Biochemistry Review: Notebook #1.
January 28th, 2002.
Dr. Julin
Course Goal: Central Dogma (Def. Dogma: a central idea with no supporting data available at the time.)
1958: Francis Crick
The Flow of Biological Information
Dna->Dna: Replication
Dna->Rna: Transcription
Rna->Protein: Translation
Currently, information also was seen to flow from RNA->Dna (retrovirus) Rna->Rna (plant virus). However, information has not been observed to travel from proteins to dna, or from proteins to Rna.

Rna and Dna Structure and Chemistry. Molecular details are necessary.
What are the Mechanisms. The Enzymes?
Four houses of knowledge in all.
1953: Watson and Crick propose the structure of DNA.
Prior work:
1860s: Frederick Meesher discovers an acidic substance in the cell nucleus. Dubs it “Nucleic Acid.”
Late 1800s-early 1900s: Organic Chemists are analyzing biological chemicals. The basic structures of the monomer units were determined (Polymer chemistry had not been developed at the time).
1919: The “Tetranucleotide” structure is proposed. Known: there were four bases: AGCT. Does it come in groups of 4? A small and monotonous view of nucleic acids.
At the time, the focus was on Proteins.

Meanwhile: Genetics was developing. Fruit Fly experiments yielded Inheritance rules.
1940’s-50’s. A Critical Experiment. Ostwald Avery conducts an experiment in Bacterial Transformation. Taking Streptococcus bacteria, he mixed live, nonvirulent ones with dead (virulent) bacteria. The result was that some of the live bacteria became virulent.
Avery grew virulent bacteria, then broke open the cells. He attempted to isolate the “Transforming Principle” or active component.

The purified element was found to be DNA!
This result was very interesting, as it suggested that DNA carries genetic information.

Skeptic: Were there impurities in the DNA? How could this be possible if the DNA were monotonous (wrong assumption)?

1952: Hershey-Chase results supported the same conclusion via a completely different approach.

DNA is important.
Erwin Chargaff isolated and identified DNA from many organisms, and determined the %A, %G, %C, and %T. He found a few interesting things:
1. The percentages varied between different organisms. They were not near 25% each, either.
2. Isolating DNA several times from different tissues or at different stages of growth of the same organism yielded the same percentage results.
3. Always, the %A=%T and the %G=%C.
The third result is called Chargaff’s Rule.

Day 2: Structures of the DNA.
T in DNA is U in RNA.
adenosine-5’-monophosphate “AMP”
hydrolysis yields phosphoric acid, twice. This has a LARGE free energy change, and is used by the cell as a convenient method of energy storage.
Lec.2
ketone-enol tautomerization can occur to the bases in DNA. this takes place whenthe hydrogen attached to the adjacent keto carbon moves to the ketone oxygen O=, then the double bond moves between the two carbons.this can happen to Guanine. Initially Watson and Crick had the wrong form for Guanine, which is why they were having trouble matching it to the other chemical, cytosine.
Structure of the Polymer.
The 5’ CH2OH becomes an ester and attaches to the 3’ Phosphate group. “Phosphodiester linkage”
RNA and DNA degrade slowly, but faster at alkaline pH, because the phosphodiester linkages get hydrolysed.
Maurice Wilkins and R. Franklin used x-ray crystallography (Fiber Diffraction) to determine that natural DNA had a helical structure.
1980s 6-8bps of DNA were synthesised and tested in the lab via crystallography.
Any sequence of bases produced the same 3d structure.
Information in the molecule was carried by the base on one strand specifying the base on the other strand.
The structure of natural DNA is usually B-Form DNA.
B-DNA is a double helix, antiparallel strands, right handed. (align your fingers in the upward spiraling direction direction. If your thumb points up, it’s right handed.)
There are 10 base pairs per helical turn, a 36 degree turn per base pair.
The base pair planes are perpendicular to the helix axis.
There is a minor groove and a major groove. Most proteins approach on the major groove, because this side has more room to access the bases.
Class 3
Structural Variability in polynucleotides.
There are multiple forms of DNA.
Although B-form DNA is the most common form, there is another form “A-form DNA.” This pattern appears in “low humidity” conditions (dry). in (wet) conditions, B form DNA is predominant.
1. Conditions
2. Sequence
3. Chemical Properties.

