Purines:
-Delocalization of electrons:
- transient dipole and
attraction between bases
Base stacking
They called bases Because N1 and N3 of pyrimidine, and N1, N3, and N7 of purine can accept protons
State three similarities between DNA and RNA
State seven differences between them
Both have adenine, guanine, cytosine • The nucleotides are linked together by
phosphodiester bonds.
• Main function involves protein biosynthesis.
DNA
- Bases are A, G, C and T
- Pentose sugar is deoxyribose
- Present in nucleus and mitochondria but never in cytoplasm
- Consist of 2 helical strands
- There are A, B, C, D, E and Z forms of DNA
DNA
- Large molecules
- One strand 3’- 5’ carries genetic information
- DNA can form RNA by the process of transcription
- Purine and pyrimidine contents are almost equal
- Alkali hydrolysis does not give 2’-3’ cyclic diesters
RNA
- Bases are A, G, C and U
- Pentose sugar is ribose
- In addition to nucleus, RNA is found in the cytoplasm
- Single stranded
- There are tRNA, mRNA, rRNA, snRNA etc
RNA
- Only m- and rRNA are large molecules
- mRNA transcribed from DNA carries genetic information
- RNA does not give rise to DNA except only in the presence of reverse transcriptase
- Purine and pyrimidine nucleotides not equal
- Alkali hydrolysis gives 2’-3’ cyclic mononucleotides.
State seven differences between mRNA and tRNA
mRNA
- Large molecular wt
- Most heterogenous
- Acts as a template for protein synthesis
- Carries codons
- Shape and size is not constant
- The cap structure is found in the 5’ end
RNA
- Poly A tail is found on 3’ end 8. Precursor is pre-mRNA
- Unusual bases are not found
- Stem and loop structure is not found
tRNA
- Low molecular wt
- Only about 20 different forms, less heterogenous
- Acts as a carrier of amino acids
- Carries anticodons
- Shape and size is constant for all tRNAs
- No cap structure.
- 3’ end always carries CCA sequence where specific amino acids bind
- Pre-tRNA precursor
- Unusual bases such as pseudouridine, thymine etc. are found
- Stem and loop structure is a consistent feature.
Recombinant DNA technology involves techniques in manipulating DNA such as,
What is a restriction enzyme or restriction endonuclease
What is it’s function
Involves techniques in manipulating DNA:-
• Molecular Cloning.
• DNA sequencing.
• Polymerase Chain Reaction (PCR).
• Nucleic acid blotting and hybridization.
(Southern, Northern analysis).
Production of proteins.
Creation of Knock-out, Knock-in and Transgenic mice.
Nucleic acid Microarray (simultaneous monitoring of expression level of each gene in a cell.
restriction enzyme, restriction endonuclease, or restrictase is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites.
A restriction enzyme is a protein isolated from bacteria that cleaves DNA sequences at sequence-specific sites, producing DNA fragments with a known sequence at each end.
The function of restriction endonucleases is mainly protection against foreign genetic material especially against bacteriophage DNA. The other functions attributed to these enzymes are recombination and transposition.
Enzymes are the agents of metabolic function
• Enzymes endow cells with the remarkable capacity to exert kinetic control over thermodynamic potentiality
True or false
What are the properties of enzymes?
Explain catalytic power under enzymes and give an example of an enzyme with catalytic power
Enzyme catalyzed reactions are much faster than the corresponding uncatalyzed reaction
E+S forward arrow and backward arrow ES forward arrow and backward arrow EP forward arrow and backward arrow E+P
) High reaction rates
(2) Catalyze reactions at physiological conditions (milder reaction conditions)
(3) Have a high degree of specificity (e.g. only A is converted to B)
(4) Can be regulated (e.g., A is only converted to B under certain conditions)
Catalytic Power
• Enzymes can accelerate reactions as much as 1016 over uncatalyzed rates!
• Urease is a good example: – Catalyzed rate: 3x104/sec
– Uncatalyzed rate: 3x10-10/sec – Ratio is 1x10 to the power 14
Succinyl CoA transferase is 1x10 to the power 13
What is the international classification of enzymes? (State the class of enzyme and the type of reaction they catalyze) Substrates are converted to products by enzymes and products Can be converted to substrates by enzymes true or false What is the function of DNA ligase?
Oxidoreductases:transfer of electrons(hydride ions or H atoms)
Transferases:group transfer reactions
Hydrolases: hydrolysis reactions (transfer of functional groups to water )
Lyases:Addition of groups to double bonds or formation of double bonds by the removal of groups
Isomerases: transfer of groups within molecules to yield isomeric forms
Ligases: formation of C-C,C-S,C-O,and C-N bonds by condensation reactions coupled to ATP cleavage
DNA ligase joins pieces of DNA together, mainly joins Okazaki fragments with the main DNA piece.
DNA Ligase enzymes seal the breaks in the backbone of DNA that are caused during DNA replication, DNA damage, or during the DNA repair process.
