Molecular building blocks of life II Flashcards
DNA functions
genetic code
storage in cell
meiosis
genome integrity
replication
transcription accessibility
scientists demonstrating DNA as a transforming molecule
Griffiths 1928
Avery, macleod mccarthy 1944
hershey and chase 1952
Hershey and chase experiment 1952
- mix marked phages w bacteria
- agitate in blender, separating phages outside of bacteria
- centrifuge
- measure radioactivity in pellet/ liquidq
nucleic acid composition
heterocyclic base
sugar
phosphate
purines
adenine
guanine
N9>C1
pyrimidines
cytosine
thymine
uracil
N1> C1
nucleoside
base + sugar
cytodine/ deoxycytidine
pyrmidine N1 attaches to sugar C1
adenosine/ deoxyadenosine
purine N9 attaches to sugar C1
phosphodiester bond formation
phosphate oxygen lost
hexose hydroxyl lost
water produced
polymeric structure of DNA/ RNA
linear polymer formed by 3’-5’ phosphodiester bonds
acidic/ - charge sugar phosphate backbone
written 5’ (phosphate) > 3’ (hydroxyl) direction
DNA vs RNA
DNA: 100* more stable, resistant to hydrolysis, long-term info storage
RNA: base-catalyzed hydrolysis of RNA backbone, temporary info
Chargaff’s rules
- [A] = [T] / [G]=[C]
- [A] + [T]/ [C]+[G] varies depending on species
DNA structure
RH double helix
2 anti-parallel strands w complementary base-pairing
H bonds between bases
meridian angle
60 degrees
rise per base
0.34 nm
DNA spacing
3.4 nm
alpha helical radius
1nm
alpha helical diameter
2nm
number H bonds per G-C
3
number H bonds per A-T
2
H bond energy
5 kj/ mol
C-H covalent energy
418 kj/mol
DNA structural stability
hydrophobic effects
Bp H bonding
cooperativity
-charge
hydrophobic effects
hydrophobic bases inside
charged backbone inside
Van der waals stacking forces
4 kJ/mol
major groove
22 A wide
info rich
txn factors
read sequence w/o unwinding
seq specific DNA binding
minor groove
12A wide
info poor
binding alters DNA
A-DNA
RH
2.3 A/ b
25.3 A pitch
19 degree tilt
2.6 nm diameter
B-DNA
RH
3.4A/b
35.4A pitch
1 degree bp tilt
2.4 nm diameter
Z-DNA
LH
3.8 A / base
45.6 A pitch
9 degree bp tilt
1.8nm diameter
watson and crick base-pairing consequences
bulges
tertiary interactions
matched/ mismatched bp
semi-conservative replication proof
meselson and stahl 1958
Meselson and stahl semi conservative proof
15N medium placed in 1st generation 14 N medium and replicated
each generation microfuged
dNTP precursors
(DNA)n + dNTP > (DNA)n+1 + PPi
semi-conservative replication process
- new DNA chain assembled on existing DNA template
- catalysis of phosphodiester bond formation - primer enables synthesis initiation
- 5’-3’ direction - mistake correction as mismatched nucleotides removed via 3’-5’ exonuclease activity
oriC
origin of replication
circular 4.6*10^6 genome
5 copies of DNAa binding sequence
AT-rich tandem array of 13mers
DNAa assembly stimulates unwinding of AT-rich array
DnaB helicase action
recruited by DnaA
loaded around ssDNA
ATPase-dependent translocation
strand exclusion model
single stranded binding protein
SSB
loaded onto SSDNA
wrapped around SSB tetramers
prevents secondary structure formation
pre-priming complex stages
initiation
loading
activation
initiation
DnaA assembly stimulates AT-rich array unwinding
recruits DnaB/helicase
loading
DnaC/ loading factor complexes w C-terminus of DnaB
after helicase closure, DnaC hydrolyzes ATP and dissociates
activation
DnaG synthesizes RNA oligonucleotides in DNA replication
DnaG primase
synthesizes RNA primer
recruited by DnaG
topoisomerase II
catalyzes untangling of DNA duplexes
1. cleavage of both strands
2. passage separate duplex molecule through break
3. break resealed
topoisomerase I
catalyzes relaxation of supercoiled DNA
1. cleavage of one strand
2. passage of cut end under other strand
3. reseals break
DNA polymerase III core components
alpha polymerase unit
exonuclease domain
sliding clamp
klenow fragment
sliding clamp
35A diameter hole accomodates dsDNA
keeps polyym III in contact w DNA ^ processivity
1-5Kb added before enzyme falls off
okazaki fragments length
1-2 Kb long
DNA polymerase III holoenzyme
2 DNA polymerase follow single DnaB helicase to coordinate synthesis of leading/ lagging strands
trombone model
looping of lagging strand, releases after 1000nt’s
new loop then formed (lengthened and shortened)
DNA damaging agents
DNA replicative stress
0 radicals/ ionizing radiation
polyaromatic hydrocarbons/ UV light
chemotherapeutics
DNA replicative stress
base mismatches
mismatch repair
ionizing radiation effect on DNA
ssDNA breaks
abasic sites
8 onoguanine
chemotherapeutics damage
breaks intra-strand cross-links
dsDNA break repair
homologous recombination
polyaromatic hydrocarbons damage
DNA adducts/ intrastrand crosslinks
nucleotide excision repair
dideoxy sequencing
2’,3’ dideoxy analogues spike DNA polym reactions > truncated products
new DNA strands separated and electrophoresed
4 reactions dd(A/T/G/C)TP
sequence read from gel electrophoresis
pros of dideoxy sequencing w fluorescent ddNTPs
enabled genome sequencing
cons of dideoxy sequencing w fluorescent ddNTPs
limited to small genomes
slow
expensive
capillary sequencing
Sanger method
capillary tube filled w viscous gel
automation
NGS pros
next generation sequencing
quick/ cheap/ large genomes
impacts of individual genome sequencing
bacteria/ virus (drug resistance)
crops (high yield)
humans (genome seq at birth, cancer genome project, disease susceptibility, pharmacogenomics)