Today is Monday, January (!) 5 th, 2015 (!) Pre-Class: Name as many enzymes as you can that are involved in DNA replication. Get a small paper towel, too

Today is Monday,January (!) 5th, 2015 (!)

Pre-Class:Name as many enzymes as you can that are involved in DNA

replication.

Get a small paper towel, too.

In This Lesson:Unit 6

DNA Structure and Replication(Lesson 1 of 3)

Today’s Agenda

• Midterm Analysis.• DNA history.• DNA structure.• DNA replication.– Also known as a look into the details of S phase.

• A DNA pickup line.

• Where is this in my book?– Chapter 16.

By the end of this lesson…

• You should be able to describe the structure of DNA in detail.

• You should be able to narrate the replication of a DNA molecule including all enzymes used therein.

• You should be able to describe the cell’s mechanism for detecting and repairing mutations.

Midterm Analysis

• Now that everyone’s done with the midterm, you’re each going to review your own work to see what went right and what may have gone wrong.

• On the accompanying “worksheet,” answer the questions honestly and completely.

• You’ll turn it in today but will get it back when the AP Exam and final are on the horizon.

Let’s Begin at the Beginning

• Challenge Questions!• DNA Base Pairs worksheet.

DNA Worksheet

• Problems 1 and 2 on your DNA worksheet:1: A C1: T G

2: G A A G G C G T T

DNA Worksheet

• Problems 3, 4, and 5 on your worksheet:3: T T G C A A G T C

4:

5. Always three rings (keeps the same width).

DNA Worksheet

• Problems 6 and 7 on your worksheet:6: Sugar/Phosphate Backbone

7: A ↔ T, C ↔ G7: Purines – 2 rings, Pyrimidines – 1 ring

The Historical Perspective

• Who discovered DNA?– You’re probably thinking…Watson and Crick?

• That’s accurate…but only…kind of.• The elucidation of DNA and its structure is a

fantastic study of science at work, and the way “standing on the shoulders of giants” that have come before lets us as humanity leap forward into the bold future of biology.– Preach!


• 1869: Friedrich Miescher– Miescher discovered DNA

while analyzing pus from discarded bandages.

– He named the substance “nuclein” but did not realize he was looking at the origin of evolution.• Must have been like when you

realize you saw a celebrity after he’s gone.

Johann Friedrich Miescher

http://upload.wikimedia.org/wikipedia/commons/b/bc/Friedrich_Miescher.jpg


• 1908: Thomas Hunt Morgan– Remember him?– Morgan worked with Drosophila

and found that genes were located on chromosomes.

– However, Morgan did not know whether it was the DNA or the histone proteins that are the actual genes.

T.H. Morgan


• 1928: Frederick Griffith– Griffith worked with pneumonia-causing

Steptococcus bacteria in an attempt to find a cure.

– He mixed harmless live bacteria with harmful dead (killed by heat) bacteria.

– The once-harmless bacteria somehow took up the harmful “transforming factor” of the harmful bacteria.• Something could be passed on, but it was unknown

what that “transforming factor” was.

– Mice injected with the harmless live bacteria (after mixing) were killed.

Frederick Griffith(in the hat)

The Historical PerspectiveGriffith

• Griffith’s experiment:

Live, Pathogenic

Bacteria

Live, Non-Pathogenic

Bacteria

Killed Pathogenic

Bacteria

Mix of Killed Pathogenic and

Live Non-Pathogenic

Bacteria


• 1944: Oswald Avery, Maclyn McCarty, Colin MacLeod– Refined the results of Griffith’s work by purifying protein and

DNA from Streptococcus and running the same experiment.– Results? Proteins had no effect on mice, but DNA did.

http://media-1.web.britannica.com/eb-media/12/160212-004-5DF19B15.jpg http://www.nlm.nih.gov/visibleproofs/media/detailed/vi_a_204.jpghttp://profiles.nlm.nih.gov/ps/access/CCAACA.jpg

Oswald Avery Maclyn McCarty Colin MacLeod

The Historical Perspective• 1947: Erwin Chargaff– Discovered what is now known as

