CMB Lehrer Replication Lecture 2018-1

CMB 621 – Fall 2018

DNA Replication – Part 1

Repair and Recombination

Axel Lehrer Assistant Professor Tropical Medicine, Medical Microbiology and Pharmacology John 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? HISTORY 1928 - Frederick Griffith Streptococcus pneumoniae HISTORY 1944 - Avery, MacLeod and McCarty HISTORY 1952- 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 Utilized in some viral replication methods as well… Rolling Circle Replication

Figure 15.7

3 Figure 6-4 Essential Cell Biology (© Garland Science 2010) Where is the beginning site of DNA replication?

G S 1 (DNA synthesis)

G2 Cytokinesis MITOTICMitosis (M) PHASE Origin of Replication

-Dictated by a specific-sequence motif

Also influenced by chromatin conformation 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… 14 Copyright © 2010 Academic Press Inc. Initial Denaturation E. coli Ori Recap

• Multiple binding sites at OriC • Recruitment of DnaA creates 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

Figure 5-27 Molecular Biology of the Cell (© Garland Science 2008) 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? Prokaryote E. coli has 1 ori or Eukaryote Humans have or approximately both? 30,000 - 50,000

Otherwise 30 days

Figure 5-26 Molecular Biology of the Cell (© Garland Science 2008) 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-phase leads to the degradation of initiator factors until the next round of the cell cycle

While canonical human oris have been hard to elucidate, some appear very similar to the yeast ORC sequence

Figure 5-36 Molecular Biology of the Cell (© Garland Science 2008) 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

Figure 5-14 Molecular Biology of the Cell (© Garland Science 2008) https://youtu.be/d_9VBgrDLUg We know it proceeds in a bi-directional fashion…

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

Figure 5-25 Molecular Biology of the Cell (© Garland Science 2008) Type I DNA topoisomerases

Reversible nucleases that transiently attach themselves to one 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

Figure 5-22 Molecular Biology of the Cell (© Garland Science 2008) Side Note Topo I TA Quick Cloning SSBP - Stabilizing Proteins RPA = replication protein A

Figure 5-16 Molecular Biology of the Cell (© Garland Science 2008) SSBP helps to minimize inhibitory hairpin structures and mutations, and exposes unpaired bases

Figure 5-17 Molecular Biology of the Cell (© Garland Science 2008) Now the DNA template strand is available for complementary synthesis…

How does DNA pol know where to start synthesis? The leading strand only needs 1 primer for synthesis

The lagging strand requires ribonucleotide primers at intervals of 100-200 nucleotides (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?

Figure 5-11 Molecular Biology of the Cell (© Garland Science 2008) Direction of Synthesis 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 Primase

S Associates as a trimer with DnaB (helicase)

S Tends to initiate synthesis at CTGs

S 3 domains S Zinc BD S Helicase BD S RNA polymerase DNA Primase Regulation

Redox 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… 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 5-18b 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

Figure 5-18c Molecular Biology of the Cell (© Garland Science 2008) 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 Note which phosphate resulting pyrophosphate group is incorporated? Figure 5-4 Molecular Biology of the Cell (© Garland Science 2008) Energetically, 3’ to 5’ synthesis will not suffice

Figure 5-10 Molecular Biology of the Cell (© Garland Science 2008) 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 kb Eukaryotic Okazaki = 0.1 - 0.2 kb

Notice that a bubble consists of forks that are inverted mirror images of each other

Figure 6-12 Essential Cell Biology (© Garland Science 2010) The Replication Fork Is Asymmetrical

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

No problem here though Notice the problem of the divergent polymerase movement?

The replisome actually does stay intact… how? Sliding Trombone Model

Figure 5-19a Molecular Biology of the Cell (© Garland Science 2008) https://youtu.be/-mtLXpgjHL0 https://youtu.be/4jtmOZaIvS0 Questions?

Can we map it all out?

Where are we? Primer Removal E. coli model

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 RNA polymerase Pol III falls off and replaced The primer then has to be removed: by Pol I Pol I has 5- 3 exonuclease activity with which it cuts out the primer – as it Pol I does that it fills in the gap with DNA removes RNA primer In eukaryotes, FEN1 removes the and replaces it with DNA primer and new DNA is laid down by Pol d (it created a flap for FEN1)

NOTICE DNA ligase then repairs the gap THIS!!!! Prokaryote DNA Pols

S Pol I S Last pol, it removes previous Okazaki primer S 20 bases/sec, synthesizes the first ~ 400 S Involved in DNA repair as well

S Pol III S Major pol for synthesis, ~1,000 bases/sec

S Pol II S Involved in repair, a back up for pol III Eukaryotic Pols

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 Examples of Eukaryotic DNA Pols Eukaryotic DNA Pols

S Eukaryotic DNA Pols

S We’ve mentioned processivity, which means?

We also need to address fidelity, which is?

How does fidelity relate to 3’- 5’ exonuclease activity?

Limiting Mutations

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 5-8 Molecular Biology of the Cell (© Garland Science 2008) DNA Polymerase is Self-Correcting

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. Figure 6-13 Essential Cell Biology (© Garland Science 2010) 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 – 2017 S 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

Again, untangles inappropriate ds complexes during transcription

Fluoroquinolones inhibit its function in prokaryotes

Note that Type II topos are generally more 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 Player Comparison Associated bits… E. coli DNA Adenine Methylase (DAM)

Nascent strands remain unmethylated for about 10’, why? Figure 5-28 Molecular Biology of the Cell (© Garland Science 2008) 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

DNA methylation status also allows us to selectively digest DNA

http://www.genomics.agilent.com/article.jsp?pageId=388&_requestid=517169 Moment of Reflection

Now you can see why

G1 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 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

• 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 Mutagenesis Directed Evolution

Note that you would need to use a faulty DNA pol in order to create an altered target sequence

This same method was used to create some GFP color variants

www.invitogen.com/site/us/en/home/Products-and-Services/Applications/Cloning/gene-synthesis/directed-evolution.html GFP variants exist for different colors

This agar plate was inoculated with 8 different strains of bacteria, each expressing a different GFP protein variant

http://www.tsienlab.ucsd.edu/Images.htm Rat Brainbow random neuronal expression of GFP variants

http://www.cell.com/cell_picture_show-brainbow2