Nucleic Acids, DNA Replication & Repair Flashcards
What is a tautomer?
Structural isomers that can interconvert between different forms quickly
What are nitrogenous bases?
- Aromatic, hydrophobic, Carbon based ring systems
- Purine or pyramidine
A* (Tautomer of Adenine)
H moves from amino group (imino form); binds with C
C* (Tautomer of Cytosine)
H moves from amino group (imino); binds with A
T* (Tautomer of Thymine)
H moves creating alcohol group (enol); binds with G
G* (Tautomer of Guanine)
H moves create alcohol group (enol); binds with T
Common Pyramidine bases
- Cytosine (2-oxy-4-amino pyrimidine)
- Uracil (2-oxy-4-oxy pyrimidine)
- Thymine (2-oxy-4-oxy-5-methyl pyrimidine)
Common Purine Bases
- Adenine (6-amino purine)
- Guanine (2-amino-6-oxy purine)
What is the acid dissociation constant?
o Quantitative measure of the strength of an acid in solution
o Larger pKa = smaller extent of dissociation
What is tautomerism?
Conformational change of Nitrogenous base between its 2 conformational states.
UV Absorption of Nucleotide bases
- Rings of bases made up of alternating single and double bonds.
o Such systems absorb in the UV spectrum - Each of 4 nucleotide bases has a slightly different absorption spectrum
o Spectrum of DNA is the average of them - A pure DNA solution appears transparent to the eye
o Absorption doesn’t become measurable until 320nm
o Peak at about 260nm followed by a dip between 220 and 230
o Then solution becomes opaque in the far UV range. - When DNA helix is denatured to become single stranded – absorbance is increased about 30%.
o Called HYPERCHROMIC EFFECT
o Reflects interaction between electronic dipoles in stacked bases of native helix.
What is a nucleoside?
Comounds formed when a nitrogenous base is linked to a pentose sugar
What is the structure of nucleosides?
- Base linked to sugar via glycosidic bond (N9 in purines; N1 of pyrimidines)
- Carbon of the glycosidic bond is anomeric.
- All beta formations
- Named by adding -idine to root name of pyrimidine (cytidine), and -osine to root name of purine (adenosine).
- Conformation can be syn or anti
o Rotation around the glycosidic bond is sterically hindered; syn goes left over sugar molecule; anti goes right (forms ‘S’)
o Pyrimidines usually anti and not syn as the 2’-O atoms sterically hinders the ring position above the ribose.
Nucleoside triphosphates
- More than 1 P group attached
• Hydrolysis of phosphoanhydride bonds releases energy (Like in ATP)
- Cyclic nucleotides are signal mols and regulators of cellular metabolism and reproduction.
What is a nucleotide?
Nucleoside bonded to phosphate group C5 of pentose sugar
What are 4 common ribonucleotides?
- AMP – adenosine 5’-monophsophate
- GMP – Guanosine 5’-monophosphate
- CMP – Cytidine 5’-monophosphate
- UMP – Uridine 5’-monophosphate
What are the functions of nucleotides?
- Nucleoside 5’-triphosphates are carriers of energy
o ATP is central to energy metabolism
o GTP drives protein synthesis
o CTP drives lipid synthesis
o UTP drives carbohydrate metabolism - Cyclic nucleotides are signal mols and regulators of cellular metabolism and reproduction
- Bases serve as recognition units.
DNA vs RNA
- Backbone is the same, nitrogenous bases change
- DNA is more stable than RNA
o Presence of 2’-OH makes RNA susceptible to alkaline hydrolysis
o Gives RNA a short half-life – allows cell to regulate gene expression (protein synthesis) at a pre-translational level - Presence of Uracil in RNA
Why does DNA have Thymine & not Uracil?
o Cytosine spontaneously deaminates to form uracil at a finite rate in vivo.
o i.e. loses anime group.
o C in DNA strand pairs with G in other strands, whereas U would pair with A.
o Conversion of a C to a U could potentially result in a heritable change of C:G pair to a U:A pair.
o Such a change in nucleotide sequence would constitute a mutation in the DNA
o Repair enzymes recognize these ‘mutations’ and replace these U’s with C’s.
- Thymine is methylated, so anything that doesn’t resemble that is replaced with thymine -5-methyl.
Secondary structure of ribosomal RNA
- High intrastrand sequence complementarity leads to extensive base-pairing
- Secondary structure features seem to be conserved, whereas sequence is not
o There must be common designs and functions that need to be conserved
Secondary and tertiary structure of tRNA
o Extensive H-bonding creates four double helical domains, three capped by loops, one by a stem
o Only one tRNA structure is known
o Phenylalanine tRNA is ‘L-shaped’.
o Many non-canonical base pairs found in tRNA.
What is Chargaff’s rule?
The total number of purines in a DNA molecule must = the total number of pyrimidines
Structure of Nucleic acids
- Very stable because of:
o H-bonds between complementary bases and sugar phosphate backbone and H20
o Electrostatic interactions: neg charged phosphate groups repel one another and interact with Mg2+
o Van der Waals and Hydrophobic interactions: stacking of base pairs - Helix is anti-parallel
- Helix dimensions conserved. Each turn is about 10bp (base pairs)
- Major and minor grooves:
o ‘tops’ of bases (as we draw them) line the ‘floor’ of major groove
o Major groove is large enough to accommodate an alpha helix of a protein
o Regulatory proteins (transcription factors) can recognize the pattern of bases and H bonding possibilities in the major groove.