Chargaff’s Rules. In DNA:• % of Adenine ≈ % of Thymine• % of Cytosine ≈ % of Guanine

Erwin Chargaff

Relative Proportions (%) of Bases in DNA

Organism A T G C

Human 30.9 29.4 19.9

19.8

Chicken 28.8 29.2 20.5

21.5

Grasshopper 29.3 29.3 20.5

20.7

Sea Urchin 32.8 32.1 17.7

17.3

Wheat 27.3 27.1 22.7

22.8

Yeast 31.3 32.9 18.7

17.1

E. coli 24.7 23.6 26.0

25.7


• 1952: Alfred Hershey and Martha Chase– The classic “blender

experiment.”– The two used a type of virus that

infects bacteria (bacteriophage) and created two groups:

– One labeled with 35S in protein.– One labeled with 32P in DNA.• Each of those isotopes is

radioactive and thus detectable.

Chase & Hershey

The Historical PerspectiveHershey-Chase

Bacteriophages are grown in radioactive cultures and tagged. 35S

32P [protein] [DNA]Viruses infect

cells by injecting DNA.

35S (protein) is found

outside the cells.

Viruses infect cells by

injecting DNA.

32P (DNA) is found inside

the cells.


• The Hershey-Chase experiment thus answers the question of what was the “transforming factor” from Griffith’s work.– Since marked protein never made it into the cell, it

couldn’t be the protein in the chromatin.– Since DNA did make it into the cell, that must have been

what allowed the harmless bacteria to transform.• So where’d the blender come in?– The blender was used to agitate the virions (virus

particles) off the cells to allow for study of just the cells’ contents and not the tagged viruses.


The Historical Perspective• 1953: Watson and Crick (and Franklin and

Wilkins)– Rosalind Franklin and Maurice Wilkins used X-

ray crystallography to learn about the structure of DNA.

– The image to the right shows a uniform X-shape, suggesting a helix shape with a consistent width. So where do Watson and Crick come in?Fr

anci

s Cr

ick

Rosa

lind

Fran

klin

Jam

es W

atso

n

Mau

rice

Wilk

ins


• Watson and Crick used the work of Franklin and Wilkins to create models of DNA, eventually figuring out its structure.– They still deserve credit,

but Wilkins and Franklin deserve just as much.

Da Structurez

• So what comes out of all that work?• The classic DNA structure: a double helix.– Meaning it looks like that spiral staircase in Gattaca.

Coincidence?• I think not.

http://media-cache-ec0.pinimg.com/736x/1a/02/bc/1a02bc5d5428bbdc64e793f61bf2b809.jpg

DNA Structure Review

• DNA is a nucleic acid, a long string of nucleotides.

• DNA takes the shape of a double-helix.

• There are four kinds of nucleotides:– Adenine– Cytosine– Guanine– Thymine

http://ghr.nlm.nih.gov/handbook/illustrations/dnastructure.jpg

Nucleotide Structure Review

• Each nucleotide has a:– Sugar molecule with 5-carbons (pentose)

• Deoxyribose in DNA• Ribose in RNA

– Phosphate group• Phosphorous-based molecule

– Nitrogenous base (makes the nucleotide unique)• Adenine• Thymine• Cytosine• Guanine


http://www.biologyjunction.com/images/nucleotide1.jpg

AdenineThymineCytosineGuanine

SKETCH ME! I’m useful!SKETCH ME! I’m useful!


• More “scientific”

Nucleotides and Nucleosides

• Just so you know, you’ll occasionally hear of a nucleoside.

• The only difference between a nucleoside and a nucleotide is that a nucleoside is just a sugar and nitrogenous base – no phosphate group.

DNA Structure Review• Surrounding the base pairs and

forming the sides of the “ladder” is a sugar-phosphate backbone.

• The backbone is made of a sugar (deoxyribose) and a phosphate group, alternating and in reverse order from the other strand.– Backbone is linked by

phosphodiester bonds.– The end of DNA with the phosphate

on top is the 5’ (“five prime”) end.– The other end of the backbone is

the 3’ (“three prime”) end.


