Lecture 16

Question 1

What is the end problem?

Answer 1

A problem for linear chromosomes. How do you finish off the end of a linear chromosome? Trouble is with the lagging strand end RNA primer; if you chew it up, it won’t replicate to the very end.

Question 2

What is the solution to the end problem?

Answer 2

Telomeres!

Question 3

What is a telomere?

Answer 3

A whole bunch of repeats of a very short sequence (6 bases).

Question 4

What is the process of adding a telomere?

Answer 4

Elongation and Translocation, with a 3’ overhang. Special proteins are put on 3’ overhang to cap and protect.

Question 5

What is telomerase?

Answer 5

The enzyme that adds telomeres to the lagging strand of DNA to solve the end problem. The enzyme is a reverse transcriptase enzyme (still 5’ to 3’). It is a ribonucleoprotein, meaning it has a protein component and an RNA component.

Question 6

Why is telomerase important for dividing cells?

Answer 6

Without this, every generation of telomeres would get a bit shorter and eventually eat into coding info. This leads to premature aging because the cell may not be able to divide as many times.

Question 7

What is the role of telomerase in cancer cells?

Answer 7

Non dividing cells do not have telomerase expressed. Rapidly dividing cancer cells need telomerase on. If it is on when it should not be, this can help contribute to the development of cancer. There may be mutations that compromise this.

Question 8

In a human cell, how many times does the DNA backbone break spontaneously each day?

Answer 8

0.5 per day. That’s one for each of the 50 trillion nucleated cells in your body.
Know how to do this calculation.
Important info: on average, each phosphodiester linkage is hydrolyzed once per 30,000,000 y. Our diploid genome has 6 billion base PAIRS per cell.

Question 9

Can error every be eliminated completely?

Answer 9

No- can only be reduced. Every 100-1000 times you’ll get a wrong base in there. Chemical systems comply with the laws of thermodynamics, and are controlled by probability

Question 10

How does the engineer’s triangle relate to accuracy in DNA replication?

Answer 10

Need to pick two between cheap, fast, and good. Cells spend energy and time in exchange for accuracy. Use energy in the form of ATP for this. Time as well. Want to make sure you’re getting the job done right.

Question 11

When can mutations arise in DNA replication?

Answer 11

Before or during DNA replication.

Question 12

What are the sources of DNA sequence errors?

Answer 12

Base disincorporation (eg dU instead of dT)
Chemical mutagenesis
Ionizing radiation (UV, x rays, cosmic rays- like the atomic bomb and okazaki)
Genetic Mutagenesis (eg retrovirus integration (won't talk about this))
Spontaneous lesions (eg backbone hydrolysis (how often backbone breaks) or deamination (cytosine spontaneously deanimates and becomes uracil- can cause problems with base pairing))

Question 13

What are the kinds of mutations that occur?

Answer 13

Base substitutions (2 types)
1) Transitions (substitution where one base is changed for another.  Purine for another purine or pyrimidine for another pyrimidine.)
2) Transversions (same as transversions, except the change is purines to pyrimidines or vice versa)
Insertions and deltions (indels) small or big
Breaks in the backbone
-can get rearrangements of chromosome (inversions, translocations)

Question 14

When must mutations occur?

Answer 14

During DNA or RNA replication. There is no alternative compatible with the laws of physics and chemistry. Consequently, descent can occur only with modification. (With inheritance, there will be change)

Question 15

Darwin combined three different ideas provide an incredibly powerful explanation of biological diversity.

Answer 15

Descent with modification:
1) Variation: not every individual in a population is the same.
2) Heritability: these differences can be transmitted between generations
Natural selection:
3) Differential survival: heritable differences increase or decrease the number of offspring that an organism has.
Different variants will have different fitness.
Descent w/ modification does lead to variation within populations. Did not know why this is true. Our contemporary understanding of DNA and RNA chemistry, obtained over the last fifty years, shows that descent with modification must occur, and that it must introduce variation into populations.

Question 16

Does genome size affect rates of mutation tolerated?

Answer 16

As genomes get larger, lower rates of mutation are tolerated. Viriods, the smallest, have the highest mutation rate.

Question 17

What are the three flavors of mutation reduction?

Answer 17

1) Inherent accuracy (error rate is 10^-3 to 10^-4) (tries to get it done right the first time)
2) Proofreading (10^-6 to 10^-7)
3) Surveillance and repair (10^-9 to 10^-10) (where we end up at: one in our genome in each cell division won’t really mess us up)

Question 18

What is a tautomer?

Answer 18

Keto/enol or amide/imidic acid interconversion
Naturally occurring mutations by tautomerism include pairing of imino tautomer of adenine with cytosine instead of thymine. If not corrected, DNA pol will incorporate a C instead of a T in the new daughter DNA strand. A transition mutation of AT to GC will be formed in dna. Ratio of occurrence is 1:1x10^-4

Question 19

DNA Pol I proofreads the nascent strand.

