Genetic information Flashcards
what excludes incorrect dNTP
steric collisions
at what rate does Pol II add an incorrect dNTP
1 per 100,000 bp
by how much does proof reading in the cell drop the error rate of mutations
by 100x
features of Pol III
3’-5’ exonuclease activity
can remove the last nucleotide if it was incorrect
exonuclease will cleave the nt at the phosphodiester terminal, releasing a dNMP
functions of Pol I
5’-3’ polymerase
3’-5’ exonuclease - proof reading
5’-3’ exonuclease - nick translation
function of 5’-3’ exonuclease in Pol I
can remove the nucleotide in front of it
why can’t rNTPs be directly incorporated onto growing DNA strands
extra OH in ribose causes a steric clash
structure of okazaki fragments
RNA at the 5’ end
Nick at the 3’ end
which Pol binds nicks
Pol I
what removes RNA primers
Pol I 5’-3’ exonuclease
when does Pol I detaches
after 1000bp
leaves behind a nick
what degrades RNA
RNAse H
what are psuedo-okazaki fragments
leading strand also consists of fragments that need to be joined together
what does the synthetic pathway for synthesising dTTP include
dUTP
what does Pol III do that needs to be corrected and what does it cause
incorporates a U instead of a T every 300 times (every 1200 bp)
needs to be corrected, leaving nicks
fragments every 1200bp of DNA
what addition of U is not offensive
U added to the opposite of A is not a problem
what addition of is a problem
U formed by the deamination of C
leads to a mutation
what removes every U, offensive or not and what does it produce
Uracil-N-glycosylase
baseless nucleotide
what is the function of apyrimidinic endonuclease
cleaves phosphodiester backbones of baseless nt
what enzyme removes and replaces the baseless nt and fills the nick
Pol I
what fills the DNA nicks left behind by the Pol’s and what can’t it do
DNA ligase
cant do RNA-DNA
what is an origin of replication (ori) and where is it located
circular chromosomes and plasmids
region of repetitive ds DNA rich in A-T
what binds to 9bp repeats and why
DnaA
causes the DNA to super coil at 9bp repeats
opens up the 3-13bp repeats
function of DnaC
binds to ssDNA and loads a DnaB helicase onto 3’ strand
Dna C detaches
function of DnaG primase
after 65 nt have been unwound by helicase
DnaG bind them to form a primasome
features of primase
RNA polymerase
self priming, adds 5’ RNA to 3’ DNA
around 10 nt in length
no proof reading
activity is increased in the presence of helicase - co-operativity
function of single stranded binding protein
binds to exposed ssDNA and prevents re-annealing
features of single stranded binding protein (SSB)
encoded by ssb gene
forms a tetramer
not sequence specific
leaves base exposed when bound
binds to co-operatively to ssDNA
what do the first primer and SSB trigger the arrival of
Pol III holoenzyme at the 3’ end
structure of Pol III holoenzyme
3x Pol III core
3x Tau proteins
Clamp loader: accessory proteins - binds SSB
function of the clamp loader and Pol III core
The clamp loader loads a β clamp onto the
DNA. Pol III core binds to the β clamp.
function of the clamp loader
Binds β clamp proteins
* Transfers the β clamp
onto DNA at primer 3’ end
what detaches the loader clamp loader
ATP hydrolysis
features of the β-clamp
Encoded by dnaN gene
* Forms a ring dimer
* Not sequence specific
* Binds Pol III core and imparts processivity
→ Goes from 10s of bp to >50,000
function of Pol III in replisome assembly
travels to replication fork
synthesises the leading strand and displaces the SSB
as it catches the helicase, a replisome forms
Pol III holoenzyme and primasome occupy how much space
around 50nm around the replication fork
function of DNA gyrase
binds to remove positive supercoiling
what happens as helicase unwinds the duplex
primase re-binds and synthesises a new primer
step 2 of the lagging strand synthesis
A β clamp is added to the primer by the clamp loader.
step 3 of lagging strand synthesis
As more ssDNA is created, the lagging strand starts to loop back, reversing the primer polarity
step 4 of lagging strand synthesis
A Pol III core binds once enough ssDNA has emerged for the β clamp to reach it
step 5 of lagging strand synthesis
First Okazaki fragment starts. DNA is pulled by helicase and Pol III. The lagging strand loops out, picking up SSB.
step 6 of lagging strand synthesis
Okazaki fragment lengthens.
