MCM helicase

I.

Eukaryotic DNA Replication

a. Two different polymerases do the lagging strand synthesis and
the leading strand. Helicase unwinds out in front. Primase is in
both the Eukaryotes and prokaryotes and does both RNA and
DNA synthesis. SSB proteins are used. Lagging strand: RFC loads
PCNA to the end of the primer, PCNA displaces DNA pol alpha,
and dna pol delta binds to pcna and elongates the okazaki
fragment. Then primer removal mechanisms.
b. Differences in Euk and Prok: Eukaryotes dont have
Polymerase I that can do nicked translation (needs
something to come behind it and check for errors); they have
two different polymerases for the leading and lagging strand.
Eukaryotes use RFC as a clamp loader and loads on the clamp
and polymerase
i. they have two mechanism to remove RNA primer and fill in
the gap:
Eukaryotes use a holoenzyme during initaiton and use
RNase H (hybrid) and protein FENI (flat endonuclease) for
synthesis.
1. RNase H recognizes hybrid and digests the RNA but it
cant cleave off the last RNA because it cant cleave
an RNA DNA bond. FEN1 (flap endonuclease)
removes the last nucleotide (requires an overhang)
2. RNase H is bypassed: Helicase unwinds the strand
and FEN1 cuts it off
3. Similiarities: Fen1 is in both.
c. Cell Cycl: G0 or G1, then S, then G2, then M
d. In G1 the orgin is marked by the origin recognition complex:
you have your ORC that interactions with proteins to make a pre
intiation complex, then cyclins phosphorylate the complex and
triggers DNA replication into S phase
e. Telemeres: Chromosomes get shorter with each round of
replication and if they keep getting shorter you are eventually
going to be losing regions of gene expression.
i. Telomerase (ribo protein complex) makes telomeres.
The end of chromsomes have sequences of repeats (series
of Ts.) TTTAGG is repeated multiple times at the end of a
chromosome. Telomerase is a RT that catalyzes the
addition of these repeats VIA DE NOVO synthesis.
Telomerase carries its own RNA template which is
complementary to the repeat sequences and allows
temomerase to make telomeres without DNA template.

Telomerases RNA template binds the LEADING strand with

complementary sequence and uses its RT ability to
synthesize the DNA complimentary sequence and then
remove its RNA, leaving a 3 end overhang. The lagging
strand can then be extended normally. Telomeres remain
stabilized because the fold into four stranded
oligonucletodies and make almost every single H
bond. Potassium is usually in the center. There is evidence
of this in vivo because there is a telomere end binding
protein that, when it was crytstalized, found it bound both
SS DNA and there is a cleft that binds quadraplex DNA
indicating that quadraplexes exist in vivo. Chrosomes also
connect to one another and it is suggested that these
types of structure may link chrosomes together for
chrosome chrosome communication. The 3 end overhangs
have to protect because SS ends are bad. Pot 1 binds the
end of DNA and TRF2 supports the invasion of the
SS tail back into the DNA and the tail gets buried in
the loop and the loops are stabilites by telomere
binding proteins (Seen in EM)
ii. How do you prove the RNA in the telomerase serves
as the template for DNA synthesis
1. Change the sequence and show DNA sequence
changes

II.

Prokaryotic DNA replication

a. DnaA is an ATPase that forms oligomers in the presence of ATP
(it changes its conformation and opens up another
binding site for DnaA) . DnaA binds five sites on the DNA.
It forms a RH helical filament. When ATP is bound, more DNA A
is recruited, increasing positive writhe, which put it into a
toroid and opens the DNAs compensation of negative
supercoiling (this region that opens is AT rich). This
allows helicase (B) to be loaded onto the DNA by DNA C.
DNA and RepA
i. Think about experiment with footprinting assay for
DS before it is open and SS when its open
ii. On DS you could do a control of just DNA and then
DNA+DnaA, then increasing concentration of ATP w/ DnaA
iii. DNA A and then increasing the concentration of DnaA in
the presence of ATP to show a more distinct protected
region. Then do a SS where the bands will only appear in
the open region (the AT rich orgin). Could then use

III.

