CMB 621 – Fall 2018
DNA Replication – Part 1
Repair and Recombination
Axel LehrerAssistant Professor
Tropical Medicine, Medical Microbiology and PharmacologyJohn A Burns School of Medicine, UH Manoa
Before we tackle DNA replication…
How do we even know it is the heritable material
passed through generations?
HISTORY1928 - Frederick Griffith
Streptococcus pneumoniae
HISTORY1944 - Avery, MacLeod and
McCarty
HISTORY1952- Hershey and Chase
Why is DNA replication important to study and
understand?
In vivo Importance
S Essential for vertical propagation of information
S May fix mutations
S May create mutations
-promote fitness & diversity
-may result in cell death
-may be neutral
Also utilized in horizontal DNA transfer
Copyright © 2010 Academic Press Inc.
Figure 15.7
Utilized in some viral replication methods as well… Rolling Circle Replication
Watson and Crick
Figure 6-4 Essential Cell Biology (© Garland Science 2010)
1958 - Meselson and StahlSemi-Conservative Replication
0
1
2
3
Where is the beginning site of DNA replication?
G1
G2
(DNA synthesis)S
Cytokinesis
Mitosis
MITOTIC(M) PHASE
Origin of Replication
-Dictated by a specific-sequence motif
Also influenced by chromatin conformation
14Copyright © 2010 Academic Press Inc.
E. coli Origin of Replication
•Note the AT-rich sequence (69%+)•Note the recognition binding sites for initiator proteins•Above is but one such motif discovered…
Initial Denaturation
Figure 5-27 Molecular Biology of the Cell (© Garland Science 2008)
• Multiple binding sites at OriC
• Recruitment of DnaAcreates torsional strain at adjacent AT-rich motifs
• Denaturation allows for DnaC (helicase loader/inhibitor) to deliver DnaB (helicase)
• Helicase expands the replication bubble and DnaG (primase) allows for fork establishment
E. coli OriRecap
Is the ori fixed?
What would happen if the ori picked up a mutation?
Side Note Plasmid Oris
S The particular ori found in a plasmid dictates the copy-number
S Early generation plasmids contained an ori that gave low copy-numbers per cell
S Contemporary plasmids contain a high copy-number ori that maintains plasmids at 25-50 per cell… why not go higher?
Figure 5-26 Molecular Biology of the Cell (© Garland Science 2008)
Prokaryoteor
Eukaryoteor
both?
E. coli has 1 ori
Humans have approximately 30,000 - 50,000
Otherwise30 days
Eukaryotic oris are found in clusters, ranging from 10-300 kb
apart
Different oris are utilized at different periods of the S phase
Euchromatin oris are activated earlier than heterochromatin, as shown by
examining replication of X chromosomes and comparing the
timing of replication for housekeeping vs. less active genes
Figure 5-36 Molecular Biology of the Cell (© Garland Science 2008)
Timing of Replication in
Yeast
Kinase activity at the S-phaseleads to the degradation of initiator factors until the next round of the cell cycle
While canonical human orishave been hard to elucidate, some appear very similar to the yeast ORC sequence
Ori is denatured to reveal a replication bubble, which then allows 2 forks to
become established…
Prokaryote Eukaryote
Ori, Initiator Proteins, Bubble, Forks…
What drives separation of the fork?