3’ and 5’? Huh?• 3’ and 5’ get their names from the

pentose sugar’s carbon atoms.• Each carbon in pentose is

numbered and has a specific job in the formation of DNA.– Carbon 1 = base attachment– Carbon 2 = oxygen (ribose) or not

(deoxyribose)?– Carbon 3 = another nucleotide

attachment– Carbon 4 = completes ring– Carbon 5 = phosphate attachment

• This is important.http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/P/Pentose.gif http://www.synapses.co.uk/genetics/pentose1.gif

DNA Structure Review

• DNA stands for Deoxyribonucleic Acid.

• By hydrogen bonds, cytosine bonds to guanine and adenine to thymine.

• A ↔ T• C ↔ G


One more time, because “important.”

Oxygen, not a zero.

DNA Unwound

Deoxy-ribose

P

Thymine

Deoxy-ribose

P

Cytosine

---H---

---H---

---H---

5’

5’

3’

3’

Thymine

Deoxy-ribose

P

Guanine

Adenine Deoxy-ribose

P

Deoxy-ribose

P

Deoxy-ribose

Adenine

PD Bond

PD Bond

PD Bond

PD Bond

Antiparallel Strands

• As seen in the image to the right, the two strands of DNA run antiparallel to one another.– One is “upside down.”

• At the 5’ end of each DNA strand there is a phosphate group.

• At the 3’ end of each DNA strand there is a hydroxyl (-OH) group.

Bonding in DNA

• The two complementary strands of DNA are linked by hydrogen bonds.– Base to base.

• Each nucleotide in a sugar-phosphate backbone is linked by a phosphodiester bond.– Phosphate group to 3’ C.

Phosphodiester Bond Hydrogen Bond

Linking Nucleotides

• Phosphodiester bonds, linking nucleotides, are formed…how?– By dehydration

synthesis, of course!

– More on this later.

Purines and Pyrimidines

• Adenine and guanine are purines and have a double-ring structure.

• Cytosine and thymine are pyrimidines and have a single-ring structure.– A purine always bonds to a

pyrimidine.– This ensures that the width of the

double helix is constant.• How can we remember this

one?

DNA Replication

• There comes a time in (almost) every DNA molecule’s life when it needs to be replicated (copied).– That time would be S phase.

• Here’s the general process:– Unwind the double-helix.– Break the hydrogen bonds (“unzip” the DNA).– Use enzymes to replace base pairs on each side.

Replication

---H---

---H---

---H---Deoxy-ribose

P

Thymine

Deoxy-ribose

P

Cytosine

Deoxy-ribose

Adenine

Thymine

Deoxy-ribose

P

Guanine

Adenine Deoxy-ribose

P

Deoxy-ribose

P

DNA Helicase breaks H-bondsStrands move apart

P

Deoxy-riboseThymine

Deoxy-riboseGuanine

P

Deoxy-riboseAdenine

P

Deoxy-ribose

P

Cytosine

Deoxy-ribose

Adenine

Deoxy-ribose

P

Thymine

P

DNA Polymerase makes new DNA

H H

H H

H H

Looks like this… [IMPORTANT]

Note that even though there

are two strands

forming down here, each is only “half”

new.

The “old” strand is

sometimes known as the

template strand

because it’s a model for the

new one.

Looks like this… [IMPORTANT]

http://online.santarosa.edu/homepage/cgalt/BIO10-Stuff/Ch10-Protein_Synthesis/DNA-Replication-Animation.gif

KindaPOGIL

• We’re going to do a segment of a POGIL now.• DNA Structure and Replication [Model 2 only]


• One more experiment, but it’s a good one.– It’s actually known as “The Most Beautiful Experiment in

Biology.”• In 1958, Meselson & Stahl set out to determine how DNA is

copied. – Conservative

• “Parent” DNA exists in whole after copying.

– Semi-conservative• “Parent” DNA is divided into two whole strands – one in each of the

daughter molecules.