I.

sequencing ladder to determine at which sequence the

DNA A is binding
Reverse Transcriptase
a. RNA virus: Reverse transcriptase recognizes SS RNA and makes a
DS hybrid. RT has RNA H activity (Like Euk DNA
polymerase) that cuts up the RNA and then synthesizes DS DNA
that gets intergrated into the genome.
b. Transcription and translation only come off of DS DNA

Mismatch Repair
a. Most common are GT mismatches and IDLs
b. Post replication DNA MMR can decrease mutation rate by 3
orders of magnitude
c. Method
i. Recognize/ find the mismatch and bind specifically
ii. Excise the mismatch
iii. Resynthesis
iv. Ligate
v. STRAND DISCRIMATION
d. How does Mut S recognize mismatch
i. Idea: Mismatch in the DNA makes the NDA more easy to
bend/ more flexible there and change in bending could
explain the mechanism. But we know that erros that are
more deleterious thermodynamically are least repaired.
Evolutionary maybe the least deleterious are better
repaired because they are more common
ii. Experiment: compare conformations of Mut S bound to
mismatch DNA and homoduplex- atomic force microscopy
experiment
1. Show that removing bent population prevents
repair
iii. As you search you bend the DNA (most stable
conformation is bent)- when you find the mismatch it goes
into Initial Recognition Complex where kinking plays a
critical role. Then you must change to unbent complex via
ATP induced conformation??
iv. In homoduplex you never make the IRC because you dont
interact with the phenylalanine.
v. There are two parts to the model: 1. it searches by
bending the DNA comes from the observation that
nonspecific compelxes were bent. 2. IRC comes from
two observations: specific complexes existed in bent
and unbent state AND crystal structure showed

specific interaction that was in a kinked state. 3.

URC is the ultimate recognition complex comes from
the observation that bent and unbent were seen in
the specific complex and only bent was in
nonspecific- this state is unique to the mismatch.
The idea that this might be correct comes from the
mutagenesis assays that showed that decreased
loss of repair is associated with loss of the unbent
state.
vi. Experiment with blocked and unblocked DNA for sliding
clamp:
1. Unblocked DNA: slides off DNA much faster
2. Blocked: stays on DNA much longer
3. ATP facilitates faster release of clamp?
e. E. COLI METHYL DIRECTED MMR
i. MutH identifies mismatch (hemi-methylated GATC) strand
discrimination signal
ii. MutS binds mismatch and recruits MutL via ATP (Mut S has
two jobs: bind mismatch and it is an ATPase to signal MutL)
iii. Formation of MutS/L complex activates Mut H (Helicase)
endonuclease activity via ATP hydrolysis (Mut H only
recognizes hemi methylated GATC site and nicks that
strand)
iv. UvrD helicase loads on to the DNA and unwinds it and
exonuclease follows behind to remove the mistmatch
sequence. REPLICATIVE poly fills gap.
v. Ligase ligates.
f. Eukaryotic Mismatch Repair
i. Mut S binds mismatch and induces a conformational
change via ATP that forms a sliding clamp state that then
recruits MutL alpha which has the endonuclease activity.
MutS structure: is a dimer and has a DNA binding domain that
lock in and pinch the DNA and the DNA is bent. It functions
asymmetrically, only one domain interacts specifically with the
DNA. There are different conformations of the domain that make
nonspecific interactions. There is a phenylalaine that interacts.
Phenylaline stacks with the thymine in the mistmatch
(RESPONSIBLE FOR SPECFICITY), if you mutate it you
cant repair. ONLY PHENYLALALINE AND GLUTAMATE
INTERACTIONS ARE SPECIFIC
ii. ALL THE REST ARE NONSPECIFC WITH THE BACKBONE OR
THE SUGARS.

iii. PCNA interacts with MutS/MutL and activates latent

endonuclease activity and nicks the daughter strand

II.