Lagging strand loop gets longer.
step 7 of lagging strand synthesis
First Okazaki fragment finished.
Primase re-binds helicase, then adds a second primer.
step 8 of lagging strand synthesis
Pol III core and β clamp detach from DNA, releasing completed fragment.
what happens after lagging strand synthesis is completed
Primer 2 gets a β
clamp. Looping process
repeats for primer 2
how is the lagging strand edited
Pol I binds the end of the first okazaki fragment and replaces the RNA with DNA - leaves a nick
DNA ligase seals the nick
what is Ter and Tus
To prevent the forks overshooting there are 23 bp sequences called Ter, which bind the Tus protein
why can Tus only be displaced by the replication fork in one direction
the helicase would stall
when can DNA gyrase no longer bind
when the forks are within 200bp long
what relieves positive supercoiling when DNA gyrase can’t bind, which decatenates the molecules
toposisomerase IV
what does OriC contain
GATC sequnces
substrates for DNA adenosine methylase (dam)
what does hemimethylated GATC bind to
SeqA protein
why does initiation only occur once per cycle
prevents DnaA from binding to OriC
why are GATC sites methylated by dam very slowly (13 mins)
newly synthesized dsDNA remains hemimethylated and new initiation is prevented
what are the requirements for PCR
Taq enzyme
primers
template DNA
dNTP’s
what are the conditions required for denaturing annealing and extending
denaturing - 95C
annealing - 55C
extending - 72C
function of Pol 1/2/3/4/5
I - DNA repair and replication
II - DNA repair
III - principal DNA replication enzyme
IV - DNA repair
V - DNA repair
features of Pol I
one gene
109kDa
~400 copies per cell
10 nts/s - 20-100 nts at a time
too slow - >100 hours per genome
features of Pol III
22 genes
10E6kDa
~ 10 copies per cell
~1600 nts/s - >50,000 nts at a time
40 minutes per genome
difference between fast and slow stop mutants
fast - stops replication immediately
slow - allows current round of replication to finish but new one can’t start
what is a temperature sensitive mutant
allow proteins to be switched on and off by changing the temperature
how are DNA strands coiled
plectonomically
how does helicase function
uses ATP hydrolysis
3bp/ATP
hexamer ring surrounds a ssDNA
both helicases move towards the 3’ end of the strand they’re clamped to
what does unwinding at one part of the duplex cause
torsional strain elsewhere
supercoiling
what equation describes DNA topology
Lk = T + W
what does Lk, T, W mean
Lk - linking number (fixed value in circular DNA)
T - twists, number of duplex turns
W - writhe, number of duplex self crossings
what does a Lk > Lk° and Lk < Lk° indicate
Lk > Lk° - there is positive supercoiling
Lk < Lk° - there is negative supercoiling
in relaxed DNA what does σ equal
σ = 0
what type of coiled is purified cellular DNA and give the σ value
negatively supercoiled
σ = -0.06
when is eukaryotic DNA negatively supercoiled
around histones when forming a nucleosome
how does positive supercoiling affect replication
needs to be removed for replication to continue
what type of enzymes cause a change in Lk
topoisomerases
difference between type I/II topoisomerases
type I - cleaves backbone of one strand, allowing duplex rotations and loss of negative supercoils
type II - cleaves backbone of both strands, using ATP and introduces a negative supercoil
outline the mechanism of topoisomerase I of supercoil removal and how does it affect the Lk
phosphodiester bond is transferred to tyrosine residue on enzyme - breaks one DNA strand
unbroken strand passes through the gap
phosphodiester bond is transferred back to DNA - reforming the backbone on the other side
Lk - +1
outline the mechanism of topoisomerase II of positive supercoil removal
horizontal section is cut
5’-P is linked to tyrosine
vertical section passed through
backbone reformed
Lk - -2
how would you distinguish between linear and supercoiled DNA in agarose gel
supercoiled DNA travels faster than linear DNA
what is a catanene
when circular DNA molecules are replicated and the two daughter rings interlock
what cleaves a catanene
topoisomerase IV
what is the end result of semi-discontinuous replication
two non-identical dsDNA molecules
how did okazaki investigate the synthesis of DNA using radioactivity
E.coli culture infected with
