DNA Structure & Replication Flashcards
What is DNA?
DNA stands for deoxyribonucleic acid. It is the genetic material and a polymer of nucleotides arranged in an antiparallel helical structure.
The components of DNA are one of four nitrogenous bases; a 5-carbon neutral sugar and a phosphate.
What are the three main experiments regarding DNA?
1928, Fred Griffith - the principle of transformation.
1943, Oswald Avery - DNA is the transforming principle.
1952, Hershey & Chase - DNA is the hereditary material.
What is Streptococcus pneumoniae?
Also known as ‘pneumococcus’, it is a pathogenic bacterium. Studied by Fred Griffith in 1928.
Describe the two forms of pneumococcus.
The rough form is non-pathogenic as it has no capsule.
The smooth form is pathogenic as it has a polysaccharide capsule which protects the bacterium from an animal’s immediate immune response. This can be killed by heat to become non-pathogenic.
Describe Fred Griffith’s experiment.
Injected mice with a mixture of dead smooth and live rough pneumococci. Resulted in mice dying. Live smooth bacteria could be recovered from these dead mice. This showed a transformation of live rough cells by the dead smooth cells.
What were Avery, MacLeod & McCarty investigating?
They were investigating what caused the transformation in DNA; that DNA is the transforming principle.
Describe Avery, MacLeod & McCarty’s experiment.
They took the crude extract from dead, smooth pneumococci and mixed it with a rough strain in vitro to transform it.
Proteases were used to form a protein-free extract which still transforms the live bacteria.
Treatment by DNase to destroy DNA resulted in no transformation.
What is a phage? Give an example.
A phage is a bacterial virus, a combination of DNA and protein.
The main components of a phage include DNA, a protein coat, a sheath, and a core.
An example is a T2 phage which attaches to and infects E. coli.
The phage replicates in the infected E. coli until E. coli lyses and progeny phage is released.
Describe Hershey & Chase’s experiment.
Radiolabelled phages were prepared. DNA was labeled with 32-P and protein was labeled with 35-S. DNA contains phosphorus but not sulfur and protein contains sulfur but not phosphorous. This was used to infect E. coli.
Injected material had to be separated from the rest of the phage that was bound to the outside of the bacterium.
Interrupted infection by shearing. The culture was centrifuged to form a supernatant containing ‘empty’ phages and a pellet containing bacteria.
35-S was present in the supernatant and 32-P was present in the pellet.
Therefore, the material that entered the bacteria was the DNA and not the protein.
Where is chromosomal DNA present?
It is present in eukaryotic nuclei and prokaryotes (e.g. bacteria).
Where is extra-chromosomal DNA present?
It is present in mitochondria and chloroplasts.
What contains ds DNA?
All cell types: eukaryotic nuclei; prokaryotes (bacteria); mitochondria; chloroplasts; plasmids, and viruses.
What doesn’t contain dsDNA?
Some viruses contain ssDNA (e.g. phage φX174).
HIV contains ssRNA.
Reovirus contains dsRNA.
What are the typical colours that represent each atom type?
Carbon is yellow or grey.
Nitrogen is blue.
Oxygen is red.
Phosphorous is orange.
What is a phosphate?
A chemical group consisting of a phosphorous bound to 4 oxygens (3 single bonds and 1 double bond).
At pH7, protons are in solution leaving a negative charge at each single bonded oxygen.
Why is DNA an acid?
The acidic protons (H+) from the phosphate group become labile in pH7 and go into solution.
Why is the sugar a ‘deoxy’-ribose sugar?
Usually there is a hydroxyl group present at each carbon, however, at carbon-2 there are only 2 hydrogens present.
Deoxy on the carbon-2 results in the sugar being a 2-deoxyribose sugar.
Why is RNA more reactive than DNA?
RNA has a ribose sugar in contrast to DNA which has a deoxyribose sugar. The hydroxyl makes RNA more reactive which is why RNA doesn’t stay stable for long.
What is a purine and a pyrimidine and what is the difference?
They are the two types of bases. A pyrmidine is a single 6 member ring with 2 nitrogens. A purine consists of 2 rings (6 member ring coupled with 5 member ring) with 4 nitrogens.
Are largely unsaturated and have delocalised electrons making them depolarised molecules.
What bases are purines?
Adenine and guanine.