What’s the difference between cofactors and coenzymes and give an example each
Name four metal inorganic elements that serve as cofactors for enzymes
Name four organic cofactors or coenzymes and an example of each of the chemical group transferred and their dietary precursor in mammals
What’s a prosthetic group
Give an example
Cofactors may be metal ions
(such as Zn2+ required for the catalytic activity of carboxypeptidase A)
Coenzymes are organic molecules
(such as the NAD+ in YADH)
Copper is a cofactor for Cytochrome oxidase
Potassium is a cofactor for pyruvate kinase
Magnesium is a cofactor for glucose-6-phosphate,pyruvate kinase
Selenium-glutathione peroxidase
Nickel (II) ion for Urease
Coenzyme
Biocytin
Chemical group transferred- Carbon dioxide
Dietary precursor- biotin
Coenzyme A
Acyl groups
Pantothenic acid
Nicotinamide adenine dinucleotide
Hydride ion
Nicotinic acid or niacin
TetrahydrofolAte
One carbon groups
Folate
Other cofactors
• Known as prosthetic groups
• Permanently attached with their protein • Often by covalent bonds
• Example: Heme in hemoglobin
Explain specificity as a property of enzymes
What controls this property
What is stereospecificity
Why does it arise
The same set of non-covalent interactions that enable a protein to fold are involved in stabilizing
the interaction between the substrate and enzyme. True or false
What is the difference between induced fit mechanism and lock and key mechanism
Enzymes selectively recognize proper substrates over other molecules
• Enzymes produce products in very high yields - often much greater than 95%
• Specificity is controlled by structure - the unique fit of substrate with enzyme controls the selectivity for substrate and the product yield
Stereospecificity
Enzymes are highly specific in both in binding chiral substrates & in catalyzing their reactions.
Stereospecificity arises because enzymes virtue of their inherent chirality.
Proteins consists of only L-amino acids.
Induced fit mechanism is most prevalent in enzymes. The enzyme active site adapts its structure to interact with the substrate & transition states.
The specific action of an enzyme with a single substrate can be explained using a Lock and Key analogy first postulated in 1894 by Emil Fischer. In this analogy, the lock is the enzyme and the key is the substrate. Only the correctly sized key (substrate) fits into the key hole (active site) of the lock (enzyme).
How do enzymes accelerate reactions
- Chemical reactions between the substrate and functional groups on the enzyme can provide alternative, lower-energy reaction pathways.
(Example:group transfer through an intermediate with the group transiently covalently attached to the enzyme) - Binding energy, DGB, is a major source of free energy used by enzymes to lower the activation energies of reactions.
Enzyme Kinetics • Enzymes accelerate reactions by lowering the free energy of activation • Enzymes do this by binding the transition state of the reaction better than the substrate
An enzyme helps catalyze a reaction by decreasing the free energy of the transition state. As a result, more product will be made because more molecules will have the energy necessary for the reaction to occur and the reaction will occur at a faster rate
Enzymes perform the critical task of lowering a reaction’s activation energy—that is, the amount of energy that must be put in for the reaction to begin.
Define rate or velocity of a reaction,rate constant,rate law,order of a reaction,molecularity of a reaction
Rate:change in concentration of a reactant or product in a given time
Rate is equal to the rate constant x the concentration of reactants
How much the concentration of a reactant decreases over a given period of time or the concentration of a product increases over a given period of time
Appearance of a product and disappearance of a reactant
So rate is equal to Delta(the triangle sign) concentration of the product divided by Delta (triangle sign) time and also equal to negative (delta concentration of the reactant divided by delta time )
It is measured in mol per Litre per unit time(check how it’s actually written)
Rate constant : The rate constant, or the specific rate constant, is the proportionality constant in the equation that expresses the relationship between the rate of a chemical reaction and the molar concentrations of the reacting substances.(check the rate constant for first order,second order,third order reactions and zero order reactions)
Rate law: A rate law shows how the rate of a chemical reaction depends on reactant molar concentration. For a reaction such as aA → products, the rate law generally has the form rate = k[A]ⁿ, where k is a proportionality constant called the rate constant and n is the order of the reaction with respect to A. And if it was a product B it’ll be k[B] raised to the power m.
Order of a reaction: The Order of reaction refers to the relationship between the rate of a chemical reaction and the concentration of the species taking part in it. The overall order of the reaction is found by adding up the individual orders. For example, if the reaction is first order with respect to both A and B (a = 1 and b = 1), the overall order is 2. We call this an overall second order reaction.
The order of reaction is defined as the dependence of the concentration of all reactants in a chemical reaction on the rate law expression
The molecularity of a reaction is defined as the number of reacting molecules which collide simultaneously to bring about a chemical reaction. In other words, the molecularity of an elementary reaction is defined as the number of reactant molecules taking part in the reaction.
a single-step chemical reaction is said to have a molecularity of 1 if just one molecule transforms into products. We call this a unimolecular reaction. An example is the decomposition of N2 O4. N2 O4 (g) → 2NO2 (g)
is also defined as the number of reactant molecules taking part in a single step of the reaction.
What is the The Michaelis-Menten Equation and what does the theory assume?
Breakdown of enzyme substrate complex to form product sis assumed to be slower than what two things ?
When the S in the equation is low,what happens to the equation rate and when it’s high what happens
What does the Michaelis-Menten equation describe ?
When is Km = [S] ?
What is Michaelis constant?
What is a first order,second order,zero order reaction
What does a Small Km mean?
What does a high Km mean?
Equation. v= Vmax[S] divided by Km + [S]
v- velocity of the reaction
Vmax-maximum rate achieved by the system or maximum velocity
[S]-concentration of a substrate S
Km-Michaelis constant (has a unit which is taken according to the unit of the substrate so if unit is mmol/L the. The Km unit is mmol/L)
• Louis Michaelis and Maude Menten’s theory
• It assumes the formation of an enzyme- substrate complex
• ItassumesthattheEScomplexisinrapid equilibrium with free enzyme
• Breakdown of ES to form products is assumed to be slower than
(1) formation of ES and
(2) breakdown of ES to re-form E and S
Combination of zero-order and first-order kinetics
• When [S] is low, the equation for rate is first order in [S]
• When [S] is high, the equation for rate is zero-order in [S]
• The Michaelis-Menten equation describes a rectangular hyperbolic dependence of Vo on [S]
When V = Vmax divided by 2
Km is defined as the concentration of substrate at which enzyme is working at half of maximum velocity. It is also a measure of the affinity that the enzyme has for its substrate.