iv. Mut S Exonuclease comes and removes mismatch. Poly

resynthesizes.
v. Ligase ligates
vi. Orientation of pcna is asymmetric allows for strand
discrimation
g. Differences: No helicase in eukaryotes- the exonuclease
functiosn as both a helicase and exonuclease
Nucleotdie Excision Repair
a. NER: (lecture 2)
b. B does not bind in the absence of A, it requires A. Once B
and A bind, they recruit C and D and C has an
endonuclease activity. B and C then excise the DNA
containing the damage. 12-13 bp fragment. Once it is
nicked that strand needs ot be removed, so Uvr D is
recruited and unwinds the strand with the erro and DNA
poly I fills in the gap and liagse seals it
c. Find the error. How do we find it. Most interactions are
non specific. Specifity is the ratio of the bidning affinity
to the specific site/ nonspecific site. Looking at kinetics of
binding: as DNA got longer, it binds to the specific site
faster than possible than 3 dimmesional diffusion (3
orders of magnititude faster). What happens is the
concentration of nonspecific sites are so much greater,
the protein binds and diffuses along the DNA and scan
along it looking for its site looking for it.
d. Experiment: Rate of binding as a function of length:
(length over which it can slide also depends on the salt
concentration)- nonspecific sites facilitate the movement
so they go faster with longer dna one dimensional over
three dimensional. Maximum is the rate of sliding versus
probablitiy of falling off. If you increase the salt
concentration it knockes the protein off bc electorstatic
reactions.
e. Models for ATP activation of MMR
f. Translocation:
i. Translocation requires hydrolysis to promote movement
and molecular switch doesnt (difference between the size
of strands they tested)
ii. Experiment-EM with time depdence: Atp vs. Atp then Atp
gamma S
g. DNA bending
i. Mut S/Mut L interact with Mut H through bending the DNA.
ii. Experiment: Footprinting under different conditions
1. Region around MM is protected by Mut S without ATP

2.
3.
4.
5.

III.

IV.

V.

Mut L doesnt bind DNA alone

Mut S protection nis reduced if ATP is presence
Mut S + Mut L + AtP results in extensive protection
Control: homoduplex showed no changed with any of
these: complex formation dpedends o npresence of a
mismatch
Base Excision Repair
a. Steps
i. Removal of incorrect base by an appropriate DNA Nglycosylase to create a AP site
ii. AP endonuclease nicks the 5 side of the AP site to
getnerate 3Oh terminus
iii. Extension of 3OH by a DNA polymerase: This polymerase
has TWO DOMAINS: POLYMERIZATION DOMAIN AND A
DOMAIN TO REMOVE THE RIBOSE GROUP)
b. Only repairs alkylation alkyl transferase
c. Glycosylase recognize the base by flipping the base into a
pocket and having specific interactions with it to catalyze
hydrolysis
d. Difference between prok and euk: more proteins and more bases
nicked in eukyarotes
Transcription Coupled Repair: Pro
a. Similar to NER but when RNA poly hits a lesion that it stalls at it
recruits the enzymes that are involved in NER by backing up
b. Saw that transcribed genes were better repaired
Other Repair:
a. Most common are oxidation of G and hydrolysis of C to U
b. Spontaneous base los leaving AP site
c. Spontaeous deamination C-U
d. Effects of Sunlight:
i. Pyrimidines (T or C) can make dimers and NER is needed in
humans but in bacteria they have photoylase:
1. That hole is where the thymine dimers bind and flips
the dimer out of the helix and fits it into the pocket.
Chromophores on enzyme absorb the light. If
you put photolyase in the dark it will bind BUT not
repair so you know light is required. Chromophore
absorb the light, transfer to the FADH- and
then that transfers an electron to the dimer
and that breaks the cyclobutaine bond
(pyrmiindine dimer) and then does a second
breakage and now you have an extra electron
to transfer back to FADH to reduce it

VI.