What bases are pyrmidines?
Cytosine, thymine and uracil.
What base is specific to RNA?
Uracil.
What base is specific to DNA?
Thymine.
What is a (deoxy) nucleoside?
Base + (deoxy) ribose sugar.
What is the bond between a base and a ribose sugar?
β-glycosidic bond between the nitrogen from the base and the carbon-1 from the ribose.
What is the name of the nucleoside for all the bases? (i.e. A, G, C, T, U)
A - adenosine G - guanosine C - cytidine T - thymidine U - uridine
How is the phosphate group bound in a nucleotide?
Linked on the 5-carbon position.
What is a (deoxy) nucleotide?
Base + (deoxy) ribose sugar + phosphate group
What does dA stand for?
deoxyadenosine (base + sugar), a nucleoside
What does dAMP stand for?
deoxyadenosine 5’-monophosphate (base + sugar + phosphate), a nucleotide
How are nucleotides joined together?
The phosphate on the 5’-carbon links to the 3’-carbon between two adjacent nucleotides.
This makes a 3’-5’ phosphodiester link (not a bond).
How are nucleic acids polymers? What is an important feature of nucleic acids?
They consist of many polynucleotides. They have polarity.
What is an oligonucleotide?
A short stretch of of nucleotides.
What is polarity?
There is a directionality to the strand. One end differs to the other.
How are polynucleotides polar?
One end has a 3’-OH group and a 5’-phosphate group.
What is the ‘Sequence Hypothesis’?
Sequences of bases in DNA is the genetic information and it determines the sequence of amino acids in proteins.
What are the forces that determine macromolecular structure?
Covalent bonds, non-covalent bonds and the hydrophobic effect.
What are the non-covalent bonds involved in determining macromolecular structure?
Electrostatic interactions, hydrogen bonds and van der Waals interactions.
Describe covalent bonds.
Very strong; single & double bonds; short; C–C = 1.54Å; shared electrons; 85-175 kcal / mol
Describe electrostatic interactions.
Weak to strong; depend on distance; depend on dielectric constant (ionic strength of solution it takes place in); 'salt bridge'; commonly weak bonds; 1-50 kcal / mol
Describe hydrogen bonds.
Weak; longer than covalent bonds; 2Å; directional; donor-----acceptor; 1-5 kcal / mol
Describe van der Waals interactions.
Also known as London dispersion forces;
weak;
attraction between induced partial charges;
0.5-1 kcal / mol
Describe the hydrophobic effect.
Disorder is more ‘favourable’ than order (entropy);
normally, water is very disordered;
water becomes ordered on ‘hydrophobic’ surfaces (this is unfavourable);
therefore, it is favourable to bury hydrophobic surfaces so they don’t make water ordered
What are the main investigations essential to determining the structure of DNA?
Pauling & Corey’s unsatisfactory model of 3 strands and phosphates on the inside;
E. Chargaff’s relative base composition that the amount of purines = pyrimidines so must come in pairs;
R. Franklin & M. Wilkin’s x-ray diffraction of a DNA fibre.
Describe the B form of DNA.
Right handed double helix; hydrophobic bases on the inside; hydrophilic backbone outside; antiparallel helix; polymer nucleotides joined by 3'-5' phosphodiester links; complementary base pairs form hydrogen bonds between strands; A * * T, G * * * C; base pairs are perpendicular to axis
How many base pairs are there per turn (360°)?
10
How long is each turn in DNA (360°)?
34Å
How long is the diameter of DNA?
20Å
What are the two types of opening in DNA?
Major groove and minor groove
What is the importance of the major groove?
Proteins bind in the major groove and can read the base sequence.
How is DNA package in a eukaryotic nucleus?
DNA wraps twice around each histone to form chromatin which is packed into nucleosomes and after, fibres, and then further condensed into a chromosome.
Describe the properties of circular DNA.
Big circles, e.g. bacterial genomes;
Smaller circles, e.g. mitochondrial DNA, chloroplast DNA, some plasmids, and some phages and viruses
How are DNA circles packaged?
They are supercoiled to be less viscous and more compact
What is the name of DNA circles that differ in supercoiling?
Topoisomers
What is the name of enzymes that interconvert topoisomers?
DNA topoisomerases, e.g. DNA gyrase
What is hyperchromism?
When single stranded DNA absorbs more light.