Figure 5-14 Molecular Biology of the Cell (© Garland Science 2008)
Helicase = Mcm2-7
ATP is utilized
Denatures ~ 1,000 bp/sec
Composed of 6 identical subunits(in bacteria)
These units have 3 different conformations
https://youtu.be/d_9VBgrDLUg
Figure 5-25 Molecular Biology of the Cell (© Garland Science 2008)
We know it proceeds in a bi-directional fashion…
But, intact dsDNA in front of fork builds torsional strain…
Figure 5-22 Molecular Biology of the Cell (© Garland Science 2008)
Type I DNA topoisomerases
Reversible nucleases thattransiently attach themselves toone strand of DNA
Thereby creating a nick
Torsional strain naturally resolves itself
The energy of the phosopho-diester bond is retained in the transient complex
Therefore no energy is needed and the rxn is rapid
Side Note Topo I TA Quick Cloning
Figure 5-16 Molecular Biology of the Cell (© Garland Science 2008)
SSBP - Stabilizing ProteinsRPA = replication protein A
Figure 5-17 Molecular Biology of the Cell (© Garland Science 2008)
SSBP helps to minimize inhibitoryhairpin structures and mutations, and
exposes unpaired bases
Now the DNA template strand is available for
complementary synthesis…
How does DNA pol know where to start synthesis?
Figure 5-11 Molecular Biology of the Cell (© Garland Science 2008) Direction of Synthesis
The leading strand only needs 1 primer for synthesis
The lagging strand requiresribonucleotide primers at intervals of 100-200nucleotides (eukaryotes)
Notice that it reads the template 3’- 5’… but it synthesizes the nascent strand 5’- 3’
Why is a RNA primer used for DNA replication?
Besides providing evidence for RNA-based early life
de novo (new) synthesis can be error-prone, therefore it is better to come back later, remove the primer, and
insert correct DNA bases
Primers are marked as “suspect”
If the cell used DNA primers, there is a greater chance of permanent
incorporation of the errors
By using RNA primers, these mutational hotspots will be
subsequently removed
DnaG – DNA PrimaseS Associates as a trimer with DnaB (helicase)
S Tends to initiate synthesis at CTGs
S 3 domainsS Zinc BDS Helicase BDS RNA polymerase
DNA Primase RegulationRedox in DNA primase regulates initiation (ox) and termination of priming (red)
Model for primase product truncation, where primer-template handoff to the [4Fe4S] signaling partner, polymerase α in vivo, is regulated by DNA charge transport
The Star DNA Polymerase
S Many, many different types amongst various organisms
S Its job is to produce complementary strands… with high-fidelity (usually)
S But like many DNA scanning proteins, it has a propensity of falling off, so…
Figure 5-18b Molecular Biology of the Cell (© Garland Science 2008)
Sliding Clamp = processivity
- delivered by the clamp loader (Replication Factor C 1-5 in euks)
- fixes DNA poly to the template, but releases it once the complex hits a dsDNA region in front of it
Figure 5-18c Molecular Biology of the Cell (© Garland Science 2008)
In eukaryotes the sliding clamp is called PCNA = homotrimer
Proliferating Cell Nuclear Antigen
aka – a processivity (1000x more) factor for DNA pol
https://youtu.be/5A77R3q0yZQ
DNA (and RNA) is always synthesized in the 5’- 3� direction
•Deoxy(ribo)nucleoside triphosphates are the building blocks
•Hydrolysis of the phosphoanhydride bond releases part of the energy for the synthesis
•The additional energy comes from the breakdown of the resulting pyrophosphate
Note which phosphate group is incorporated?
Figure 5-4 Molecular Biology of the Cell (© Garland Science 2008)
Figure 5-10 Molecular Biology of the Cell (© Garland Science 2008)
Energetically,3’ to 5’ synthesiswill not suffice
Could you explain the components and process?
Minimal Rates:
Prokaryotic synthesis proceeds at 500-1000 bases per second
Eukaryotic synthesis proceeds at ~50 bases per second
in vitro Taq synthesizes at 10- 45 bases per second
Figure 6-12 Essential Cell Biology (© Garland Science 2010)
One strand (leading) is made continuously and the other (lagging) is made discontinuously…
Therefore replication is considered semi-discontinuous
Prokaryotic Okazaki = 1 - 2 kbEukaryotic Okazaki = 0.1 - 0.2 kb
Notice that a bubble consists of forks that are inverted mirror images of each other
At the replication fork the two newly synthesized strands are of opposite polarity…this clearly leads to logistical problems here since synthesis
only proceeds in one direction
The Replication Fork Is Asymmetrical
No problem here though
Notice the problem of the divergent polymerase
movement?