– Dispersive• “Parent” DNA is fragmented among the two daughter molecules.

• Which one do we now know to be correct?

DNA Replication Models

• Compare the models:

The Historical Perspective• So how did Meselson and Stahl pull

this one off?– They started by “labeling” nucleotides in

the parent bacterial DNA with 15N (“heavy nitrogen”).

– New nucleotides were labeled with 14N.

http://www.g2conline.info/content/c16/16448/16448_stahloffice.jpg http://library.cshl.edu/static/oh4/images_still/MatthewMeselson.png

Franklin Stahl

Matthew Meselson

The Historical Perspective• After one replication, they found that the density of

the nucleotides was between that of 15N and 14N.– So it can’t be conservative – that would have two

separate densities (15N and 14N).

The Historical Perspective• After two replications, they found that the density of the

nucleotides was between that of 15N and 14N OR equal to that of 14N.– So it can’t be dispersive – that would have one density slightly higher

than the first replication.

Back to Replication…

• So how would you guess DNA replication is actually achieved?– As in, we’re talking about molecules here. How is

the molecular “work” done?• Yep, enzymes. The repeat offenders of

“getting stuff done.”• Can you remember any enzymes involved in

replication?

Replication Enzymes

• Helicase• DNA Polymerase III (abbreviated pol III)• DNA Polymerase I (abbreviated pol I)• Ligase• Primase

• Technically, more than a dozen enzymes participate in replication.– Many are smaller enzymes complexed into larger ones.

• Also, the polymerases listed above were identified in prokaryotes.– The eukaryotic enzymes are very similar and discoveries are still being

made.

Enzymes and Energy

• Awesome. Enzymes. So?– No, really, just because you have enzymes doesn’t

mean you can magically build a giant DNA molecule.• And DNA is giant. Even though it’s only about two nm

wide, there is six feet of it in every cell!

– Something like DNA is highly endergonic and is quite unlikely to happen completely spontaneously.

• So from where is our energy coming?– Well, from where does it usually come?

ATP, GTP, CTP, and TTP

• Yep, you read that right.• DNA polymerization uses ATP, along with its close relatives GTP,

CTP, and TTP – each corresponding to a different letter.– In other words, the energy is packed with the raw materials.

• DNA bases arrive as nucleosides (nucleotides without the single phosphate), and in fact have three phosphate groups attached.– We call them nucleoside triphosphates:

Aden

osin

e Tr

ipho

spha

te

Gua

nosi

ne

Trip

hosp

hate

Thym

idin

e Tr

ipho

spha

te

Cytid

ine

Trip

hosp

hate

Formation of Phosphodiester BondsDeoxy-ribose

P

Thymine

Deoxy-ribose

P

Adenine

P

P

Deoxy-ribose

P

Guanine

P

P

Dehydration Synthesis


DNA Replication

• So we’ve met the “characters” (enzymes) and the “props” (ATP, GTP, CTP, TTP).

• Now let’s watch the play…– Heads-up: I’m going to summarize the whole

process in one big note-worthy slide first, then we’ll look into the details of the process.

– It might be a good idea to write this with space in between. You’ll need no more than 1/3 of a page unless you have ginormous handwriting.

– Watch the headings to orient yourself.

DNA Replication ProcessSummary Slide [enzymes underlined]

1. DNA helicase unwinds the double helix by breaking hydrogen bonds between nitrogenous bases.– Single-strand binding proteins prevent re-coiling.– Topoisomerase relieves physical strain in the coiled part

of the strand.

2. Primase lays down an RNA nucleotide primer.3. Pol III adds DNA nucleotide bases from 5’ to 3’.4. Pol I replaces RNA primers with DNA nucleotides.5. Ligase joins disconnected fragments of DNA.

Step 1: Unwind the DNA• [DNA pickup line]• For protection, the DNA

molecule is highly coiled.– A consequence, however, is

that it also can’t be copied – enzymes cannot access it.