2. Alkyltransferase transfers the alkyl group form

the DNA to the protein on the cytestine group
(in the enzyme) which kills it. Suicide enzyme.
Types of Techniques we use.
a. Gel Shift Assay:
i. Label DNA with radioactivity
ii. If a protein binds to DNA it will travel slower (Higher band
shift)
iii. DS non denaturing polyacrylamide gel
iv. Can be used for competitive inhibition
b. DNA Footprinting:
i. Label only ONE strand (label only one primer when you run
a PCR reaction) and find single hit conditions
ii. DS assay: Run with DNase and on a denaturing
polyacrylamide
iii. Limititaitons: Lower resolution than chemical
footprinting (cant tell if it binds to one side or both)
and if there is weak binding you would get
separation
iv. SS assy: Run with KMnO4 (attacks SS thymines)and on a
denaturing polyacrylamide gel
v. Usually do these experiments as a concentration of protein.
vi. Can get a Kd by plotting extent of protection vs.
concentration and get a binding constant/ binding affinity
vii. Assumption: extent of protection is directly
associated to amount of binding
viii. If you run a sequence ladder you can get the
location and sequence of binding
c. Nitrocellulose filter binding
i. Binds proteins not nucleic acids
ii. Label DNA on ONE strand and use filter paper, buffer, and
vaccum
iii. Control is to put just the DNA on the paper and see what
the background is.
iv. Add protein to DNA and increase concentration of protein.
v. Limitations: non equlibirium method: if you have a
weak interaction with rapid off rate the DNA can
dissociate during the wash and then you could lose
it
d. Flourescence polarization/anisotropy
i. If you excite the flurophore with polarized light, only those
with the dipole in the same orientation of the light get
polarized. If you emit the light slower than the rate of
tumble then you get then you have randomized it and it

will have low antisopry. If you emit light faster than the rate
of time then you get more in the channel in the same
direction= anisotropy
ii. Control: DNA alone to see what inherent anisotropy it has
and then do it with protein- slows down tumbling, so you
increase anisotropy
iii. Plot anisotropy vs. protein concentration
iv. Limitation: Size of DNA and flurophores have to
match the size of DNA
e. Atomic Force Microscopy
i. A small cantilevelr that moves on the surface of the
protein/ DNa and as it moves up and down you get a
topographic image. Laser is being shot and as the
cantilever changes angles it is reflected into the photiode.
ii. You can measure how far the protein is from the end of the
DNA so you can know if its at a specific or nonspecific site
if you know hwere you put the mistmatch and you can
know how much it bends the DNA.
iii. Limitations: cant determine which end of the DNA
you are are so you might count some nonspecific as
specific sites. It has a small scanning area so size
matters and its slow.
iv. Plot the number of counts vs. DNA bending angle to get
you average and distribution.

Eukaryotic chromosomes are linear whereas pro are circular.

Translesion synthesis polymerases: polys designed to go agross
specialized lesion
In the sun you get thymine dimers. There is a specialized polymerase that
synthesizes with high fidelity across those and it has active site has space for
thymine dimer as opposed to just one nucleotide.
Pcna helps you find where to go- weak interactions keeps proteins near
replication fork so when something happens repair can occur

I.

II.

Transcription For E. Coli

a. RNA polymerase is six subunits, one of which is sigma
i. Sigma is responsible for specific binding to protons
b. The holoenyzyme is binds the promoter
c. Binding of the holoenzyme promotes the melting of DNA and
opening a bubble at the promoter= open complex
d. Initation happens and short aborted fragments are synthesized
e. When RNA is long enough it knocks off sigma and you get into
elongation phase: Sigma sits on the exit channel of RNA
polymerase so it binds competitively with RNA.
f. Translocation of the bubble down the DNA (helicase is unwiding
and the DNA is rewinding in the back) until you reach the
termination sequence
g. Form a hairpin and if there is a poly U string you terminate.
h. RNA poly has exonuclease activity so if it runs into a
lesion it will back up, extrude the 3 end of the RNA so its
not in the active site and forms a dead end complex- Gre
A and Gre B rescue this strand BY HELPING CLEAVING
THAT 3 end into a 3OH so it can rextend AND they help
with fidelity
i. They stick their finger into the secondary channel and their
finger has an Apsartic acid that brings and kelates a
second Mg that helps facility the cleavage.
i. Transcription Termination:
i. Hairpin in RNA secondary structure followed by U residues
ii. Pauses occur if its just a hairpin: pauses allow for
regulation and proteins to be recruited- rho is an
example of this- most promoters code for operons that
have stop sequences between each gene, a pause would
give the body enough time to produce a protein to inhibit
termination and all for the regulation of more downstream
genes to be produced
iii. Termination efficiency can be determined on denaturing gel
and comparing the amout npercent terminated vs
readthrough
Transcription For Eukaryotes:
a. Initiation complex for RNA poly II
b. TBP binds and recognizes the promoter box and bends the DNA.
It then recruits TFIIB and brings RNA polymerase and makes the
preinitation complex.

