CMB 621 – Fall 2018 DNA Replication – Part 1 Repair and ...lesaux/621/ewExternalFiles/AL Lecture...

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?


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


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?


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


We know it proceeds in a bi-directional fashion…

But, intact dsDNA in front of fork builds torsional strain…


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


SSBP - Stabilizing ProteinsRPA = replication protein A


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?


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


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

We’ve mentioned processivity, which means?

We also need to address fidelity, which is?

How does fidelity relate to3’- 5’ exonuclease activity?


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


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

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


Type II Topoisomerase -Gyrase

ATP hydrolysis allows for dimerization and alternate conformations

= breaking of a duplex, and pass through occurs


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…


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

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