BCEM 393 - Nucleic Acids, DNA Replication & DNA Repair

nucleic acid

consists of bases linked to a sugar-phosphate backbone, form of linear information

each monomeric unit contains a sugar, base, and phosphate

sugar

furanose ribose or deoxyribose

3’OH and 5’OH are involved in formation of the nucleic acid backbone

backbone

sugars are linked by phosphodiester bridges between the 3’OH of one sugar and the 5’OH of an adjacent sugar

3’-to-5’ phosphodiester linkage

directionality 5’ to 3’

why is RNA less stable than DNA

2’OH in RNA can hydrolyze the phosphodiester backbone (acts as Nu-)

RNA degrades over time

purines

adenine, guanine

double ring structures

N9 forms a glycosidic bond with the sugar’s C1

pyrmidines

cytosine, uracil, thymine

single ring structures

N1 forms a glycosidic bond with the sugar’s C1

nucleotide

consists of a base, a pentose sugar, one or more phosphates (nucleoside joined to phosphoryl group by ester linkage)

eg. adenylate, adenosine 5’-triphosphate

nucleoside

contains only the base and pentose sugar

eg. adenosine

double helix

two DNA chains of opposite directionality intertwine to form a right-handed double helix

sugar-phosphate backbones are on the outside, bases are on the inside

bases are nearly perpendicular to the axis of the helix with adjacent bases separated by 3.4A, ~10.4 bases per turn

helix is ~20A wide

3 H bonds

guanine and cytosine

2 H bonds

adenine and thymine

reverse watson crick

rotation of a base, sugars end up on opposite sides, often form in RNA or at the end of DNA

wobble base pairs

tautomerization - movement of double bond

leads to AC, GT & GU pairing

Hoogsteen

rotation around C1-N glycosidic bond - minor and major groove twists kinda

because DNA is restricted in a helix

hydrophobic effect

hydrophobic interactions drive bases to inside of the helix, more polar residues (phosphodiester linkages) outside

base stacking

stacked bases attract each other through van der Waals

major and minor grooves

proteins recognize groove patterns of H-bond donors/acceptors

different between AT and GC

exist because glycosidic bonds of each base in a pari are not diametrically opposite each other

supercoiled DNA

would migrate faster down because it’s more compact

RNA structures

can be complex 3D structures

allows RNA to act as catalysts (ribozymes, spliceosomes, hairpin)

DNA polymerase

catalyzes addition of deoxyribonucleotides to a DNA strand

binding a dNTP induces a conformational change in DNA polymerase (induced fit)

DNA pol I (e.coli)

primer removal (5’-3’ exonuclease activity)

DNA repair (3’-5’ exonuclease activity)

DNA pol III (e.coli)

replicative polymerases, 3’-5’ exonuclease domain

Mg²⁺

metal ion cofactor that stabilizes negative charges on phosphates

strand-elongation reaction

catalyzed by DNA pol III, releases pyrophosphate

pyrophosphate catalyzed to inorganic phosphate - drives reaction (-DG)

requires dNTPs and Mg²⁺

new DNA strand is assembled on a pre-existing strand

requires a primer to begin synthesis

elongation is in 5’ to 3’ direction

can correct mistakes using 3’ to 5’ exonuclease activity (proofreading)

polymerase proofreading

3’ to 5’ exonuclease domain removes incorrect nucleotides from the 3’ end of the growing strand by hydrolysis

mismatch results in a stall

pause gives additional time for the incorrect region to flop into the exonuclease active site where it is removed

origin of replication (oriC)

a region of DNA with special features and is the start site of DNA replication

DnaA

binds to oriC to initiate the pre-priming complex

single strand binding protein (SSB)

prevents two strands from reannealing

DnaB

helicase that separates duplex DNA

hexamer - hydrolysis of ATP causes ratcheting of the subunits of the hexamer, pulling ssDNA through the center

primase

RNA polymerase that makes a short primer

DNA polymerase III

simultaneously synthesizes the leading and lagging strands at the replication fork

• sliding clamp (B₂) improves processivity

• 1000 nt/sec

leading strand

made continuously by pol III in 5’ to 3’ direction

lagging strand

is looped out, starting from an RNA primer, pol III adds ~1000 nt in 5’ to 3’

releases sliding clamp, new loop formed, adds sliding clamp again

primase adds RNA primer, pol III makes a new Okazaki fragment

pol I fills gaps between fragments and removes RNA primer with 5’ to 3’ exonuclease activity

ligase seals fragments

ligase

joins the 3’OH and the 5’ phosphate group of two fragments using ATP hydrolysis

mechanism of ligase

• catalytic lysine attacks the alpha phosphate of ATP (innermost), generating lysyl-AMP adduct and releasing pyrophosphate

• adenlyated ligase transfers AMP from lysine to the 5’ phosphate at the nicked backbone

• 3’OH at the nick site attacks adenylated 5’ phosphate, making a phosphodiester bond and releasing AMP

mutagens

chemical agents that alter DNA bases through oxidation, deamination, alkylation, UV radiation, a-radiation, x-ray exposure

guanine oxidation

OH radical reacts with guanine to form 8-Oxoguanine

8-Oxoguanine base pairs with adenine

following replication of an 8-oxoguanine:adenine mismatch, one strand will have an AT base pair instead of a GC

deamination

removal of an amino group through reaction with H₂O, occurs about 500x per cell per day

cytosine deamination

cytosine deaminates to uracil

adenine deamination

deamination of adenosine (adenine) results in hypoxanthine

hypoxanthine base pairs to cytosine

following replication, AT —> GC

5-methylcytosine deamination

cytosine is methylated at C5 to regulate gene transcription

deamination of 5-methylcytosine results in thymine

guanine alklyation

alkylation is addition of hydrocarbon to the base

aflatoxin B₁ epoxide alkylates N7 of guanine

bulky group added, stalls the polymerase during replication

GC —> AT transversion

thymine dimer

UV light covalently links adjacent pyrimidines along the DNA strand

results in a pyrimidine dimer that creates a bulge in the DNA double helix

DNA repair

damage and repair is occurring constantly, repair often restores the genetic information

otherwise, cell may use approximate repairs and/or undergo apoptosis

repair - recognize damage —> remove damage —> repair damage

mismatch repair

prokaryotes —> eukaryotes (well conserved)

DNA pol III associates with proteins involved

mismatch recognized by MutS, MutL binds and recruits MutH (endonuclease)

Exonuclease I excises incorrect region

DNA pol III fills gap, DNA ligase seals the backbone

direct repair

studied in E. coli, conserved in fungi, plants, some animals, but does not occur in mammals

repair without removing fragments of DNA

photochemical cleavage of pyrimidine dimers by DNA photolyase

uses energy of visible light (blue light absorbed by cofactor MTHF) to break cyclobutane ring

nucleotide excision repair

conserved from E. coli to higher eukaryotes, present in mammals

recognizes distortions in helix

UvrABC excinuclease cuts DNA at two sites

DNA pol I fills gap, DNA ligase repairs the phosphodiester backbone

pathway important in humans because we don’t have photolyase

base excision repair

for non-helix-distorting damage

defective base flipped into DNA glycosylase active site, glycosidic bond cleavage

AP endonuclease nicks phosphodiester backbone

deoxyribose phosphodiesterase removes deoxyribose phosphate unit

DNA polymerase I inserts correct nucleotide

DNA ligase seals strand

why does DNA have T and not U

cytosine deaminates to uracil A LOT so cell needs to detect and remove uracil (base excision repair)

methyl group on thymine helps the cell to find and detect the product of cytosine deamination and distinguish it from thymine