Chapter 28 Flashcards
Most tantalizing comment by Watson and Crick about DNA structure
immediately suggested a method for replication
error in one in every ? base pairs
billion
A-dna
shorter, wider, right handed. present in dehydrated samples. 1 bp repeating unit. 11bp / turn.
when the out of plane carbon is on the same side as the 5-OH in a sugar
endo
n-glycosidic torsion angles
always anti, except in Z-DNA
z-dna
left handed. 2 bp repeating units. 12 bp/turn. 0 degree propeller twist.
Linking number, twisting number. writhing number.
linking number is number of right turns. twisting number is number of helices. Writhing number is measure of coiling. Twisting + Writhing = Linking.
More twisting or linking number results in?
positive supercoiling. makes more compact.
topological isomers
molecules with same linking number but different twist and writhe.
puts pressure on DNA for unwinding
negative supercoiling
How do plasmids replicate and separate strands
introduce negative supercoiling by topoisomerases
type one topoisomerases
break one strand of DNA and act to relax supercoiled DNA. increases linking # by 1
type two topoisomerases
break both strands of dna and use atp to introduce supercoiling. decrease linking # by 2.
Topo 1 mechanism
1) DNA binds topo 2) OH of tyr attacks phosphoryl group to from phosphodiester linkage between enzyme and dna 3) 5’ end of DNA is cleaved. 4) Topo controls unwinding, driven by energy of supercoils 5) 5 OH’ of free DNA attacks phosphotyrosine to reseal backbone
Topo 2 mechanism
dimer binds two DNA dupexes. One to be cut is called G and the one that passes through is T. ATP binding brings domains together to trap T and cleave G. T is released and ATP hydrolysis preps for new T.
human topo 1 is inhibited by ?
camptothecin (antitumor drug)
bacterial topo 2 is inhibited by ?
Nalidixic acid and ciprofloxacin
synthesis always occurs in ? direction
5 to 3
? classes of DNA polymerases that either replicate or repair
7
Polymerase 1
erases primer and fills in gaps on lagging strand
polymerase 2
DNA repair
polymerase 3
primary enzyme of synthesis
Klenow fragment of E. coli DNA polymerase 1
first DNA polymerase structure determined. contains finger, thumb, and palm domains; Proofreading domain; 3 to 5 exonuclease.
polymerization reactions use ? to catalyze ?
two mg++ to catalyze activation of 3’ OH to attack the alpha-phosphoryl of the dNTP. One mg++ binds the primer and the other binds the dNTP.
DNA polymerase fidelity starts with ?
recognition of the shape of a correct base pair. Not just based on ability to H-bond
All watson-crick base pairs have ? located in minor groove
h-bond acceptors
Selection of correct dNTP gives error rate of about ?
1 in 10^4
function of 3 to 5 exonuclease ?
to proofreed newly synthesized strand and hydrolyze errors.
Error in 3 to 5 exonuclease increases DNA polymerase fidelity to ?
1 in 10^7
accessory proteins required for DNA replication
primase (produces primer), ligase (joins DNA pieces), helicase (unwind duplex DNA), single stranded dna binding proteins (prevent unwound dna from folding on itself)
DNA ligase mechanism
NAD+ donates E-AMP. E-AMP reacts with pyrophosphate. Transfer of AMP to 5’ P to activate. 3’ OH attack releases AMP.
PcrA is an example of a ?
helicase
part of helicase involved in DNA binding
P-loop
PcrA moves in ? to ? direction. Opposite of other helicases
3 to 5
rate of replication by polymerases
about 2000 bp per second
processive
ability of enzyme to catalyze consecutive reactions without releasing substrate
DNA sliding clamp
encloses dna to increase processivity
complete holoenzyme DNA poymerase 3 contain ? polymerase cores
two
replisome mechanism
lagging strand loops out as polymerase synthesizes about 1000 bp, then lagging polymerase unclamps from DNA and reloads
dna gyrase function
release excess supercoiling
completion of lagging strand requires
1) dna polymerase 1’s 5 to 3 exonuclease activity to remove and replace primers. 2) Dna ligase to seal okazaki fragments
origin of replication occurs in these regions
dense A and T
E. Coli origin of replication
OriC
mechanism of replication initiation at OriC
DnaA binds and recruits helicase DnaB to begin unwinding. Primase (DnaG) is then able to synthesize RNA primer. DNa pol 3 assembles on complex. DnaA is disassembled to prevent additional rounds of replication.
DnaA structure and function
P-loop NTPase, hexamer. Broken down by ATP hydrolysis.
protein kinase that activates replication origins
Cdk2
eucaryotic replication initiation by ?
DNa pol alpha
eucaryotic replication polymerase
dna pol delta (primary) and epsilon
telomere
a region of repetitive nucleotide sequences at each end of a chromatid, which protects the end of the chromosome from deterioration or from fusion with neighbouring chromosomes
DNA repair polymerase in humans
beta
replication leaves a ?’ sticky end that functions to ?
3’ sticky end that functions to form a loop structure that protects from degradation
repeating 3’ sticky end in humans
AGGGTT
telomerase
makes telomeres. a reverse transcriptase that extends 3’ ends by providing an RNA template strand. potent target for anti cancer therapy.
huntingtons disease
trinucleotide repeat disorder. more than 35 “CAG” repeats
G paired with A undergoes t? to T
transversion
deamination of to hypoxanthine would lead to ?
transition of A to G
5-BU
a uselful mutagen that binds guanine
aflatoxin reacts with ? to form reactive epoxide that reacts with N-7 of A or G
cyt P450
UV induces?
cross links on same strand of DNA
? cross link both strands by reaction with 2 bases
Psoralens
Photolyase
directly repairs thymine dimers. contains FADH2 and a chromophore
mismatch repair mechanism
MutS recognizes mismathces and uses MutL to transduce signal to MutH to “nick” the new strand. exonucleases create a gap that is filled by DNA pol
Base excision repair mechanism
1) dna glycosylase hydrolyzes glycosidic linkage between the base and sugar leaving an AP hole 2) AP endonuclease nicks DNA 3) phosphodiesterase removes sugar phosphate 4) DNA pol 1 inserts base 5) DNa ligase seals nick
deamination of cytosine produces ? that would cause ? if replicated
uracil that would cause transversion if replicated
uracil DNA glycosidase recognizes ? and removes ?
G:U pairs and removes U
Nucleotide excision repair directly removes
a segment of DNa
DNA repair enzymes are ? suppressor genes
tumor
in xeroderma pigmentosum, mutations in homologs of the UvrABC proteins cause >
affected individuals to be extremely sensitive to UV light
why is cancer treated with chemical agents that damage DNa when cancer is already the result of damaged DNa?
they are more readily killed by these chemicals as they lack some DNA repair mechanisms
ames test
use bacteria to see if something damages DNA
Recombination
allows genetic information to be shared or exchanged between DNA molecules
steps of holiday recombination model
1) nick 2) invasion 3) sealed by ligase 4) branch migration
recombination in E. Coli
RecBCD complex initiates nicking. RecA generates holliday junction. RuvABC resolves junction
RecA
initiates strand inversion for recombination. helix structure with DNA binding groove
Cre recombinase (Important)
Used to remove a gene from a chromosome. drives recombionation at LoxP sites. Catalyzes recombination (does not require atp). homologous to Topoisomerase 1: Uses P-Tyr covalent intermediates to exchange strands. isolated from bacteriophage
Cre recombinase mechanism
1) cleavage of two different DNA duplexes 2) new bond formations between two different strands to form holliday junction 3) isomerization 4) cleavage 5) bond formation