Sugar Pucker: An organic chemistry property of sugars; a steric phenomenom in 5-member rings.
when the sugar is exactly in sp3 hybridization, then an eclipsing interaction occurs between the adjacent carbons. This can be avoided by stretching the bond a little. this distortion is called sugar pucker.
In DNA, the 2’ and 3’ are the most important carbons.
if c2’ is moved up relative to c5’, then the B-DNA structure is formed. (THIS CLASS)
c3’ up results in A-form DNA.
the A-type DNA has 11 base pairs per turn, and the helix pitch is 28 angstroms. A-form helices like to form in double-stranded RNA.
Proteins actually bind in the minor groove in A-form DNA. It is unclear if A-form DNA is used in cells.

Z-form DNA is left-handed, and has a different arrangement. This form of DNA was first seen in crystal form. The sequence crystalized was d(CGCGCG) in 1980. The nice feature of this sequence is that two of them could form identical DNA fragments. When crystallized, it was a big surprise when the researchers discovered the form of the molecule.
In A and B-DNA, all the bases are in the anti form.
In Z-dna, all the Cs are in anti (with the base to the side of the sugar), but the Gs are in the syn (the base is “above” the sugar) configuration.

Take a single polynucleotide chain:
It is possible for different structures to form depending on the sequence. If there is a palindromic complement sequence, for example, a “Hairpin” structure can appear.

The stability of the hairpin depends on the number and type of base pairs present in the stem and the size of the loop.
the best loop has 5 to 7 base pairs.
Less than three is too strained, and more than seven makes the chain unstable (and it difficult for the bases to find each other.
dsDNA: an “inverted repeat” or “palindrome” sequence can cause a “cruciform” shape to appear once the DNA is heated. This shape is less stable, but entropy makes it happen sometimes. Several base pairings were lost in the cruciform shape, so it has more energy in it than the normal base paired sequence.

In supercoiled DNA, however, the cruciform sequence becomes energetically favorable as well.

There are a few other unusual structures that may occur. “Hoogstein” base pairs can make a triple-helix, when a normal dA/DT helix gains an additional strand of (dT). This form is called “Triplex DNA” or H-DNA. It’s favored at low pH, ‘cause the C gets protonated. It is unknown if this has a biological function.

If poly-G is taken at high concentrations, it can form the “G tetraplex,” a four-stranded segment of DNA. This might occur in telemeres, which are G-rich sequences of DNA on the ends of the linear Eukaryotic chromasomes.

Lecture 4, Feb 6, 2003.
RNA is always single stranded in the cell. It has a secondary structure, which is the pattern of hydrogen bonds that base-pair inside the same molecule. It can form helical structures (mostly A-form RNA).
the Tertiary structure is how it arranges itself in three dimensions. This would include “bulges,” “bulge loops,” and “hairpins.”
Also note that sometimes a G=U base pair appears in RNA

DNA denaturation
dsDNA->(denaturation)->ssDNA
ssDNA->(renaturation)->dsDNA
There is a chemical equilibrium.
Heating will denature the DNA, because thermal energy overcomes the hydrogen bonding and stacking interactions.
Stacking interactions are van-der-waals bonding between the adjacent base pairs (above and below). DNA “melting” is observed between 70 and 100 degrees. Melting is measured by uv absorbance. dsDNA absorbs less at 260nm than ssDNA.
the “Hyperchromic” effect.

Temperature and pH are ways to manipulate DNA structure. For example, at pH=10, G and T are deprotonated, and hydrogen bond formation is completely lost.

What other condtions?
At Tmelting, [dsDNA]=[ssDNA]/2.
When concentration is increased, Tm increased.
Ionic substances stabilize the doublestranded form of the DNA
Typically, DNA is used in a buffered salt solution
the metal cations counteract the bad charge interactions between the phosphate backbones of the DNA.
A low salt concentration results in a low Tm (<50mM), while a high salt concentration (100-1000mM .1-1M) is good for the dsDNA.
with PCR it’s important to check your salt concentration.
the size of the dna also plays a factor.
base composition. If there is more G=C bonding, the Tm is larger.
Stacking interactions are also important for the stabilization of the sequence.
there is a table of Tms for each different sequence.