First order:rate is dependent on the concentration of one reactant
Second order:dependent on conc of two reactants
Zero order: independent on conc of the reactants
Km is a constant
• Km is a constant derived from rate
constants
• Km is, under true Michaelis-Menten conditions, an estimate of the dissociation constant of E from S
• Small Km means tight binding; high Km means weak binding
How can you reach Vmax?
Is it practical or theoretical and why?
What is turnover number
What is the formula
The theoretical maximal velocity
• Vmax is a constant
• Vmaxisthetheoreticalmaximalrate of the reaction - but it is NEVER achieved in reality
• To reach Vmax would require that ALL enzyme molecules are tightly bound with substrate
• Vmax is asymptotically approached as substrate is increased
The turnover number
(also known as the molecular activity of the enzyme)
A measure of its maximal catalytic activity
• kcat, the turnover number, is the number of substrate molecules converted to product per enzyme molecule per unit of time, when E is saturated with substrate.
kcat =Vmax divided by [ET]
ET-given enzyme concentration
Vmax- max reaction rate
kcat-turnover number
Unit of kcat is per second so sec raised to the power -1 or 1 divided by sec
• If the M-M model fits, k2 = kcat
kcat = Vmax/Et
K2 is the catalytic rate constant
What is the catalytic efficiency of an enzyme
What is Double-Reciprocal or Lineweaver-Burk Plot(look at a pic of how it looks like)
State the equation issued in this plot
Catalytic efficiency of an enzyme
Name for kcat/Km
• An estimate of “how perfect” the enzyme is
• kcat/Km is an apparent second- order rate constant
• It measures how the enzyme performs when S is low
• Catalytic efficiency cannot exceed the diffusion limit - the rate at which E and S diffuse together
The double-reciprocal (also known as the Lineweaver-Burk) plot is created by plotting the inverse initial velocity (1/V0) as a function of the inverse of the substrate concentration (1/[S]). The Vmax can be accurately determined and thus KM can also be determined with accuracy because a straight line is formed. biochemistry, the Lineweaver–Burk plot (or double reciprocal plot) is a graphical representation of the Lineweaver–Burk equation of enzyme kinetics,
1/V =Km/Vmax[S] + 1/Vmax
State the optimum pH of pepsin,catalase,trypsin,Fumarase,ribonuclease,Arginase
State and define the types of enzyme inhibitors
State the classes of inhibition
P-1.5 C-7.6 T-7.7 F-7.8 R-7.8 A-9.7
Reversible versus Irreversible
• Reversible inhibitors interact with an enzyme via noncovalent associations
• Irreversible inhibitors interact with an enzyme via covalent associations
Classes of Inhibition
Two real, one hypothetical
• Competitiveinhibition-inhibitor(I) binds only to E, not to ES
Example: Malonate is a strong competitive inhibitor of succinate dehydrogenase
• Uncompetitiveinhibition-inhibitor (I) binds only to ES, not to E. This is a hypothetical case that has never been documented for a real enzyme, but which makes a useful contrast to competitive inhibition
• Noncompetitive(mixed)inhibition - inhibitor (I) binds to E and to ES
What changes in competitive inhibition in the M-M equation and the changes in non competitive or mixed inhibition
How is enzyme activity regulated
Why are regulatory enzymes important?
Kmchanges while Vmax does not
They increase Km by interfering with the binding of the substrate, but they do not affect Vmax because the inhibitor does not change the catalysis in ES because it cannot bind to ES.
At any given time, only the competitive inhibitor or the substrate can be bound to the enzyme (not both). That is, the inhibitor and substrate compete for the enzyme. Competitive inhibition acts by decreasing the number of enzyme molecules available to bind the substrate.
Km Unchanged Vmax Reduced
In non-competitive inhibition, the inhibitor binds to an allosteric site and prevents the enzyme-substrate complex from performing a chemical reaction. This does not affect the Km (affinity) of the enzyme (for the substrate).
Noncompetitive inhibition, a type of allosteric regulation, is a specific type of enzyme inhibition characterized by an inhibitor binding to an allosteric site resulting in decreased efficacy of the enzyme. An allosteric site is simply a site that differs from the active site- where the substrate binds.
Two ways that this may occur:
1) Control of enzyme availability
Depends on rate of enzyme synthesis & degradation
2) Control of enzyme activity
Enzyme-substrate binding affinity may vary with binding of small molecules called allosteric effectors (ex: BPG for Hb)
Allosteric mechanisms can cause large changes in enzymatic activity
important in controlling flux through metabolic pathways
1. Allosteric enzymes
2. Regulation by covalent modification: Covalent modifications are enzyme-catalysed alterations of synthesised proteins and include the addition or removal of chemical groups. Modifications can target a single type of amino acid or multiple amino acids and will change the chemical properties of the site
3.Regulation by Feedback Inhibition : Feedback inhibition occurs when the end product of a reaction interferes with the enzyme that helped produce it. The inhibitor does this by binding to a second active binding site that’s different from the one attached to the initial reactant. The enzyme then changes its shape and can’t catalyze the reaction anymore. example:
Conversion of L-threonine to L- isoleucine catalyzed by a sequence of five enzymes, E1-E5
L-isoleucine is an inhibitory allosteric modulator of E1
Regulatory enzymes, which facilitate the transferring of phosphate groups to the specific substrates, are called kinases
Regulatory molecules. Enzymes can be regulated by other molecules that either increase or reduce their activity. Molecules that increase the activity of an enzyme are called activators, while molecules that decrease the activity of an enzyme are called inhibitors
What is DNA replication
What is the importance of high fidelity of replication ?
What should germ cells be protected against?
What should somatic cells be protected against?
In all cells, DNA sequences are maintained and replicated with high fidelity.
•The mutation rate, approximately 1 nucleotide change per 109 nucleotides each time the DNA is replicated, is roughly the same for organisms as different as bacteria and humans.