T4 bacteriophage
add 3H-TTP (tritiated thymidine)
new DNA strands will be radioactive
map out sizes of radioactive ssDNA over time
what happens to the 3H-TTP
after 2s its removed
‘chased’ by normal TTP
the radioactivity moves down the tube by 60s
how do we know that okazaki fragments also contain a RNA primer
because tiny fragments are left over after using DNase on okazaki fragments
what do eukaryotic DNA polymerase not contain
5’-3’ exonuclease
where do origins of replication arise from
autonomously replicating sequence
name sequence repair mechanisms
direct repair
base excision repair (BER)
nucleotide excision repair (NER)
mismatch repair (MMR)
name molecular repair mechanisms
homologous recombination
non-homologous end repair
function of O6-methylguanine-DNA methyltransferase
transfers methyl/ethyl group from G to a Cys residue on itself
G is restored
function of DNA photolyase
absorbs blue light and breaks T-T internucleotide bonds using FADH
2 T nucleotides restored
function of DNA glycosylases
recognise abnormal bases and cleave them from the deoxyribose
creates an abasic site
what remains attached to DNA after DNA glycosylase cleaves the sugar-base bond
UDGase
what is UDGase responsible for
responsible for leading strand fragments
what happens to a baseless nt
recognised and phosphodiester backbone is cleaved by AP endonuclease
leads to nicked DNA
what happens to nicked DNA
Pol I nick translation restores T nt and DNA ligase seals nick
what is nucleotide excision repair (NER)
removal of oligonucleotide fragments from one strand
what is NER triggered by
physical changes in the duplex as a result of damage
what enzyme drives NER
UvrABC exinuclease (in E.coli)
how does NER excise a thymine dimer
UvrA and UvrB bind to the dimer
UvrA dissociates, UvrC binds to dimer
UvrC and UvrB move ~5nts away and cleave DNA
UvrC and UvrB dissociate
UvrD helicase displaces damaged DNA ~12/13nts
step 1-3 of methyl-directed mismatch repair (MMR)
. MutH binds to unmethylated GATC at OriC,
identifying the daughter strand.
. MutS binds to a distorted site on the duplex
MutL binds to MutS
steps 4-6 of MMR
MutL/MutS complex travels back to the origin and activates MutH
MutH cleaves daughter strand (nicked)
Specialized helicase and exonucleases remove nt until past the distortion
step 7 of MMR
Pol III fills in missing nt. DNA ligase seals nick
what protein do eukaryotes not have a homologue for in MMR and why
No homologues of MutH
they don’t use hemimethylation replication tags either
outline the sequence repair multi-pronged approach
MutT recognises 8-oxo-GTP and hydrolyses it
MutM recognises 8-oxo-G in DNA and removes it, via BER
MutY recognises 8-oxo-G opposite A in DNA and removes the A, via BER
outline the homologous recombination method for molecular repair of two strand breaks
Following replication, while the sister chromatids are still joined one can be used as a template to repair the other
outline non-homologous end-joining (NHEJ) to repair double strand breaks
A protein complex binds the naked ends of duplex fragments and recruits DNA ligase IV, which can ligate both strands – but it does it blindly, to any two pieces of DNA, with loss of some nt
features of Xeroderma Pigmentosum
Individuals show dry, parchment-like skin (xeroderma) and many freckles (pigmentosum)
Increased sensitivity to UV light
1000-fold increased risk of skin cancer
Due to inherited defects in one of eight distinct genes responsible for components of the NER complex
features of Hereditary non-polyposis colon cancer (HNPCC)
Individuals exhibit a predisposition to colon cancer (2-3% of all colon cancer cases)
Due to defects in the human equivalents of the MutS/L MMR system (MSH2 and MLH1)
Leads to the accumulation of mutations throughout the genome
what is a down mutation
to decrease promoter efficiency usually decrease conformance to the consensus sequence
structure of holoenzyme
2 α subunits
β, β’ (prime)
σ sigma (70 kD)
what do the 2 α and 2 β form and what is their function in a holoenzyme
core enzyme
The core enzyme has a general affinity for DNA- this is known as loose binding
+vely charged (Mg2+ and Zn2+ bound ions which has affinity for the –vely charged DNA
function of σ subunit
unit ensures RNA polymerase only binds at promoter sequences
1000 X binding strength