Lecture 5, Feb 11, 2003.
There is a program online that finds Tms.
Supercoiled DNA was first discovered when purifying virus DNA.
“Plasmid” analysis was later.
Electron Microscopy revealed that some of the DNA was “relaxed” but some was super-compacted into “supercoils,” a formation made of a coil of coils of the DNA.
Supercoiling occurs at places where the helix axis crosses itself. (Writhe)
Writhe is an individual crossing-point. Twist is native to the coiled DNA; whenever the two strands reach the same orientation, that is one “twist.”
The preferred conformations are the one with the least energy.
Topology:
Mathematics dealing with the shapes of objects. DNA is a knot.
Take a circular closed dsDNA. This is a Knot consisting of two loops that are twisted. (a topological linking of the circular strands).
If there is no writhe, the Linking Number for the loop will be equal to the Twist of the DNA.
the Linking Number may not be changed without cutting the DNA or treating it with some enzymes that cut it momentarily.
Twist may be converted into Writhe.
B-DNA has a positive Twist (right handed twist).
Writhe has handedness too. If the top strand goes to the right, the Plectonemic writhe is Negative. So, Writhe is the opposite of Twist.
Note: there are two kinds of Writhe. Plectonemic (which is like the buzzing toy writhe), and Solenoidal (which is like those coil wrapped loops).
In this class, take Tw+Wr=Lk as a given dogma.

B-DNA has about 10 basepairs per turn (10.5, experimentally). This means that 2000 basepairs will have 200 Twists by the 10bp/Tw rule. This DNA is fully relaxed. If its ends were joined to form a circle, (one with NO writhe), then it would have a planar shape.
Lk0 is a special linking number. It is formed when there is no writhe.
Ok! Distort the circle! Grab the two strands and seperate them so that two of the Twists disappear (21 bp become linear). Then +2 Solenoidal Writhe will appear, (-2 Plectonemic Writhe).
This situation occurs when RNA polymerase binds to a promoter region on the DNA.
Lk can’t change, but Tw was reduced (and Wr was increased).
If the molecule were completely untwisted, then the ends joined, the ring will develop Writhe so that the Tw=200, its favored value. This will cause the Writhe to be -200. (or 200 Right-Handed Plectonemic Writhe.)
Lecture 6: DNA supercoiling (ill).
An old exam is online at this point.
isolated E.Coli plasmid DNA, SV40 virus DNA... both have negative supercoiling. Why?
Negative supercoiling has a Lk<Lk0. This means that if the DNA were relaxed, there wouldn’t be enough twist in it to satisfy the 1 twist/10.5 base pairs rule.
Let’s take a DNA, 2100bp. Lk=Tw+Wr.
How many Twist would it like? 2100/10.5=200 Twist.
But it only had 190 Twist in the unwound form, what would happen?
The DNA would develop -10 Writhe (10 RH Plectonemic writhe, or 10 LH Solenoidal Writhe), then add that 10 onto the Twist to get the proper Twist (and the same Lk as before).
So, by observing Writhe, one may check the twist of a DNA molecule.
The relaxed DNA doesn’t have as much energy as one that is undertwisted, but it’s better to have Writhe than Twist, (until you get lots of writhe).
For a small molecule, changing the linking number costs a lot of energy compared to that of a large molecule.
Cruciform DNA may become favorable in the case of highly unwound DNA, since it gets base pair interactions back without changing the Twist number.
Topoisomerases are enzymes that can change the linking number of the DNA.

There are two kinds of Topoisomerases
Type I (one strand dsDNA, Delta Link=1)
1. Enzyme binds the DNA
2.Enzyme breaks one strand
3.Enzyme passes the unbroken strand through the gap.
4.Enzyme reseals the break.
Type II (two strands dsDNA, Delta Link=2)
1. Enzyme binds the DNA.
2. Enzyme breaks a dsDNA (on both strands).
3. Enzyme passes the other part of the dsDNA through.
4. Enzyme seals the dsDNA.
Two questions remain:
How can Topoisomerase temporarily break DNA?
How can DNA become negatively supercoiled?
A single molecule experiment.twist some DNA with a magnet, and observe the position of the bead. Carlos Bustamante.
Dna Gyrase introduces negative supercoils into DNA. It’s a bacterial protein/enzyme. Why? Supercoiled DNA is smaller. Supercoiled DNA (negatively supercoiled) is easier to unwind.
How does Topoisomerase work?
Type 1: Tyrosine in the active site of the protein attacks the phosphodiester bond in a substitution reaction, displacing the other side. Then, the bond is reformed, displacing the tyrosine.
DNA replication.