III.

IV.

V.

VI.

i. TBP widens the MINOR groove and bends the DNa. Two
phenylaline stick into the DNA and facilitate the binding
and bends it NINETY DEGREES
ii. TFIIB cant bind before TBP binds because that bending it
was allows TFIIB to touch the DNA in two domain spots. =
COOPERATIVITY
c. This complex then recruits TFIIH (helicase activity) and forms the
initation complex= TFIIH USES HELICASE ACTIVITY TO OPEN
THE COMPLEX
d. To get to elongation, phosphorylation of the C terminal domain
lets the polymerase go into the elgonation phase
i. Complexes can be poised and waiting for phosphorylation
ii. Phosphorylation regulates transcription quicker than
having to recruit cells.
Tryptophan biosynthesis:
a. In proteins translation and transcription are coupled so
tryptophan can be self regulated
b. RNA fold into different secondary structures
c. If you have high trp, no stalling, so 1&2 and 3&4 form. Opposite
if low trp.
d. Only occurs because of coupling of transcription and translation
E. coli Promoters
a. Sigma bind sto the promoter and is autoinhibited (until it binds
RNA poly it cant interact iwht the promoter).
b. Sequence and spacing between -35 and -10 regions determines
the strength of the promoter
c. 1 RNA in prokaryotes and 3 RNA in eukaryotes (one for m, t, r)
but prokaryotes has more than one sigma factor
d. RNA DOES NOT USE ATP TO OPE NTHE DNA- ALL THE
ENERGY COMES FROM BINDING OF RNA POLYMERASE
e. Two promotoers: TATAAT box (-10) and then -35 region
RNA polymerase Structure
a. Two structural domains (Beta and Beta primre where prime is
catalytic) with cylinders being sigma.
b. Sigam is necessary for promoter specific initiation, and it
interacts with the promoter DNA only when bound to Poly, and it
facilitates opening of the promoter DNA because it can make
those connections
c. RNA exit channel overlaps with sigma binding site so RNA and
sigma compete here
d. Two Magnesiusm in the catalytic pocket
e.
Lac operon

VII.

a. Will synthesize unless repressed. Only repressed when lactose

breaks down and binds to repressor.
b. IPTG is lactose that doesnt break down so you can get
constitutive expression.
c. Repressor binds operator and keeps RNAP from leaving
the promoter so it can only make abortive transcripts
d. Induceer: causes a structural change in the repressor
that prevents it from binding the operator (lactose is the
inducer)
e. Experiment: chemical footprinting ???
f. Inducer bindingprevents the DNA binding domains from
contacting the operator site effecitvity by going from
optium geometry to poor geometry- BENEFIT OF
BIPARTITE DOMAIN
Bacteriophage lambda
a. Two pathways: Dormant and Lytic
i. When DNA gets intergrated via integrases everything is
dormant
ii. If you make a cell sick via UV radiation you get an
induction event and its starts replicating the DNA
iii. Lytic: synthesizes DNA and makes the proteins that will be
the phage particles and assembles nad lyses.
b. Lambda repressor vs. Cro
i. Cro preferentially binds: 3,2,1
1. Wants to go to lytic pathway
2. HAS A STRONG PROMOTER
3. If Cro binds 3 it shuts off lambda repressor and when
it binds 1 it shuts off itself
ii. Lambda preferentially binds: 1, 2, 3 and has cooperativity
1. Cooperatively binds Or1 and Or2
2. Stops Cro if it binds Or1
3. When it binds Or2 it activates its own synthesis
(Activator)
4. If it binds Or3 it shuts itself off
5. It is a dimer with a protein protein domain and
a protein DNA binding domain
6. When UV radiation occurs, Rec A cuts the connector
domain and the cooperativey is lost and Cro takes
over
7. Experiment: Footpriting and binding affinity
8. GOOD ENTROPICALLY BECAUSE YOU ARENT PAYING
THE COST FOR THAT PROTEIN PROTEIN INTERACTION