•Because of this remarkable accuracy, the sequence of the human genome (approximately 3x109 nucleotide pairs) is changed by only about 3 nucleotides each time a cell divides.
•This allows most humans to pass accurate genetic instructions from one generation to the next, and also to avoid the changes in somatic cells that lead to cancer.
True or false
All organisms must duplicate their DNA with
extraordinary accuracy before each cell division. This process is called DNA replication.
.
• Maintaining order requires the continued surveillance and repair of the genetic information because DNA inside cells is repeatedly damaged by chemicals and radiation from the environment, as well as by thermal accidents and reactive molecules.
• Despite the great efforts that cells make to protect their DNA, occasional changes in DNA sequences do occur. Over time, these changes provide the genetic variation upon which selection pressures act during the evolution of organisms
• Whereas germ cells must be protected against high rates of mutation to maintain the species, the somatic cells of multicellular organisms must be protected from genetic change to safeguard each individual.
Explain DNA replication is semi conservative according to Meselson and Stahl experiment
What are the properties of DNA polymerase
DNA replication is Semiconservative
The two old DNA strands serves as a template for the formation of an entire new strand.
properties common to ALL DNA polymerases
1) Catalyze the polymerization of deoxyribonucleotides in the 5’ to 3’ direction.: DNA synthesis catalyzed by DNA polymerase. DNA polymerase catalyzes the stepwise addition of a deoxyribonucleotide to the 3’-OH end of a polynucleotide
chain, the primer strand, that is paired to a second template strand. The newly synthesized DNA strand therefore polymerizes in the 5’-to-3’ direction.
Because each incoming deoxyribonucleoside triphosphate must pair with the template strand to be recognized by the DNA polymerase, this strand determines
which of the four possible deoxyribonucleotides (A, C, G, or T) will be added.
2) Require a template.
3) Require a primer
What are the biological roles of DNA polymerase I
Explain elongation as a step in DNA replication
• removes RNA primers • fills gap with DNA • DNA repair • 5’→3’ polymerase fills gap left by repair enzymes which excise regions of DNA containing damaged or mispaired nucleotides (more later) • Processivity: usually catalyzes ~20 nt additions before falling off template. Enymatic activities of DNA Pol I • 5’ -> 3’ Polymerase • 3’ -> 5’ Exonuclease • 5’ -> 3’ Exonuclease
DNA polymerase type I cuts out primers in a 5’ to 3’ exonuclease activity to pluck out RNA primers in the leading strand . It reads the DNA strand which the primer has been plucked out from,from 3’ to 5’ and synthesizes from 5’ to 3’. It also proof reads from 3’ to 5’ and if any mistake is found it cuts it out in a 3’ to 5’ exonuclease activity .
Same for the lagging strand.
Elongation :primase makes RNA primers Which enable DNA polymerase type III to make DNA since it needs the 3’ OH of RNA primers to carry out its activity. It reads the first DNA parent strand from 3’ to 5’ and It synthesizes RNA primers in a 5’ to 3’ fashion. DNA polymerase now also reads the DNA strand from 3’ to 5’ and Makes a DNA strand in a 5’ to 3’ fashion. Or opposite fashion of the parent strand .So RNA primers are produced and the DNA polymerase follows and makes the DNA strand so it’s like RNA primer(5’ to 3’) then DNA strand(continues where the RNA primer ended which is at 3’ and continues with 5’ to 3’) . The strand that theDNA polymerase makes and that’s trans it toward the replication fork is called the leading strand. The primase comes to the other parent strand which will be in the 3’ to 5’ and read that strand and synthesize complimentary nucleotides from 5’ to 3’. So now there’s an OH at the 3’ strand and DNA polymerase continues and reads the strand from 3’ to 5 thereby continuing what the primase started and synthesizes the strand from 5’ to 3. This new strand is the lagging strand
But the difference is that there are DNA strands in between the RNA primers meanwhile in the leading strand the RNA primer is only seen before the DNa strand and it’s all DNA. No RNA in between. Okazaki fragments are seen in lagging strand. These fragments are defined as fragments where there are multiple RNA primers and multiple stretches of DNA.
DNA polymerase proof reads to prevent mistakes. It goes back the strands it has read to make sure everything has been paired correctly. It reads from 3’ to 5’. And if there are any mistakes it uses a 3’ to 5’ exonuclease activity to cut it out and put in the correct complimentary nucleotide.
DNA polymerase type I cuts out primers in a 5’ to 3’ exonuclease activity to pluck out RNA primers in the leading strand . It reads the DNA strand which the primer has been plucked out from,from 3’ to 5’ and synthesizes from 5’ to 3’. It also proof reads from 3’ to 5’ and if any mistake is found it cuts it out in a 3’ to 5’ exonuclease activity .
Same for the lagging strand. In the lagging strand though this creates gaps between the DNA strands since that’s where the primers used to be. Ligase enzyme comes on the lagging strand and fuses the DNA ends together. In HIV their T cells are infected. And replication occurs a lot. Drugs target the T cell replication process. Example of such drugs are Nucleoside reverse transcriptase inhibitors(didanosine) . These remove the 3’ OH region so the DNA polymerase III is not able to build on it to make a DNA strand .
Explain the fidelity of DNA replication
Fidelity of DNA replication
Low error frequency (≈ 10-9) accounted for by redundant safeguards.
1. Binding pocket of DNA polymerase clamps tightly around the base before catalysis occurs. Wobble pairs (Non Watson/Crick geometry) don’t fit and so catalysis can’t occur.
• But binding pocket is unlikely to be rigid enough to exclude wobble pairs every time.
2. Enol tautomers(Watson/Crick geometry but rare tautomer) are very unstable (keto/enol tautomerization equilbrium constants are in the 10-5 to 10-3 range).
• But this isn’t enough to account for the 10-9 error frequency.