How is the dsDNA (Type II) topoisomerase to work? It does the tyrosine break in two different places close to each other. Then the few hydrogen-bonding base pairs between the two sides of the dsDNA are easy to seperate temporarily, and they even like to snap back together afterwards.

The E. Coli cell.
Semi-conservative replication was discovered. The Meselson-Stahl experiment used density labeling to determine that the original two strands of DNA making the original dsDNA remained intact, although new strands were attached to each of them.
DNA sampling and the ultracentrafuge.

Biochemistry: Tuesday, Feb 24,2003.
Biochemical studies of DNA replication:
in vitro and in vivo
Arthur Kornberg purified DNA polymerase I.Pol II, IV, and V are used in DNA repair. Eukaryotes have at least 10 DNA polymerases.
Polymerization in Bacteria.
DNA Pol I is a monomeric protein of 103,000 Daltons. It has three enzyme activities.
1. DNA synthesis
2. Nuclease: 3’-5’ exonuclease (used for proofreading; cuts the DNA backwards from the direction it was synthesizing.)
3. Nuclease: 5’-3’ exonuclease (used for Nick Translation function in DNA repair.)

Function:
The DNA Pol 1 binds the dsDNA, then a dNTP. if it is the right one, then it will attach it to the DNA, freeing 33kJ/mol of energy as inorganic phosphate is released.
DNA Polymerase One has some processivity (the opposite of Distributive, which binds, acts once, then dissociates). Low to Moderate processivity allows it to catalyse three to two hundred reactions on average before dissociating. the exact figure depends on reaction conditions.

Nucleases:
Exonuclease I digests single-stranded DNA. It will destroy DNA longer than 10 base pairs, releasing deoxy-nucleotide-mono-phosphates (dNMPs)
If/when Pol 1 attaches the wrong nucleotide, it becomes confused and only reacts the next part very slowly. This gives it a good chance to use it’s 3’->5’ exonuclease activity to remove the mismatched base pair.
Its 5’->3’ exonuclease activity (present ONLY in DNA Polymerase 1) allows it to process dsDNA for nick translation.
Nick translation can replace RNA primers.
Note: the part of the protein responsible for nick translation is seperate from the main body of the enzyme. It is easily cleaved off with a protease (Trypsin), leaving a large fragment behind (consisting of the first two parts): the “Klenow Fragment” which can do DNA synthesis on its own. Hint: to make a complex enzyme, attach two simple enzymes to each other with a link of Amino Acids.

Finally, DNA Ligase is the protein that fixes nicks in DNA.

DNA Polymerase III is the most important DNA polymerase. It is composed of many parts. The Enzyme CORE is made of an alpha subunit (synthesis), an epsilon subunit (Proofreading exonuclease function), and a theta subunit (function unknown).
There are three parts to the enzyme. The CORE, the Clamp Loader, and the Clamp (beta clamp). The enzyme has two cores, two tau subunits (stick the cores together), one clamp loader, and two clamps in its final form.
One of the experiments mixed circular DNA with a core, the beta-complex, and the gamma complex.
I already know this.

-- Next section:
Fidelity:
there is one error in every 10^10 base pairs replicated. This is impressive.
there are three steps in avoiding errors.
1. DNA polymerase “selects” the right dNTP often.
2. the Proofreading function
3. Mismatch repair (occuring after replication)
Base selection errors occur once every ten thousand base pairs processed.
The enzyme can correct 99.9% of these errors with proofreading.
The mismatch repair takes care of another 99% of the errors that slipped through.
10^5*10^3*10^2 gives an error of once in 10^10.

In Vitro Replication:
there were different DNA mutants. The bacteria were screened so that their enzymes were nonfunctional above 42oC.
Many TSmutants are around today.
There are “Quick Stop” and “Slow Stop” mutants. Quick stop mutants stop dividing instantly when the temperature is raised. Slow stop take a little while before they stop replicating.
This was used to identify different enzymes.