What are the canonical base pairs?
The base Paris that occur most frequently
i.e. A:T & G:C
(A:T - 2 H Bonds
G:C - 3 H Bonds)
Secondary structure of DNA
- Can be denatured or renatured
- Heating DNA to >8- deg C causes a 30-40% increase in its UV absorbance
- The hyper chromic shift reflects the unwinding of the DNA double helix
- DNA can also be denatured using an alkali
Tertiary Structure of DNA
Supercoiling:
o In duplex DNA, 10 bp/turn of helix
o Circular DNA sometimes has more or less than 10bp/turn
- A supercoiled state
o Enzymes called topoisomerases or gyrases can introduce or remove supercoils
o Can be negatively coiling or positively coiling (clockwise or anticlockwise)
What is the structure of Eukaryotic Chromosomes?
- Human DNA total length is ±2m
- This must be packaged into a nucleus that is about 5 micrometers in diameter
- Represents a compression of >100 000x
- Made possible by wrapping the DNA around protein spools called nucleosomes and then packing these in helical filaments.
5 Features of DNA Replication
- Replication begins at specific regions known as origins of replication
- DNA replication is bidirectional
a. 2 replication forks move in opposite directions - Nucleotides are added at the 3’-end of the strand (Replication occurs 5’ – 3’)
a. Replication fork moving left to right
b. Sense strand = 5’-3’ (left to right)
c. Antisense strand = (3’-5’ from left to right) - The double helix must be unwound – by helicases
a. Use ATP and disrupt H base pairing that holds the 2 strands together
b. Unwinding will result in introduction of positive supercoils that will ultimately become too tightly supercoiled for unwinding to occur.
c. Action of gyrase (a topoisomerase) introduces negative supercoils at the expense of ATP by breaking the phosphodiester bond, introduction a negative supercoil and then sealing the breakage.
d. Single stranded DNA binding proteins bind to the single DNA strand to prevent re-annealing. - DNA replication is semi-discontinuous
a. Leading strand copies continuously
b. Lagging strand copies in segments (Okazaki fragments) (1000 to 2000 nucleotides long) which must be subsequently joined
DNA polymerase I
- Functions in repair and replaces RNA primers with DNA during replication using 5’-3’ polymerase activity
- Serves proof-reading function
DNA polymerase III
- Used in DNA replication
- Contains 2 polymerases embedded in a particle with 9 other subunits
- ‘Core enzyme’ has 3 subunits:
o Alpha subunit is polymerase
o Epsilon is 3’-5’ exonuclease
o Theta stabilises Epsilon - Beta subunit dimer forms a ring around DNA = sliding clamp
Structure of the replisome
- DNA-unwinding proteins
- Priming complex (primosome)
- DNA polymerase III holoenzyme comprising 2 replicative polymerases
Replication Initiation in E. coli
- DnaA protein binds to repeats in ori (replication fork), initiating strand separation
- DnaB (a helicase delivered by DnaC) further unwinds the DNA
- Primase then binds and construct the RNA primer
Replication elongation & termination in E. coli
- Topoisomerase II (DNA Gyrase) relieves supercoiling that remains
- Elongation involves DnaB helicase unwinding, single-stranded binding proteins to keep strands separated, and DNA polymerase replicating each strand
- Lagging strand is looped around and replication occurs 5’-3’
- DNA Pol III unclamps and reclamps periodically on the lagging strand when it encounters the primer of the Okazaki fragments
- DNA Pol I excise the RNA primer and replaces it with DNA
- Termination: The “ter” locus, rich in Gs and Ts signals the end of replication
- A Ter protein is also involved: a contrahelicase which prevents unwinding.
What are the point mutations?
- Transitions:
o Purine replaced with purine
o Pyrimidine replaced with pyrimidine - Transversions:
o One purine replaced by pyrimidine
o One pyrimidine replaced by purine
What can cause mutations?
Environmental factors or errors during synthesis:
- Replication errors
- Deletions/Insertions
- UV-induced base alterations
- Strand breaks
- Covalent cross linking
What are 2 major forms of spontaneous DNA damage?
- Deamination of:
a. Cytosine: forms Uracil which base pairs with Adenine
b. Adenine: forms hypoxanthine which base pairs with Cytosine
c. Guanine: forms xanthine which base pairs to Thymine - Depurination (loss of purine base)
a. result of cleavage of bond between purine bases and deoxyribose, leaving an apurinic (AP) site in DNA
What doe alkylating agents do?
- Attach alkyl groups to DNA bases
What are 3 repair mechanisms for damaged DNA?
1) UV damage: Photolyase
a. UV Damage causes thymine dimerization.
b. Photolyase requires light, unlinks dimer
2) DNA replication: Mismatch repair
a. Endonuclease cuts new unmethylated DNA strand
b. Exonuclease removes the new unmethylated DNA strand including mismatched base pair
3) Chemical modification: Excision repair
a. DNA glycosylase removes the damaged base by cleaving the glycosidic bond, creating an apurinic/apyrimidinic acid (AP) site
b. Apurinic/apyrimidinic endonuclease cuts the DNA strand
c. Excision exonuclease removes AP site & several nucleotides
d. DNA polymerase I and DNA ligase repair the gap