Describe the basic process of hybridisation.
First strands are denatured/melted which causes them to separate;
if they are cooled quickly the strands stay apart;
if they are cooled slowly the strands have a chance to re-join which is known as renaturation or annealing.
What is the Central Dogma?
DNA makes RNA makes Protein
What are proteins?
Polymers of amino acids;
complex 3-dimensional fold;
structural and functional
What are enzymes?
Proteins that catalyse biochemical reactions
What are the enzymes that manipulate DNA?
Nucleases; ligases; polymerases; kinases & phosphatases; methylases & demethylases; recombinases / topoisomerases
What are nucleases?
Enzymes that cleave DNA
What are the two types of nucleases?
Exonucleases which cut small bits of the ends of DNA;
endonucleases which cut internally, some cut randomly and some are sequence specific
What are blunt ends?
Formed when endonucleases cut the sugar-phosphate backbone on both strands of the DNA in the same position.
What are sticky ends?
Formed when endonucleases cut the sugar-phosphate backbone on both strands of the DNA in different positions to form a staggered cut / overhangs
What are restriction endonucleases?
They recognise inverted repeats called ‘palindromes’ because they bind to DNA as dimers;
they have a protective role in microorganisms but not recognising foreign DNA resulting in foreign DNA being cut up and eliminated
What is recombinant DNA?
When DNA fragments of different origins are ligated together via sticky ends, this is the basis of genecloning.
What is a thermostable DNA polymerase?
An enzyme the ligates DNA strands together but does not denature at high temperatures; vital in the Polymerase Chain Reaction (PCR)
What are the requirements for PCR?
Template DNA;
primer;
free nucleotides;
Taq polymerase (a thermostable DNA polymerase)
What is the process of PCR?
- Form PCR mixture of template/target DNA, Taq polymerase, free nucleotides and DNA primers;
- Heat to 95°, DNA denatures as hydrogen bonds are broken causing DNA strands to separate;
- Cool to 60°, primers form hydrogen bonds and anneal to complementary sequences in target DNA;
- Heat to 72°, optimum temperature for Taq polymerase to function and so begins polymerisation in which nucleotides are added to 3’ of primers.
End of Cycle 1 forms two double stranded copies of target DNA;
lasts several hours;
typically has 20-35 repeated cycles
What are primers?
Typically ~20 nucleotides long;
complementary in sequence to the ends of target DNA
What is the process of gene cloning?
- Isolate target DNA that codes for specific gene from cell;
- Use PCR to produce many copies of target DNA;
- Produce restriction fragments with sticky ends via digestion by restriction endonucleases;
- Isolate linearised vector from E. coli via digestion by same endonuclease used to form restriction fragment to produce identical sticky ends;
- Ligate fragments and vectors together to form recombinant plasmid;
- Insert recombinant plasmid into bacterial cells via transformation;
- Through a selection and/or screening process, identify bacteria containing target gene
How is DNA replicated?
Semi-conservative replication;
one strand originates from original parent and the other is newly synthesised
What research supports the theory that DNA replicates via a semi-conservative process?
Meselson and Stahl, in 1958, used density labelling of DNA together with equilibrium centrifugation;
- Grow E. coli in heavy medium (15NH4Cl)
- At time 0, shift to light medium (14NH4Cl)
- Prepare DNA after 0, 1, 2 generations,
- Centrifuge in CsCl
Results
One generation after density shift (after one round of DNA replication), two daughter DNA strands with one heavy strand and one light strand of DNA forming a single band of DNA with intermediate density.
After two generations, two bands of DNA with one light and one intermediate density.
Who discovered the first DNA polymerase and when was it discovered?
Arthur Kornberg (1958) extracted an enzyme from E. coli dubbed DNA pol I
What does DNA polymerase do?
(DNA)n + dNTP (DNA)n+1 + PPi
Adds a deoxynucleosidetriphosphate to the DNA to become one nucleotide longer and subsequently loses a pyrophosphate
What happens to pyrophosphate when it is produced from DNA polymerization?
The pyrophosphate is rapidly hydrolyzed to form inorganic phosphate by an enzyme called pyrophosphatase.
PPi —> 2Pi
This allows the polymerase reaction to occur in one direction.
What does DNA polymerization require?
dNTPs (deoxynucleosidetriphosphates) + Mg2+
template strand
primer with free 3’-OH
Describe DNA pol I.