Plasmids:
These are small circular pieces of DNA. Plasmids evolved as a portable way to transfer information between bacterial cells. Plasmids contain information on replicating themselves, and sometimes on making other proteins that can help a bacteria. The best plasmids carry proteins that make their hosts invincible against different kinds of poisons. This allows the creation of diseases that resist medicine through targeted evolution.
It is possible to make artificial plasmids that cause the bacteria to produce any protein you would like.
Bacterial DNA Replication:
Start: Clone the OriC. A plasmid of 5000 basepairs can act as a model of the bacterial chromasome. The proteins are added one by one.
There are four genes that you have to know:
DnaA finds the OriC while it holds the ATP. It aggregates in a large clump and unwinds a little piece of the DNA.
DnaC protein is the loader for DnaB. It has switched activity by ATP. First binding ATP, it attaches to DnaB, opening it. It can’t hydrolyze the ATP until DnaB is attached to some ssDNA. Once the ATP is hydrolyzed, then it dissociates, attached to the ADP.
DnaB Hexamer binds the unwound DNA and unwinds even more (after DnaA is all attached). DnaB is the DNA Helicase that unwinds all the DNA for E. Coli. Every ATP it hydrolyses results in a step forward of four base pairs unwound. Tremendous amounts of ATP are needed for replication.
and DnaG “the DNA Primase”
DnaE is the alpha subunit of Pol3.
Meanwhile SSB “Single-stranded binding protein” stabilizes all the ssDNA that is present in the cell. (a tetramer)
DNA Gyrase is needed to reduce the writhe in the dsDNA loop, to make it possible to unwind it all the way. It keeps reducing the Linking Number as the DnaB helicase reduces the Twist.
Finally, Lk=0 and replication is complete.

DnaB carries the primase and the tau subunit of DNA Polymerase 3.

RNA primers are removed via nick translation by DNA Polymerase 1. Then DNA ligase fixes the nicks.

Termination is helped by the tus-ter system. the ter region is 100kbp. It binds the Tus protein, “Ter Utilization Substance”
The ter-tus system only lets the replication fork pass in one direction, so both replication forks eventually meet inside the ter-tus region. ter is placed opposite to the oriC.
The final link is removed by Topoisomerase 4 in E. Coli.

Linear chromosomes have ends. The ends can not be copied. What is the solution? the Telomer enzyme puts a repeating sequence “TTGGGTTGGGTTGGG-OH” on the 3’ end of the molecule. The telomerase adds the same sequence on the end of the molecules over and over. Telomerase is a ribonucleoprotein. It contains an RNA template which has the repeating sequence on it. With a modified template sequence, the telomere ends could be altered.

Viruses such as HIV are “retroviruses.” they are made from RNA, but translate themseleves to DNA and add themselves to the cell code. There are also things called retrotransposons which are inside the DNA. When transcribed they can add copies of themselves to other parts of the genome. They are similar to the telomerase protein.

If there is no active telomerase, then the chromosome loses 100-200 nucleotides with every cell division. Only cancer cells can divide indefinitely, and they always have an active telomerase protein, even if they shouldn’t.

Inhibitors can be used to battle telomerases. There are different drugs and possibilities.

Part 5: Genetic Damage
DNA damage can be repaired in several ways.
Reactions with H2O
Hydrolytic Deamination.
100-500 C->U changes everyday.
Chemical Mutagens.
Nitrous acid
Bisulfite
both help change C->U.

Depurination is fastest with A and G. It removes the nucleotide from the DNA entirely.
this occurs 2000-10,000 times everyday in each human cell.
Abasic sites freeze DNA Polymerase 3.
Also, the strand is easier to break.
Dimethyl sulfate kills G.
also, superoxide (O2.- and hydrogen peroxide mess up DNA.)
the mitochondria in human cells make .OH it is super dangerous.
exposure of H2O to x-rays can also cause .OH to appear.

uV light damage can mae pyrimidine dimers (2+2 cyclo-addition)
(or the 6,4 photoproduct.)