3. DNA polymerases have “editing exonuclease activities” that allow them to erase mistakes and try again
4. Cells contain mismatch repair systems that come along after DNA polymerase to clean up any residual errors
How is replication fidelity increased by mismatched repairs
What is the structural basis for proofreading
What is a replisome
What’s a lagging strand and a leading strand
What is processivity
What is fidelity
Explain mismatch repair mechanism
State the proteins involved
DNA methylation
DNA methylation is delayed after replication
Switch between Polymerization and Editing Modes
in DNA Polymerase: Structural Basis for “Proofreading”
Replisome: replicating machinery that moves DNa along at the replication fork
It consists of DNA polymerase III
Primase,sliding clamp,single stranded burning protein,clamp loader,Helicase
A strand that is synthesized discontinuously in the 5’ to 3’ direction away from the replication fork
A strand that is synthesized in the 5’ to 3’ direction towards the replication fork. It is synthesized continuously
Number of catalytic turnovers per binding event ie. the number of nucleotides incorporated before DNa poly erase dissociates from DNA
DNA polymerase I had a processivity number of 10 to 100 nucleotides
DNA poly erase III has a processivity number in the thousands because of the beta sliding clamp(ring shaped protein that encircles the DNA polymerase of the lagging strand and keeps it from falling off when it restarts DNA synthesis at a new Okazaki fragment.
Fidelity is the frequency of errors. Fidelity is high due to the 3’ to 5’ exonuclease activity of DNA polymerase I and DNA polymerase III
They both have a net error of 1 error per 10 raised to the power 6 to 10 raised to the power 8 nucleotide additions.
During DNA replication DNA polymerase may add wrong base during elongation. The wrongly added base results in mismatched nucleotides because of the presence of this mismatch the DNA strands get distorted. Mismatch repair is the mechanism by which mismatched nucleotides are removed .
Mut S,Mut L,Mut H
Mut S (mismatch recognition protein) recognizes mismatched nucleotides in the DNA.
Mut L binds to the Mut S protein
Mut H binds to Mut S and Mut L complex
The parent strand is methylated but the new daughter strand is not methylated
The GATC sequence in the parent strand is methylated
This structure is called hemi methylated DNA meaning one strand is methylated while the other strand is not.
The mismatch proteins can recognize methylated and non methylated strands
Mut H protein is endonuclease and Mut L activates the endonuclease activity
Mut H searches for the nearest GATC sequence with methylated adenine
During the search,the DNA is looped out.
When the sequence is found,the Mut H protein cleaves at the daughter or new strand
Helicase II unwinds the cleaved strand
The unwound strand is removed and cleaved by exonuclease enzyme
DNA polymerase III replicates the DNA and adds correct nucleotides and the gap is sealed by DNA ligase
Explain DNA polymerase proof reading
Explain the exonuclease activity of polymerase I and it’s biological significance
DNA polymerase has three domains namely the palm domain (responsible for exonuclease and proofreading activity),fingers domain(polymerization activity), thumb domain(for structural integrity part and holding DNA in the proper shape )
During nucleotide addition when they encounter a wrong nucleotide the polymerase chnages the structure and brings the newly added wrong nucleotide to the palm domain or exonuclease activity domain and this domain cuts the wrongly put nucleotide and the original conformation is gotten back and they start adding the nucleotide sequences again
Biological significance:
- Allows the replacement of damaged or abnormal DNA sequences
by “Nick translation”
- Also allows the removal of RNA sequences embedded in DNA(removal of replication primers)
Explain the two problems posed by the properties of the known DNA polymerases and state the solution
Why is DNA replication semi discontinuous?
Synthesis of DNA on the lagging strand requires continuous synthesis
In bacteria, the Okazaki fragments are about 1000-2000 bp in length true or false
Two problems posed by the properties of the known DNA polymerases
1.
The directionality problem. How can DNA polymerase replicate both strands behind each replication fork, when all polymerases operate in the 5’ to 3’ direction?
Solution - semidiscontinuous DNA synthesis
2.The priming problem. Since all DNA polymerases require a primer (usually of at least 10 nucleotides in length), where do the primers come from?
Solution - primers are made of RNA
Semi discontinuous:
During DNA replication one of the two strands specifically the leading strand is replicated continuously in the 5’ to 3’ direction while the other strand lagging strand is replicated discontinuously or in pieces from the 3’ to 5’ direction
This is important because DNA polymerase (the enzyme that synthesizes a new DNA strand using a template strand) can only add nucleotides to the 3’ end of a polynucleotide strand
Overall DNa replication is semi discontinuous
So when the Helicase unwinds the DNA it exposes the strands as Leading strand template and lagging strand templates
DNA polymerase attaches to the 3’ end of the RNA primers and the polymerase adds dna nucleotides in the 5’ to 3’ direction
DNA ligase eliminates the nick or the gaps formed when the RNA primers are removed and replaced with DNA nucleotides
Primase synthesizes short RNA primers on each template strand from the 5’ to 3’ direction
Explain the replisome of E. coli
Explain the sub units of DNA polymerase III
Helicases
Unwind DNA at the replication
fork in a reaction coupled to ATP Hydrolysis
2) Single-stranded DNA
binding proteins (SSB)
Bind and stabilize the DNA in a single stranded conformation
after the melting by helicases 3) The Primosome Synthesizes RNA primers
of the lagging strand Contains Primase
4) DNA Polymerase III : The replicase
5) DNA topoisomerase II Relaxes supercoiled DNA that forms ahead of the replication fork. Decatenates
the final product
6) DNA Polymerase I Replaces RNA primers with DNA by nick translation
7) DNA Ligase Joins the Okazaki
fragments
DNA Polymerase III Core comprises of three sub units namely:
Alpha sub unit:Catalytic site for polymerization
Epsilon sub unit ;3’->5’ editing exonuclease or proofreading activity
Theta sub unit: structural role or stimulate the exonuclease activity
It has two catalytic cores
DNA Polymerase III: is a multisubunit Enzyme
- gamma Complex:
4 polypeptides
ATP-dependent conformational changes facilitates the loading of the β clamp onto DNA or it places the processivity sub unit ie. the beta clamp on the DNA - beta Subunit or processivity sub unit:
A homodimer
ATP-dependent processivity
factor = β clamp Pol.III Core is poorly processive by itself. Beta clamp is responsible for holding the catalytic core on their template strand
The clamp controls the association of core enzymes with DNA .
