CBG Lecture 3: DNA Replication Flashcards
what must be overcome wrt DNA structure in order for DNA replication to proceed
high fidelity copy: as digital info
double helix needds to be melted
need to synthesis 5’-3’ and 3’-5’ simultaneously
very long molecule=avoid tangling
what does ease of melting DNA depend on
AT:CG ratio as C:G base pair 3 H bonds harder to melt
where is DNA replication initiated
at origins or replication
how is DNA replication initiated
by proteins melting AT rich origins of replication
at around 90degrees using helicase
where does replication occur
at repication forks, which contain 2 molecules of DNAP and a large number of other essenial proteins
what direction is DNA synthesized in
5’ to 3’ so the leading strand is synthesized continuously, the lagging strand is synthesized in Okazaki fragments
what are the main things replication should achieve
digital info (high fidelity copy) long molecule (avoid tangling) doouble helix (must be melted, must synthesize 5'-3' and 3'-5' simultaneously
how many origins of replication are there on a prokaryotic chrom
one
how many origins of replication are there on eukaryotic chrom - why
have more than one ori per chromosome, as they have a larger genome
how to tell from microscope where DNA replication is happening
you can see a replication bubble, between two replication forks
what do initiator proteins allow
DNA to melt a
how is DNA replicated
semiconservatively by DNAP
what experiment showed DNA semiconservative replication
Meselson-Stahl
used density gradient centrifugation
15N grown E.coli transferred to 14N medium.
after 1 gen in 14N, single band of DNA between 15N and 14N density
after 2 gens, 2 bands, one at intermediate density,, and one at 14N band
what is the Klenow Fragment
give features of it
in prokaryotes
part of the bacterial DNAP1 repair polymerase (in E.coli)
it is the large fragment of DNAP1
present at high copy number (easy to extract as lots of it)
has a primer removing exonuclease domain removed by hydrolysis
shaped like a right hand
has 5’ to 3’ polymerase activity
has 3’ to 5’ exonuclease proofreading activity
outline structure of Klenow fragment
shaped like a right hand
has fingers, thumb and palm
has primer-removing exonuclease domain which can be removed by hydrolysis
palm region has 2 AS
what active sites are present in the Klenow fragment
has 2 active sites
polymerase site 5’ - 3’ which synthesizes DNA then has 3’ - 5’ exonuclease which breaks down DNA in opposite direction
secondary AS used for proofreading and removing incorrectly added nucleotides
why does DNAP1 have high fidelity rate
because Klenow fragment has a secondary active site used for proofreading and removing incorrectly added nuceotides
define lesion
A potential mutation
what mutations can be introduced in DNA synthesis
transient base tautomery
what occurs during transient base tautomery? How does this happen?
if a base is in the wrong tautomeric form during DNA synthesis, it will stick to the wrong base pair and become a lesion
how is transient base tautomery prevented
the Klenow fragment (part of the bacterial DNAP-1 repair polymerase has a proofreading 3’-5’ exonuclease site
which bases are prone to transient base tautomery
all 4 bases
as all are capable of either amino or imino or keto-enol tautomery
define tautomerization
The interconversion of two isomers that differ only in the position of protons (and, often, double bonds).
what is aminocytosine
guanine base pair
what is iminocytosine
adenine base pair
what form are the N- atoms attached to C G and A in
amino NH2 form
what form is the O atom in G and T in
keto form =O
what is imino form of N?
=NH
what types of rare base pairing are there?
A(amino)=C(imino)
A(imino)=C(amino)
G(keto)=(3dbl bond)T(enol)
G(enol)=(3dblbond)T(keto)
what does deamination of cytoisine make? what can this pair with?
Uracil
pairs with A
what does deamination of 5-Methylcytosine make
thymine
how does exonuclease of DNAP work
it removes a mismatched base and exposes a ‘clean’ OH group, to which another dNTP can be attached, so polymerisation can immediately recommence after proofreading activity
why does DNAP have to be unidirectional
because during 5’-3’ synthesis, it is the dNTPs that bring in the energy via the P-P-P group,, needed to make the new phosphodiester bond in the backbone of DNA
if the strand was synthesised 3’-5’, it would be the P-P-P already on the daughter strand that provides the energy for polymerisation.
removal of a mismatched base would result in a ‘clean’ 5’ OH group which would terminate polymerisation, so youd need some complex and inefficient system to readd the P-P-P and restart polymeisation
give elongation eqtn for DNA syn
DNAn + dNTP -> DNAn+1 +PPi
what direction do DNAPs synthesise in?