• DNA helicase uncoils the helix and creates two replication forks (uncoiling spots).– Sometimes called a

“replication bubble.”http://stream1.gifsoup.com/view3/1504259/dna-helicase-o.gif

Step 1: Unwind the DNA

• Remember that we’re dealing with unthinking molecules, though. How does the DNA molecule not just re-coil?– Single-strand binding

proteins attach themselves to the nucleotides to prevent them from coming back together.

https://dr282zn36sxxg.cloudfront.net/datastreams/f-d%3A85df246e2fbc147adc76f893a37156af339c95c1a0f63df3456a110b%2BIMAGE%2BIMAGE.1 AND ALL FOLLOWING

Step 1: Unwind the DNA

• Of course, untwisting one section of the DNA will add strain to the still-coiled section.

• Topoisomerase prevents damage to the “upstream” part of the strand.

Step 2: Add RNA Primers

• DNA polymerase has a major limitation:– It needs a 3’ carbon to serve as a foundation

for the placement of the 5’ end.• It’s really that little hydroxyl group that it needs

to “plug into.”

• So even though the parent DNA molecule is ready to go, there’s no way for pol III to start adding nucleotides.

• Luckily, there’s another enzyme out there that can get things started: primase.

3’

5’

5’

3’

Step 2: Add RNA Primers

• Primase attaches to the DNA molecule and adds a short stretch of RNA to the template parent strand (5-10 nucleotides).

• These primers provide the 3’ carbon for pol III.

Step 3: Pol III Polymerization

• Pol III, which is made of a bunch of subunits, starts at the primer and adds DNA nucleotides, moving from 5’ to 3’.

Step 3: Pol III Polymerization

• Wait…what about the 3’ to 5’ direction?– As in, how does pol III polymerize the

complementary strand?• Pol III does so in short stretches.

• Let’s look at this problem with a conceptual diagram.

Why Only 5’ to 3’?Deoxy-ribose

P

Thymine

Deoxy-ribose

P

Adenine

P

P

Deoxy-ribose

P

Guanine

P

P


http://a.fastcompany.net/multisite_files/fastcompany/imagecache/inline-large/inline/2013/11/3021307-inline-fb-thumbsup-printpackaging.jpg

Deoxy-ribose

P

Cytosine

Deoxy-ribose

P

Guanine

P

P

No Dehydration Synthesis! Dehydration Synthesis

5’

3’

5’

3’

Linking Nucleotides

• Remember this?

5’ to 3’ Confusion Point• Look at the DNA molecule below.• To some of you, DNA polymerase may appear to be

running backward.– If so, it’s because you’re looking at the template strand…– …not the daughter strand.• Key: Always look at the daughter strand.• Pol III reads 3’ to 5’ but writes 5’ to 3’.

More on Replication

3’

5’

5’

3’

Replication Fork

Replication Fork

Note: The following slides concerning replication will feature close-ups of different regions of the above molecule as it is

replicated, unless otherwise noted. That’s important to know.

DNA Replication: Leading Strand

Pol III

For the leading strand, primase lays down an RNA

primer (5’ to 3’).Pol III adds DNA

nucleotides (5’ to 3’).

3’

5’

3’

5’

5’

Primase

3’

DNA Replication: Leading Strand

Pol III

As the replication fork moves toward the 3’ end,

Pol III adds more nucleotides continuously.

3’

5’

3’

5’

5’

3’

DNA Replication: Lagging Strand

Pol III

For the lagging strand, primase lays down an RNA

primer (5’ to 3’).Pol III adds DNA

nucleotides (5’ to 3’).

3’

5’

3’

5’

3’

5’

Primase

DNA Replication: Lagging Strand

As the replication fork moves toward the 5’ end of the daughter strand, a

new primer is needed.

Primase Pol IIIWe have now formed two Okazaki fragments.

3’

5’

3’

5’5’

3’

DNA Replication:Leading and Lagging Strands

3’

5’

5’

3’

5’3’

3’5’

Leading Strand

Lagging Strand

Leading Strand

Lagging Strand

Leading vs. Lagging Notes

• The leading strand is the one polymerized continuously from 5’ to 3’ in the direction of helicase’s movement.