The replisome actually does stay intact… how?
Figure 5-19a Molecular Biology of the Cell (© Garland Science 2008)
Sliding Trombone Model
https://youtu.be/-mtLXpgjHL0
https://youtu.be/4jtmOZaIvS0
Questions?
Can we map it all out?
Where are we?
DNA polymerase doesn’t start DNA synthesis de novo.
The primer is RNA (about ~11 nucleotides in eukaryotes or ~5 nucleotides in prokaryotes)
The primer is made by Primase, an RNApolymerase
The primer then has to be removed: Pol I has 5�- 3� exonuclease activity with which it cuts out the primer – as it does that it fills in the gap with DNA
In eukaryotes, FEN1 removes the primer and new DNA is laid down by Pol d (it created a flap for FEN1)
DNA ligase then repairs the gap
Pol III falls off and replaced by Pol I
Pol I removes RNA primer and replaces it with DNA
Primer Removal
NOTICE THIS!!!!
E. coli model
Prokaryote DNA Pols
S Pol IS Last pol, it removes previous Okazaki primerS 20 bases/sec, synthesizes the first ~ 400S Involved in DNA repair as well
S Pol IIIS Major pol for synthesis, ~1,000 bases/sec
S Pol IIS Involved in repair, a back up for pol III
S a (+ primase) • Primase synthesizes ~10 RNA bases, then pol synthesizes the first
~15 DNA bases• Primarily initiates lagging strand synthesis• No exonuclease activity, but ~30,000/cell
• e - Performs leading, (maybe more regulatory than catalytic?)
• d (+ PCNA) • Greater processivity than above
•Lagging strand extension, must be constantly reloaded
•Has 3’-5’ exonuclease activity
Sg - mitochondrial DNA replication
Eukaryotic Pols
Examples of Eukaryotic DNA Pols
S
Eukaryotic DNA Pols
S
Eukaryotic DNA Pols
We’ve mentioned processivity, which means?
We also need to address fidelity, which is?
How does fidelity relate to3’- 5’ exonuclease activity?
Figure 5-8 Molecular Biology of the Cell (© Garland Science 2008)
LimitingMutations
Correct incoming base is a better fit
Before covalent bond formation DNA pol undergoes a conformational change that can destabilize incorrect base pairing
3’- 5’ exonuclease activity
Figure 6-13 Essential Cell Biology (© Garland Science 2010)
There are going to be mistakes, (mutations if they are not corrected)
Mistakes are corrected by the 3’- 5�proofreading exonuclease activity of the polymerase (pol III, e and d)
Initially, the mutation rate approaches 1 per 107 nucleotide pairs
But the actual mutation rate approaches 1 per 109 nucleotide pairs -- other repair mechanisms (DNA mismatch repair) keep the mutation rate down.
DNA Polymerase is Self-Correcting
Figure 5-9 Molecular Biology of the Cell (© Garland Science 2008)
DNA polymerase - proofreading
https://youtu.be/OwZgQCOUxCk
Is there a target level of allowed mutations that provide
genetic stability
…yet still allow variation in a population either horizontally
or vertically?