The Helicase creating the replication fork is connected to two DNA polymerase catalytic sub units each of which is held on to DNA by a sliding clamp. The polymerase that synthesizes the leading strand moves continuously. The core on the leading strand is processive because it’s clamp keep it on the DNA . The clamp associated with the core on the lagging strand dissociates at the end of each Okazaki fragment and read so coated with a primer in the single stranded template loop to synthesize the next fragment reassembles for the next fragment
Helicase or DNaB is responsible for interacting with the primase DnaG to imitate each Okazaki fragment
- tau sub unit Dimerization factor or dimerizing sub unit :Holds two Pol. III catalytic cores together
Polymerase III holoenzyme functions with processivity,catalytic potency and fidelity
Explain these terms with regards to polymerase III
- Processivity: means the enzyme remain bound to the template for many rounds of nucleotide addition (probably whole synthesis) Pol III core (αεθ) alone only has processivity of 10-15 residues
- Catalytic potency: not only processive, but efficient
Result of 1 & 2: fastest known polymerization reaction in vivo - Fidelity of Pol III replication
α subunit: 5’→3’ polymerase
ε subunit: 3’→5’ editing exonuclease
Why have 3’→5’ exonuclease activity? allows enzyme to proofread newly synthesized DNA.
If a wrong (unpaired) base is incorporated, polymerase activity is inhibited & 3’→5’ exonuclease excises wrong nucleotide. 3’→5’ exonuclease activated by unpaired 3’- terminal nucleotide with free 3’OH.
Sliding b clamps ensure processivity of DNA polymerase III
True or false
How do we get clamp on and off?
What is nick translation
Summary of DNA replication paradigms • Semiconservative • Bidirectional • One origin per bacterial chromosome • Semidiscontinuous • RNA primed True kr false
True
Topoisomerases relax positive supercoils ahead of the replication fork and decatenate the final products
1.Topoisomerase required to relax positive supercoils ahead of replication fork
2.Not enough room between the converging replication forks for topoisomerase
If topo II is absent at this stage,
the daughter chromosomes can’t
3.Type II topoisomerase required to decatenate the final products
Pol I excises the RNA primers by “nick translation”(Nick translation is the name given to a reaction that is used to replace cold nucleoside triphosphates in a double-stranded DNA molecule with radioactive ones (1,2). Free 3’-hydroxyl groups are created within the unlabeled DNA (nicks) by deoxyribonuclease 1 (DNAse 1). Or nick translation is tagging DNa with fluorescence labels or labeled dNTPs . The DNA to be nick translated is treated with DNAase 1 enzyme which produces small single stranded nicks or cuts in the DNA. The nicked DNa is then treated with DNA polymerase I enzyme. DNa pol I Carrie’s out two simultaneous reactions: additions of nucleotides in 5’ to 3’ direction and removal of nucleotides in 5’ to 3’ direction. Hence the sample DNA is labeled. Once the nick translation is completed the nicks are sealed with DNA ligase enzyme
The eukaryotic replisome is homologous in many respects to the bacterial replisome!!!
True or false
Pol - the eukaryotic replicase
Pol alpha /primase - contains both primase and DNA polymerase activities
PCNA - trimeric sliding clamp Replication Factor C (RFC) - the
clamp loader
Each chromosome has multiple origins. Why?
• Time for DNA replication is limited - e.g., mammalian S phase lasts 6-8 hours.
• Eukaryotic forks move at only about 1/10th the rate of bacterial forks
• Chromosomes can be in excess of 108 bp
Completion of replication in the allotted time necessitates multiple
origins.
Origin Recognition Complex (ORC) - a complex of 6 ATPases
- the functional equivalent of DnaA
MCMs - a heterohexameric helicase
Replication Protein A (RPA) = SSB
RNase H - nuclease that is specific for RNA in RNA/DNA hybrids - excises primers
What is telomerase
What’s the clinical relevance of telomerase
Telomerase: a reverse 5’ transcriptase, with a built- etc. in RNA template Humans: TTAGGG Tetrahymena: TTGGGG 10-1000 repeats.
Telomerase–aging, cancer, and disease
Most somatic cells have low or undetectable level of telomerase activity.
Telomere length is correlated with cellular aging.
Telomerase is active in some germline, epithelial, and
stem cells (haemopoeitic cells), and in >90% of cancer cell lines.
Mutations in the RNA component of human telomerase have been linked to autosomal dominant dyskeratosis congenita and some forms of aplastic anemia.
Telomerase is required for telomere maintenance.
Telomerase is responsible for the immortal phenotype of
cancer cells.
After a few generations, telomerase-mutant mice exhibit reduced fertility, signs of premature aging, and shortened
life-span.
Psychological stress leads to reduced telomerase activity and increased telomere shortening! This presumably results in premature aging!!
Telomere length
Study conducted on mothers under stress due to need to care for a chronically ill child.
• Telomere shortening in high stress subjects is the equivalent of one decade of additional aging!