Repair Pathways:
1. Base Excision Repair
2. Nucleotide Excision Repair
3. Direct Reversal
4. Mismatch Repair (MMR)
5. Recombination repair

Nucleotide excision repair has one enzyme with a broad specificity.
it is UvrABC, the “exinuclease”
UvrA detects damage in the DNA and puts UvrB. Then UvrB gets UvrC, and both together are a nuclease that cut on either side of the damaged site of the DNA. Then DNA Polymerase 1 and DNA ligase can clean the little piece of ssDNA up!

Yeast: RAD mutants were sensitive to radiation.

Direct Repair:
This is where the enzyme does reverse process (DNA Photolyase) to the pyrimidine dimer problem.

Mismatch repair
MutS marks the mismatches, and gets MutL to pull the DNA on both sides along until it finds a MutH.
MutH likes to attach to methylated GATC sequences. MutH cuts the DNA on the unmethylated side once it touches MutL.
UvrD unwinds the DNA
and Dna Polymerase III rewrites the missing bases.
dam methylase tags the DNA so MutH knows where it should be cutting.

Recombinational Repair is the final mechanism.
This has to occur if there was a nick in the DNA while it was being replicated, and the replication fork falls off, leaving two pieces of DNA. This is a bad situation.
dsBreaks are lethal unless repaired ASAP!
recombination allows replication to resume.
It involves physical exchange of DNA strands.
Occuring during both meiosis in eukaryotes, conjugation of bacteria, and DNA REPAIR.
Strands are exchanged via strand invasion, creating a D-Loop (displacement loop).
bacteria use RecA for recombination. RecA binds ssDNA. RecAmonomers coat all ssDNA, activating it. Then RecA moves to the major groove of normal DNA and slides all around, if the homology is found, RecA causes a Strand Exchange.

When the DNA breaks, RecBCD starts chewing up the end. But when it reaches a CHI sequence, it inhibits the nuclease activity, which results in RecA binding to the newly revealed ssDNA. Then RuvC is used to cut the Holliday Junction that RecA creates when it does the dual strand-exchange.
then DNA ligase seals the breaks. Meanwhile, the replication fork has been recreated, so replication may continue.
Next.
uv Light and the SOS response.
Large doses of uv light cause a huge amount of DNA damage. this results in persistant replication blocking. The sos response first releases UvrA, which attempts recombination. If this still doesn’t work (for example, a badly damaged base pair that the replication machinery can’t pass), then it releases its final weapon: UmuC,D. UmuC,D is a Dna Polymerase with no error checking that doesn’t mind binding random bases if it can’t find a match in the other strand. It has very low processivity, but it can jumpstart the synthesis by copying over a serious error. There is usually a minute amount of UmuC,D in the cell, unless things get really bad.

LexA is the protein that inhibits the SOS reaction. ss-bound RecA cleaves LexA proteins, causing them to migrate off of the DNA. This results in more expression of the SOS proteins.
Note: Bound RecA also activates UmuC,D when they are in the cell.
UmuC,D is the Lesion Bypass Polymerase, DNA Polymerase Type 5.
the Kd for UmuC,D is very very strong, so they aren’t expressed until insane amounts of damage have occured.
Making a Gene Library.
Take human cells. Break them open, and isolate the mRNA. The mRNA codes for proteins, so use a reverse transcriptase to get the DNA that codes for the respective protein. Human DNA mixed with E. Coli, and each one gets one piece of human DNA on average. Then you can identify each protein by purifying the foreign protein from the E. Coli and sequencing it. Use the DNA code to get the sequence that made the protein, and take the complement and put a radioactive label on it. Apply this to each E. Coli cell, and the one that it attaches to is the one that is making the protein.

Making RNA:
Three steps:
RNA Polymerase makes RNA from dsDNA.
1. Initiation
only dsDNA is used. There are upstream sequences called “UAS” which activate the promoter by bonding proteins which stabilize the RNA Polymerase binding to the DNA. The main one is called the TaTa Box. This appears at -10. at -35 there’s a TTGACA sequence.
3. Dissociation. An inverted repeat sequence appears before a series of AAAAAAAs makes the bond to the RNA super-weak. the inverted repeat sequence causes the tRNA to form a hairpin, which makes the RNA Polymerase pause in its steps, letting the weak bonds break, causing it to dissociate.
Promoters: There are weak and strong promoters.

The Lac operon.