Answer 19

When you get the right base in there, will hydrogen bond with one on its template, and also base will fit before covalent bond has occurred. When you have the wrong one, base does not fit as well, and is more likely to get kicked out. Helps when pools of nucleotides are balanced appropriately.

Question 20

What does DNA Pol I in proofreading configuration entail?

Answer 20

5’ end of template strand in between fingers and thumb, nascent 3’ end in proofreading site on palm (where 3’-5’ exonuclease is). Must have a second form of checking, beyond just induced fit. Happens at the editing site. 3’ base put on, if it is the wrong base 3’-5’ exonuclease will chew it off. Problem is that the activity down there is 25 angstroms away from the mistake. Need flexibility in the DNA too. Kinetics of how the process works: put the correct one in, and everything is very stable. Put the wrong one in, and everything is less stable. Slows down a little, the DNA flops around/over, and then it can fix the mistake by chewing off whatever is there.

Question 21

DNA Pol I has two exonuclease activities.

Answer 21

5’-3’ exonuclease chews through primers and other debris.
3’-5’ exonuclease proofreads for accuracy.
Only if bases were misfired would the DNA have the time to flop down and get paired off. Then the incorrect nucleotide would get removed.

Question 22

Chemical mutagens: DNA intercalating agents cause indels.

Answer 22

EtBr slips in between strands in between bases. This will cause little local bulges and a little winding effect locally. As it’s going through replication, it’s more likely to miss and skip over a base. This could lead to either a small insertion or a small deletion (small indels)

Question 23

What is the risk with repetitive DNA sequences?

Answer 23

They are susceptible to slipped-strand misfiring, which can cause indels. When sequence is repetitive, can slip out because it’s basically the same thing. This can happen if it’s just two repeats or if it’s more. Backward slippage causes an insertion, forward slippage causes a deletion.

Question 24

Example of problems with repetition: Expansion of triplet repeats can cause several diseases

Answer 24

Huntington’s disease: incurable, progressive, neurodegenerative disease. Usually fatal.
Problem is CAG repeats. Includes glutamine (usually called polyglutamine diseases). Normal individual has 20 repeats. This is a bunch of glutamines in a row. Too many is not good for the protein- can do 20, but once you get up to 30+, it doesn’t work. Protein misfolds b/c of expanded glutamine region, and this either leads directly to cell death or it leads to aggregation, which leads to cell death. This is bad in your neurons. A lot of these genes are in our cells, we don’t know why they only affect some of our cells (i.e. neurons).
This occurs more in older people, usually around 40. Neurons don’t really divide, maybe this happens because of other damage. Mech can either lead to extras or decrease. This tends to increase by generation.

Question 25

Another example: Cancer

Answer 25

A major cause of cancer is mutation. Lots of cancers occur because of problems with DNA repair.
Gain of function mutations in protooncogenes can cause cancer. These genes generally promote cell division. Usually dominant (one bad copy causes the disease). (Example: genes promoting cell division, like telomerase)
Loss of function mutations in antioncogenes (tumor suppressors) can cause cancer as well. Usually this is recessive (one good copy is enough to stay healthy). (Breaks stop cell division- knock out both copies of tumor suppressors, etc.) (usually need to knock out both for disease to occur)
It is generally thought that it takes multiple mutational hits to cause cancer.

Question 26

DNA damage is ongoing and cumulative

Answer 26

Depurination (base loss): 20,000/cell/day
Deamination: (100/cell/day)
About 1/1000 of the genome in each human cell is damaged per year.
If you live to age 80, that’s approx 10% of the genome in each cell.
And as we age, the repair machinery becomes less efficient.
This is why many older people get cancer.

Question 27

Radiation and mutagenesis: UV cross linking produces pyrimidine dimers.

Answer 27

Skin cancer is the most common cancer in the united states. Lesions shown on slide probably account of most cases of skin cancer.
Sunlight/UV-B rays cause pyrimidine dimers. This causes DNA to scrunch up, which leads to strand slippage. Two ways to repair:
1) DNA photolyase (not in humans)
2) Nucleotide excision repair
Can get cyclobutane thymine dimer or 6-4 photoproduct

Question 28

Radiation and mutagenesis- DNA photolyase

Answer 28

DNA photolyase is a remarkable solar-powered enzyme. Repairs cyclobutane thymine dimers.
1) MTHF is a “photon antenna” that absorbs UV-A and gets photo excited
2) MTHF transfers photo excitation to FADH
3) FADH transfers an electron to the dimer, breaking the dimer into thymine monomers, then takes the electron back. Not the base-flipping mechanism of the enzyme!
(MTHF= methenyltetrahydrofolate)
Humans don’t have this

Question 29

Nucleotide excision repair

Answer 29

System recognizes helix (backbone) distortions, not specific chemical groups or adducts.
Repairs UV photoproducts, among other lesions.
Recognizes distortion, makes cuts on either side and cuts it out to fix it. Can use for several types.
UvrABC endonuclease = ABC excinuclease recognizes and cuts, UvrD takes out, Pol I and DNA ligase repair
This is what humans have.