• The lagging strand is the one polymerized in sections.– In the opposite direction of helicase’s movement.– The sections are called Okazaki fragments.

• The lagging strand is still polymerized at the same speed but takes slightly longer to finish (more on that soon).– It’s also still polymerized 5’ to 3’, just not continuously.

Reiji Okazaki

Step 4: Pol I Replaces Primers

• Pol I moves in and changes all the RNA primers to segments of DNA.

3’

5’

3’

5’

3’

5’

Pol I

Pol I

Pol I

Step 5: Ligase Polymerization• Ligase joins Okazaki fragments to seal

any gaps in the DNA.

3’

3’

5’

3’

5’ Ligase

Aside: Ligase Mechanism

• In case you’re wondering, ligase works by, essentially, this mechanism:– Ligase brings over AMP (adenosine monophosphate) attached to

an amino acid (lysine).– AMP is attached to the 5’ phosphate of the “upstream” nucleotide.– The addition of AMP causes the OH- (hydroxyl) group on the next

“downstream” nucleotide to bond with a phosphate in the upstream nucleotide.

– The adenosine molecule is then released, leaving a bond between the 3’ OH- of one nucleotide and the phosphate of another.• In short, ligase “plays matchmaker” by adding an adenine nucleoside

temporarily. It then is released as the two previously separate nucleotides decide they’re going to bond after all.

So once again, that’s…

1. DNA helicase unwinds the double helix by breaking hydrogen bonds between nitrogenous bases.– Single-strand binding proteins prevent re-coiling.– Topoisomerase relieves physical strain in the coiled part

of the strand.

2. Primase lays down an RNA nucleotide primer.3. Pol III adds DNA nucleotide bases from 3’ to 5’.4. Pol I replaces RNA primers with DNA nucleotides.5. Ligase joins disconnected fragments of DNA.

Or, in one image…

https://dr282zn36sxxg.cloudfront.net/datastreams/f-d%3A85df246e2fbc147adc76f893a37156af339c95c1a0f63df3456a110b%2BIMAGE%2BIMAGE.1

More limitations…

• The other problem occurs at the ends of a chromosome.

• Remember how pol III and pol I need 3’ carbons off of which to build?

• Yeah…

Replication at Chromosomal Ends

3’

5’

5’

3’

5’ 3’

3’ 5’

End of DNA MoleculePrimasePol III

The lagging strand has no noticeable issue.

The leading strand cannot fully replicate.

PrimasePol III Pol I

There is no 3’ end of an existing nucleotide for pol I to use.

Pol I

The Consequences• Since pol I can’t replace the RNA primer, DNA

molecules develop staggered ends.• This is a normal part of replication and it occurs at

the telomeres. Remember those?– Telomeres are the “junk” end caps of a chromosome.• They’re like the aglets (nubs) on shoelaces that prevent

unraveling.• Interestingly, they have 100 to 1000 repeat sequences of

TTAGGG. This will come into play for telomerase soon.

5’ 3’3’ 5’

Staggered End

The Consequences

• With each replication, the telomeres shorten, which is thought to be related to aging.– Each division eliminates between 30 and 200 base pairs.– The number of cell divisions that can occur before cell

cycle arrest is around 60 or so – this is known as the Hayflick limit.

• White blood cells in newborns have 8000 base pairs.

• In adults, they average 3000 base pairs.• In the elderly, there are only 1500 remaining.

http://learn.genetics.utah.edu/content/chromosomes/telomeres/

Undoing Staggered Ends

• Normally, DNA with staggered ends will trigger a checkpoint to stop mitosis.

• However, staggered ends at a telomere is normal, so proteins at the telomeres inhibit that kind of response.

• But what about cells like sperm and ova?– Once it forms, it has a whole lot of divisions ahead of it

in life.– Wouldn’t the telomeres degrade before they’re “done?”• The answer is “no,” thanks to an enzyme called telomerase.

Telomerase

• Telomerase rebuilds telomeres to prevent shortening.– It extends the 3’ end so that the usual process can

extend the other side using an RNA template of AAUCCC.• More on the next slide.