Associated Mutation Rates
S Only ~3 mutations occur in a human cell with each cell division
S Germline numbers must be low to protect the species
S Somatic cell numbers must be low to safeguard the individual
Cancer Correlation
S Vogelstein et al – 2017S 17 cancer types in 69 countries
S Found that cancer rates correlated with stem cell division rates in different tissues… across varied environs/countries
S Cancer results from accumulated mutations in driver genes that successively increase cell proliferation
S Inferred that ~2/3 of mutations are from replication
Figure 5-23 Molecular Biology of the Cell (© Garland Science 2008)
Type II Topoisomerase -Gyrase
ATP hydrolysis allows for dimerization and alternate conformations
= breaking of a duplex, and pass through occurs
Figure 5-24 Molecular Biology of the Cell (© Garland Science 2008)
Type II Topoisomerase
Again, untangles inappropriate ds complexes during transcription
Fluoroquinolones inhibit its function in prokaryotes
Note that Type II topos are generallymore active in proliferating cells
Therefore it can serve as an anticancer target = doxorubicin and etoposide
– Polymerase - all sorts, depends on the particular task
– Primase - does not proofread though
– Helicase (and loader) – (Mcm proteins in eukaryotes) unwinding enzyme
– Clamp loading protein (Replication Factor C in eukaryotes) - help guide and orient polymerase onto the DNA
– Sliding clamp (Proliferating Cell Nuclear Antigen in eukaryotes) -help guide and orient polymerase onto the DNA
– Ligase - to covalently link the sugar-phosphate backbone of the pieces together
– Single-strand DNA binding proteins (Replication Protein A in eukaryotes)
– Topoisomerase - remove torque ahead of replication fork (type I -single stranded break; type II - double stranded break
The Players
Player Comparison
Associated bits…
Figure 5-28 Molecular Biology of the Cell (© Garland Science 2008)
E. coli DNA Adenine Methylase (DAM)
Nascent strands remain unmethylated for about 10’, why?
Stalling deters inappropriate ori activation
Stalling allows for proper repair of mutations
Methylation also protects against restriction digestion from
endogenous enzymes
…why would this matter?
A Rookie Mistake
• Some E. coli lab strains have DAM or DCM
• Therefore the extracted DNA is methylated
• Unfortunately some restriction enzymes cannot bind at methylated restriction motifs
• Therefore you think you are digesting DNA… but are not
Site-Directed Mutagenesis
http://www.genomics.agilent.com/article.jsp?pageId=388&_requestid=517169
DNA methylation status also allows us to selectively
digest DNA
Moment of Reflection
Now you can see whyG1 is so essential?
-ATP-DNA pols-initiating, elongating, and supporting enzymes/ proteins-deoxy(ribo)nucleoside triphosphates
As you have seen previously, histones must also be addressed
during replication
Histone expression occurs in S phase
Histone mRNA created in other cell cycle phases is rapidly degraded
Once made, histone proteins are stable
Figure 5-38a Molecular Biology of the Cell (© Garland Science 2008)
Chromatin-remodeling proteins help facilitate replication through intact nucleosomes
Chromatin assembly factors (CAF1)associate with forks and load histones, both recycled and newly synthesized
New histones are initially acetylated (relaxed), but will be properly deacetylated(clamping) rapidly
Not Fully Elucidated…
• How histones are destabilized
• How histones are recycled and loaded
• How histones maintain epigenetic markers such as phosphorylation, methylation, acetylation, and ubiquitination…
• CAFs are associated with PCNA, therefore they are localized at the replisome, and nucleosome formation occurs just after replication
Concerning DNA replication
Can you think of any real-world applications?
Applications Involving Replication
S PCR and its descendants – amplification…
S Probe creation – arrays/chips, F.I.S.H.
S Cloning – blunting, Gibson Assembly
S Mutant generation – loss/gain/change of fxn
S Sequencing – traditional and next gen.
S Cancer & Antivirals - nucleoside analogs
A curious question:
How could you create a new DNA pol that has
improved processivity and fidelity?
Random MutagenesisDirected Evolution
www.invitogen.com/site/us/en/home/Products-and-Services/Applications/Cloning/gene-synthesis/directed-evolution.html
Note that you would need to use a faulty DNA pol in order to create an alteredtarget sequence
This same method was used to create some GFP color variants
GFP variants exist for different colors
This agar plate was inoculated with 8 different strains of bacteria, each expressinga different GFP protein variant
http://www.tsienlab.ucsd.edu/Images.htm
http://www.cell.com/cell_picture_show-brainbow2
Rat Brainbowrandom neuronal expression of GFP variants