What are the differences between replication in eukaryotes and prokaryotes
Prokaryotes
1.One specific initiation
point (Ori)
2. Three types of DNA polymerases, I, II and III
3. Diverse functional variety specially that of DNA Pol I
4. Not applicable
• Prok 5. DNA Pol I is main repair enzyme 6. Few replication forks 7. Theta structure observed 8. Accessory proteins few with limited function 9. Only unwinding takes place 10. No histones, few replication bubbles 11. RNA as primer 12. Primase makes primer
• Eukaryotes
- Multiple specific initiation points but different from prokaryotes
- Five types of DNA polymerases, α,ε,γ,β and δ
- Functional variety of DNA Pol is specific.
- DNA Pol. γ replicates mitochondrial DNA
• Euk
5. DNA Pol β is the main repair
enzyme
6. Many replication forks
7. Theta structure not observed
8. Many accessory proteins with diverse functions
9. Histone separation from DNA as well as unwinding takes place
10. Many replication bubbles 11. RNA/DNA as primer
12. Pol α/ primase makes primer
Summary of DNA Replication
• Identification of the initiation site of replication (OriC)
• Unwinding of parental DNA (DS -> SSDNA)
• Formation of replication fork
• Synthesis of RNA primer complementary to the DNA template by primase
• Leading strand is synthesized in the 5’-3’ direction by DNA Pol.
• Lagging strand is synthesized as Okazaki fragments
• RNA primers removed when polymerization is done
• The gaps are filled by dNTPs and the pieces are joined by DNA ligase which requires energy from ATP.
State five things that cause DNA damage
State various DNA damages that need to be repaired
What is nitrosonium ion
DNA damage
A gallery of horrors
– 1. UV damage
– 2. Environmental Chemicals
e.g. alkylating agents –
3. Normal Physiological agents H20 (Hydrolytic deamination)
depurination 02 Oxidation)
nitrites (Oxidative deamination) Alkylation
- 4. Replication errors (wrong base) – Et cetera
various DNA Damages that need to be repaired:
-Pyrimidine dimers
(UV light)
UV-induced formation of pyrimidine dimers in DNA is a major deleterious event in both eukaryotic and prokaryotic cells. Accumulation of cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidone photoproducts can lead to cell death or be at the origin of mutations.
- Alkylation of bases
Methylation of guanine N6 G-> O6meG - Hydrolysis of glycosidic bond
(Depurination) - Deamination of bases
Another common type of DNA damage that occurs under physiological conditions is the hydrolytic deamination of cytosine to form uracil (5).
•Spontaneous: Spontaneous deamination is the hydrolysis reaction of cytosine into uracil, releasing ammonia in the process. This can occur in vitro through the use of bisulfite, which deaminates cytosine, but not 5-methylcytosine.
•Chemically induced
•C->U
•5 meC -> T
•A->HX: Deamination of adenine results in the formation of hypoxanthine. Hypoxanthine, in a manner analogous to the imine tautomer of adenine, selectively base pairs with cytosine instead of thymine. This results in a post-replicative transition mutation, where the original A-T base pair transforms into a G-C base pair.
- Oxidative damages
•G -> 8 oxoguanine
•Strand Break
Nitrosonium ion
•Electron hungry
•Formed from: nitrates and nitrites (common food preservatives, but also naturally occurring in foods such as spinach)
•Also formed from nitrosamines (byproducts of rubber production)
The nitrosonium ion is a critical step in the creation of carcinogenic nitrosamines, as well as the deamination of DNA or amine-containing amino acid residues in proteins.
So processed foods are dangerous.
But so is water!
Why was uracil not selected as a natural base for DNA?
Explain oxidative damage in DNA
What is the consequence of oxidative damage in dna
How is oxidative damage reduced
as a natural base in DNA:
If Uracil were a natural
base, the DNA repair machinery would not know whether these Uracil are “normal” uracil that
come from incorporation by polymerase, or “non-natural”
uracil coming from deamination
of cytosines.
Since there is no way to discri- minate between these, the
genetic systems did not select uracil as a natural base (exceptions)
in DNA
’TGUA Due to incorporation of dUTP by polymerase
Source of oxidative agents
Oxidative DNA damage provides direct routes to mutations. While guanine usually pairs with cytosine, 8-oxo-7,8-dihydroguanine (8-oxoG), the most frequent type of oxidative base damage, may cause mispairing with adenine through a conformational change. This is one route to oxidative DNA damage induced mutations.
8-oxo-guanine generates replication block or G-C -> T:A transversions after DNA Replication
G-C
DNA Polymerases tend to incorporate A opposite to 8-oxoG because of the tendency of 8-oxoG to switch in the syn conformation; A is the only nt that can form a base pair
8xoG•C
H2O with syn8-oxoG whose geometry vaguely 2 resembles that of a Watson-Crick base pair
Oxidative damage, produced by intracellular ROS, results in DNA base modifications, single- and double-strand breaks, and the formation of apurinic/apyrimidinic lesions, many of which are toxic and/or mutagenic
• Respiratory Chain:
Accordingly, cells have evolved a balanced system to neutralize the extra ROS, namely antioxidant systems that consist of enzymatic antioxidants such as superoxide dismutase (SOD)(breaks down oxygen and hydrogen into hydrogen peroxide and oxygen) , catalase (CAT) (converts hydrogen,oxygen and hydroxide to water and oxygen)and glutathione peroxidases (GPxs), thioredoxin (Trx) as well as the non-enzymatic antioxidants which collectively reduce
No cellular Neutralization !!! -> Main source of Oxidative agent
Explain alkylation in dna damage
Consequences to dna ,examples of alkyl agents
Any nucleophilic atom on the DNA (e.g., N7, N6, N3, O2, O6, etc.) can be alkylated sometimes leading to changes in basepairing specificity. For example, alkylation of guanine O6 can lead to GC to AT transition mutations.
Alkylation damage of DNA is one of the major types of insults which cells must repair to remain viable. One way alkylation damaged ring nitrogens are repaired is via the Base Excision Repair (BER) pathway.