• Are you thinking what I’m thinking?– “If we force telomerase to become active all the

time, can we prevent aging?”– Well…maybe.

• More in three slides.

Action of Telomerase

5’3’

3’5’

TelomerasePrimasePol III

The telomere is shortened by a round of replication.

Telomerase rebuilds the lost nucleotides.

Shortened telomere terminal

Original telomere terminalRebuilt

Action of Telomerase

• Telomere Replication video

Telomerase and Immortality• Telomerase concentrations “in the real world” are highest in

cancer cells, which use the enzyme to prevent the telomeres from shortening to the point that the cell can’t divide anymore.– Pancreatic, bone, prostate, bladder, lung, and kidney cancers all

feature shortened telomeres.• In lab settings, however, telomerase has been used to keep

cells dividing continuously without causing cancer.• Further, telomeres are not the only influence on aging, since

mice have giant telomeres compared to much longer-lived humans– Let’s hear more from the voice of Richard Cawthon at the University

of Utah.http://learn.genetics.utah.edu/content/chromosomes/telomeres/

Aside: Dyskeratosis

• Dyskeratosis is a disease that causes premature telomere shortening.– The result? People with the disease age and die much

more quickly than those without the disease.– Other symptoms:• Early hair graying and balding.• High leukemia risk.• High cirrhosis risk.• Learning disabilities.• Bone softening.

http://learn.genetics.utah.edu/content/chromosomes/telomeres/

DNA Can’t Spell

• Okay, that’s a little harsh. DNA actually spells pretty well most of the time, but every once in a while it puts a letter or two in that shouldn’t be there.

• As you might guess, evolution also brings us a fair degree of typo recognition and error correction.– Naturally, some slip through even these layers, but

at least it’s something.• As you also might guess, it’s enzyme-driven.

Polymerization Speed

• Pol III runs at about 1000 bases per second as the main DNA builder.

• Pol I runs at about 20 bases per second.• Why the speed difference?– Pol I is actually a proofreader, too!

• We know Pol I removes primers, but it also does some error correction (known as mismatch repair).– The error rate without pol I is 1 in every 10,000 bases.– The error rate with pol I is 1 in every 100,000,000 bases.

How it works…

• Another enzyme, called a nuclease, literally cuts the erroneous nucleotides out.

• Pol I then replaces the DNA with appropriate nucleotides.

• Ligase, as usual, steps in to seal up the strand.

• Fun fact: Pol II appears to be involved in error checking in prokaryotes.

Mismatch Repair/Action of Nuclease

5’3’

3’5’

Nuclease

NucleasePol ILigase

Damaged DNA is excised by nuclease.

Pol I and ligase proceed as normal.

Remember this?

http://skreened.com/scienceforscientists/dna-checks-itself-before-it-wrecks-itself

Closure

• So…DNA.• It’s the stuff of life, right?• Worth another 80+ slides, right?• Why?• What does DNA do for you?

Closure

• DNA can also be used in an “outside the body sense.”– I mean like in a “forensics” sort of way.

• Especially since a lot of DNA in forensics is outside the body.

• Still other uses come from forensics of other creatures.• No, not “it was the giraffe in the study with the lead pipe.”

– I mean like in a “clues in DNA that tell us about the evolutionary relationships between organisms.”

• Why don’t I just stop talking so we can start Lab 3?

Closure Part Deux

• DNA Replication Fork 1 animation• DNA Replication Fork 2 animation

Closure Part Three

• Turn to your neighbor, say hello, and take turns narrating the steps of DNA replication.

• For example, suppose you and your partner are talking:– James Westfall: “Hello.”– Dr. Kenneth Noisewater: “Hey there.”– James Westfall: “So, DNA replication starts with

helicase.”– Dr. Kenneth Noisewater: “Helicase works by…”

Documents

Today is Monday, January (!) 5 th, 2015 (!) Pre-Class: Name as many enzymes as you can that are involved in DNA replication. Get a small paper towel, too