It is processive (n =~20)
Adds ~10 nucleotides per second
Synthesis proceeds 5’ —> 3’
Slow enzyme
3’ —> 5’ exonuclease activity - it digests in other direction to cut out mismatched nucleotides by switching active sites (proofreading of the enzyme)
5’ —> 3’ exo/endonuclease activity - Finds single-stranded break (nicks) and removes several nucleotides before filling it in with polymerase activity.
Describe the polA1 mutant.
Only ~1% of polymerase activity; both nuclease activities normal; DNA replication normal; UV sensitive; DNA pol II and DNA pol III first found in polA1 mutant discovered by Thomas Kornberg in 1971
What do the enzymes DNA pol II and DNA pol II require?
dNTPs + Mg2+
Template DNA
primer with free 3’-OH
Describe the properties of DNA pol II and DNA pol III
5’ —> 3’ polymerase activity
3’ —> 5’ exonuclease activity
But does not have 5’ —> 3’ nuclease activity
DNA pol II has moderate processivity (~1500) and a low rate of activity
DNA pol II is highly processive >500x10^3 and has a high rate of activity >10^3 per second
Describe the structure of DNA pol I.
Mr 103,000
single polypeptide
contains Zn2+ (important for catalysis)
3 distinct functional domains
Describe the structure of DNA pol III.
Mr 900,000 At least 10 subunits Holoenzyme α β χ γ δ δ' ε τ θ ψ Core enzyme α + ε + θ 2 β subunits form a ring which clamps around DNA
How does E. coli replicate its DNA?
Starts bi-directional replication at a unique site called the origin.
Pair of forks converge on termination site whilst unwinding the DNA so they don’t tangle together.
What occurs at the replication fork?
Unwinding of DNA
Strand separation
Replication of both strands (each acts as a template)
How does replication occur at the replication fork?
DNA polymerase can easily progress in the 5’ —> 3’ direction to form the leading strand or the continuous strand.
In the 5’ —> 3’ direction, a lagging strand or the discontinuous strand is formed via Okazaki fragments via the detaching and reattaching of DNA polymerase. DNA ligase seals gaps on the lagging strand after RNA primers have been removed by DNA pol I 5’ —> 3’ nuclease activity which also uses 5’ —> 3’ polymerase activity to fill gaps.
What is the function of primase or the primozone?
The enzyme creates a short primer on the lagging strand. However, primase does not have a 3’ —> 5’ exonuclease activity so does not correct errors, so may introduce errors in the lagging strand. Instead, primase makes an RNA primer which can be removed and correct at a later stage.
What is an Okazaki fragment?
Hybrid of RNA primer and DNA that forms on the lagging strand of the replication fork.
What are the enzymes involved in solving the topology problem?
Helicase: ATP-dependant DNA unwinding
SSB protein: binds to ssDNA (drawn as 4 dots as it is a tetramer)
DNA topoisomerases - DNA gyrase (DNA topoisomerase II): control superhelical density.
How does DNA gyrase differ between bacteria and mammalian enzymes?
DNA gyrase in bacteria is inhibited by antibiotics.
novobiocin (an aminocoumarin)
ciprofloxacin (fluoroquinolone)
selective toxicity
How many of each type of DNA polymerases are in each cell?
pol I 400
pol II 100
pol III 10
What are DNA lesions?
DNA damage
What can be a cause for DNA damage?
Cellular metabolism; chemicals; UV light; ionizing radiation; replication errors
What can repair DNA damage?
Cell cycle checkpoint; gene expression; DNA repair; cell death
How does UV light damage DNA?
Forms pyrimidine dimers aka thymine dimers, adjacent thymines under UV light can form covalent bonds to each other.
This distorts the DNA backbone.
What removes pyrimidine dimers?
uvrABC excinuclease cuts on either side of affected backbone area to form single stranded piece of DNA.
DNA pol I binds to these breaks and extends 3’-OH whilst removing mismatched bases and RNA.
DNA ligase seals the gaps.
Why is DNA polymerase I important in the repair of pyrimidine dimers?
5’ —> 3’ nuclease activity removers primers and repairs lesions.
5’ —> 3’ polymerase activity fills gaps.
3’ —> 5’ exonuclease activity corrects errors.