5’ -> 3’
what error rate does binding of nucleotides by DNAP have
1x10-4 bp-1
what is the mutation rate in inherited DNA
10-9 per base pair
how does the proofreading site of DNAP work
Iminocytosine base pairs with A, but because its a tautomer IC quickly goes back to C, which is wrong pairing
this causes C to flip into the proofreading site, so gets removed
what are the 2 strands of DNA termed in DNA synthesis
lagging and leading strand
what direction is leading strand
5’ to 3’
what direction is lagging strand
3’ to 5’
how is the lagging strand synthesis different to leading strand synthesis
lagging strand is synthesised discontinuously in discrete Okozaki fragments (5’ to 3’ chuncks)
leading strand is synthesised continuously in 5’ to 3’
what is the direction of Daughter DNA strand
5’ to 3’
which DNA will DNAP bind
ssDNA
how does DNAP bind ssDNA
through addition of erasable RNA primers
why wont DNAP bind ssDNA
due to a conflict between being able to spot a mismatched base at 3’ end DNA and being able to initiate a new strand de novo
on the 5’3’ strand, what way does DNAP bind and synthesise
it creates a 5’3’ daughter strand, therefore binds at end/bottom 3’ end working continuously upwards towards the 5’ end
compare addition of RNA primers on leading/lagging strand
DNAP wont bind ssDNA so 1 erasable primer added on leading strand and MANY on lagging strand
how long roughly is RNA primer
100bp
what part of DNAP-1 is removed to make the Klenow fragment?
how is this done
the primer removing nuclease activity of the DNAP-1 is removed during hydrolysis to make the Klenow fragment
what is primase
adds RNA primer to ssDNA so DNAP can extend the ssDNA
what deletes the added RNA primer to ssDNA
RNAse, then DNA ligase stickes the two bits together
define Okazaki fragment
short, newly synthesized DNA fragment formed on the lagging template strand during DNA replication
what are the steps for the termination of DNA synthesis
remove primer through use of RNAse
DNA ligase
if molecule linear: TELOMERES
define telomeres
piece of DNA at end of eukaryotic chromosomes that cant be replicated using DNAP
only occurs in eukaryotes with linear chromosomes, doesnt happen in prokaryotes with circular DNA
what is a replisome
replicating machine to transcribe DNA, composed of numerous proteins
what is helicase
what difficulties does helicase create?how is this overcome?
enzyme that melts DNA strands, breaks H bonds, creates replication bubble.
makes negative supercoiling behind it and positive supercoiling up stream, therefore have proteins upstream to combat this
what is DNA topoisomerase
runs ahead of helicase and nicks one of the strands of DNA to allow strain release of DNA
give some examples of proteins in the replisome
sliding clamp:increased processivity topoisomerase:strain relief helicase: melt DNA primase: add RNA primer single stranded binding proteins: stabilise ssDNA of parent and prevent self annealing of ssDNA as it is sticky and can make hairpin loops on itself
what are single strand binding proteins?
stabilise ssDNA of parent and prevent self annealing of ssDNA as it is sticky and can make hairpin loops on itself
what does a sliding clamp do? where is it found?
found on leading clamp
hexomeric donut shope
increases processivity to stop DNAP falling off DNA
always behind DNA
what is telomerase
a reverse transcriptase with an internal RNA template which extends telomeres in germline cells
how are different DNAPs specialised in E.coli
DNApol 1 - repair and Okazaki clean up
DNApol 2 - repair
DNApol 3 - replicative polymerase
how are different DNAPs specialised in humans
DNApol alpha: priming
beta: repair
gamma: mitochondrial
delta: lagging strand
epsilon: leading strand
what base tautomery exists for T+G
keto-enol
what base tautomery exists for A+C
amino-imino
which bases have amino-imino base tautomery
A+C
which bases have keto enol tautomery
T+G
what is a primosome
primase and helicase, found in bacteria
what four sections make up DNA replication, transcription and translation
- MACHINE
- INITIATION
- ELONGATION
- TERMINATION
what effect does germ line cells having telomerase have
no hayflick limit therfore can replicate indefinitely
name some cells that have their own telomerase
stem cells and cancer cells - can replicate indefinitely
what is the Hayflick limit/constant
max number of divisions a cell can undergo before death, due to the absence of telomerase which extends the telomeres
which nucleotides are telomeres rich in
GC
what happens to length of telomeres over generations
constantly get cut off end of chromosomes and eventually runs into genes/coding DNA
what does telomerase do
extends the telomeres -its a reverse transcriptase