Alkylation of guanine or other bases results in abnormal base pairing as well as the excision of these bases, which in turn leads to strand breakage.
Alkylation damage repair involves multiple partially redundant pathways, which include direct reversal by O6-methylguanine DNA methyltransferase (MGMT), the ALKB family of demethylases, and base excision repair (BER).
Alkylating agents exert cytotoxic effects by transferring alkyl groups to DNA, thereby damaging the DNA and interfering with DNA transcription and cell division.
Some favorite alkylating agents
Ethylmethane sulfonate (EMS) - a favorite among geneticists MNNG - another tool of geneticists - a nitrosamine
Cl-
Nitrogen mustard
Alkyl groups transferred are shown in red
Mustard gas
State five ways to minimize damage to the dna
State five dna repair strategies
Explain three
Three excision repair pathways exist to repair single stranded DNA damage: Nucleotide excision repair (NER), base excision repair (BER), and DNA mismatch repair (MMR). True or false
How to minimize damage to your DNA
• Avoid chemical warfare • Avoid processed foods • Avoid the highways
• Avoid sunlight
• Avoid aerobic activities
• Avoid water
But if all these precautions fail, our cells have multiple DNA repair pathways to undo the damage!!!
Some DNA repair strategies • Direct reversal of the damage • Base excision repair • Nucleotide excision repair • Methyl directed mismatch repair • SOS repair • Double-strand Break Repair • Recombination repair
-Bypass of lesions: avoids DNA replication stalls
• bypass of 8 oxoG by DNA polymerase
-> not really a “repair”, more like a quick fix..
- Direct Reversal of Damage
• Photolyase reversion of Y dimers
• Dealkylation of 1mA and 3mC by AlkB
Direct reversal of the damage
• DNA Photolyase - uses energy from sunlight to split pyrimidine dimers
– Absent in placental mammals
– In one study (Current Biology 15, 105-15), transgenic mice carrying a bacterial photolyase gene were found to be resistant to sunlight induced skin cancer
– The solution to the thinning ozone layer?!! • Dealkylating enzymes
– O6-methyl guanine methyl transferase (MGMT) - a suicidal “enzyme” that dealkylates G O6
– AlkB - promotes dealkyation of A N1 and C N3
- Base excision repair
• Uracil-N glycosylase
• 8-oxoG glycosylase - Nucleotide excision repair
• Bacteria: UvrA, UvrB, UvrC, Helicase II
(UvrD)
• DNA pol. I, DNA ligase
• Eukaryotes : Xeroderma pigmentosum proteins,
TFIIH - Methyl directed Mismatch
repair
• Dealkylation of guanines by suicidal MGMTase
Dealkylation of guanines by Methyl Guanine Methyl Transferase: Mutations of Human Homologues of O6MGMT (methyl guanine methyl transferase) linked to cancer : another proof that maintaining DNA information is required for
tumor suppression
• The “inactivated” enzyme serves as a transcription factor to induce expression of DNA repair genes ->”biosensor” for DNA repair
• MutS, MutL, MutH
For more information regarding the mechanisms of DNA repair enzymes, http://www.scripps.edu/~cliff/class/class.html
Oxidative Demethylation
by AlkB; Oxidation of 1-methyladenine and 3-methylcytosine by AlkB requires O2, - ketoglutarate (KG) and Fe(II), and generates CO2 and succinate. Oxidized methyl groups are released as formaldehyde resulting in direct reversal of the lesions to the unmodified base residues.
Base excision repair pathway
X = damaged base (e.g.,uracil,hypoxanthine,8-oxoguanine,etc)
There are many dozen such
enzymes each one recognizing
a different X (e.g., uracil-N-
glycosylase)
In bacteria, the exonuclease and polymerase functions are both provided by Pol I through the process of nick translation
Base excision repair (BER) corrects small base lesions that do not significantly distort the DNA helix structure. It is initiated by a DNA glycosylase that recognizes and removes the damaged base, leaving an abasic site which is further processed by short-patch repair or long-patch repair. BER takes place by short-patch repair or long-patch repair that largely use different proteins downstream of the base excision. The repair process takes place in five core steps: (1) excision of the base, (2) incision, (3) end processing, and (4) repair synthesis, including gap filling and ligation.
Nucleotide excision repair: In humans, this step catalyzed by XP-A, XP-C, XP-F, XP-G TFIIH (contains XP-B and XP-D) DNA polymerase b XP = xeroderma pigmentosum
The main difference between base excision repair and nucleotide excision repair is that the base excision repair pathway corrects only the damaged bases, which are non-bulky lesions, whereas the nucleotide excision repair pathway corrects bulky DNA adducts through the removal of a short-single stranded DNA segment . Nucleotide excision repair (NER) is the main pathway used by mammals to remove bulky DNA lesions such as those formed by UV light, environmental mutagens, and some cancer chemotherapeutic adducts from DNA. nucleotide excision repair (NER), damaged bases are cut out within a string of nucleotides, and replaced with DNA as directed by the undamaged template strand. This repair system is used to remove pyrimidine dimers formed by UV radiation as well as nucleotides modified by bulky chemical adducts.
What is xeroxerma pigmentosum
Methyl-directed mismatch repair in prokaryotes
Uvr genes = genes that promote UV Resistance
Mut genes = When these genes ar mutated, bacteria show increased
rates of mutations true or false
A defect that is due to a defect in nucleotide excision repair(XP group A to 6). It’s a rare skin disorder marked by extreme sensitivity to sunlight
NER is the sole pathway for repairing thymine dimers in humans
NER is also involved in repairing oxidative damage
When NER is defective,translesion bypass mechanism is used
NER is coupled to transcription and the NER pathway in humans is the same in E. coli but involves different proteins in genetic groups
