DNA structure and function, Replication, and Repair

Question 1

What are the components of a nucleotide.

Which are purines, pyrimidines

How many hydrogen bonds?

Answer 1

A base with a five carbon sugar (either 2’OH RNA, or no 2’OH, deoxyribose)
linked by an N-glycosidic bond
= nucleoside

The addition of the phosphate group to the sugar via a phosphoester bond. makes it a nucleotide.

Purines; AG
Pyrimidine: CT

3 hydrogen bonds: CG
2 hydrogen bonds: AT

Question 2

Salmonella causes food poisoning. What was a conceptual treatment?

hint, methyl groups induce expression on DNA for viruses

Answer 2

some bases are methylated, and this allows protein to recognize the DNA for expression.

Inactivation of DNA adenine methylase blocks the expression,

Gut colonizing pathogens all carry methylases, meningitis, cholera…etc

Question 3

Acyclovir - what is it a type of?

what is it used for?

Answer 3

It is a nucleoside analog that doesn’t allow for elongation, missing 3’ OH. You let the organism incorporate it into a nucleotide.

Used for Herpes Simplex virus infections.

Analog of deoxyguanosine

Question 4

AZT what is it used for? Dallas buyers club.

Answer 4

analog of thymidine

Used to treat HIV and AIDS, like acyclovir. It blocks further DNA synthesis.
Does not significantly affect host cell metabolism which is good and odd compared to the movie…

Question 5

What is the most common form of DNA?

How many bases per helical turn?

How many angstroms is each turn?

Answer 5

B form

10 bases per helical turn

34 angstroms each turn.

Question 6

Certain anti-cancer drug interferes with DNA and RNA synthesis by intercalating what?

Answer 6

the minor groove.

Question 7

What are the benefits to negative supercoils?

How are they formed?

Answer 7

Easier strand separation, energetically favorable, and the energy for strand separation is stored in the super coil.

Formed by partial unwinding of the double helix. Then restoring complementary basepairing. End result is one less helical turn. 100 basepairs and 10 helical turns. Remove a helical turn, 9 helical turns, the Molecule folds on itself.

When eukaryotic histone proteins bind, they force the DNA to wrap around them and this generates a negative supercoil.

Question 8

What are DNA topoisomerases and generally what do you need to know?

Answer 8

They introduce swivel points into the DNA. Topo 1 only does single strand breaks and strings one strand through the other. Removes positive and negative supercoiling.

Topo 2 is a double strand break, and does both as well. REQUIRES ATP AND TOPO 1 DOESN’T.

Gyrase is exclusive to prokaryotes and it can pump negative supercoils.

Question 9

How are topoisomerase inhibitors used in anti-bacterial therapy and anti-cancer therapy?

Answer 9

inhibits bacterial DNA synthesis by blocking gyrase, the tension becomes too much for strand separation. Eukaryotes don’t have gyrase so no side effects.

anticancer, remove the nick sealing portion of the topoisomerases 1 and 2, so it becomes DNA breaking agents! But this leads to cell death in eukaryotic cells as well

Question 10

Describe the structure of histones. Include linker.

What is the order of compaction?

Answer 10

An octomer, two of each. H2A, H2B, H3, H4, they’re lysine rich.

There is a DNA spacer, 20-80 bp of DNA between histones and it binds H1. This linker is allows the cores to become closer and coil.

nucleosomes = 10 nm fiber > 30 nm fiber (solenoid) = then DNA loops coiled around a protein scaffold.

Question 11

Describe the mechanism of prokaryote replication.

Answer 11

The replisome, carries out replication. Strand separation begins at the origin of replication (usually AT rich), DnaA recognizes the AT region and uses ATP to locally melt it. This forms two replication forks, helicase binds near the forks and uses ATP to force the strands open. Single strand binding proteins stabilize the single strands to keep them apart and protect them from degradation.
Topoisomerase 2 works ahead of the fork to relieve tension .

Question 12

Which polymerase catalyzes chain elongation in prokaryotes?
Which exonuclease activity does it contain.

What does DNA pol 1 do?

Answer 12

DNA polymerase III catalyzes 3’-5’ bonds with OH attack 5’ phosphate.

3’ to 5’ exonuclease activity. DNA is made 5’ to 3’ so this lets it rewind.

DNA polymerase 1 has 5’ to 3’ exonuclease activity so it removes the primers ahead of pol 3. (It also has 3’ to 5’ exonuclease activity and can synthesize DNA)

Question 13

Which molecules are ATP dependent?

Answer 13

Topoisomerase 2’s
Helicase
DNA ligase.

Question 14

What is different about eukaryote replication? Especially polymerases.

Answer 14

Multiple origins of replication,

DNA is associated to histones,

Has telomeres

Eukaryotic DNA polymerases,

Pol alpha (contains primase, initiates synthesis on leading and lagging strands)

Pol delta - elongates lagging strand (has 3’ to 5’ exonuclease activity)
-displaces the primer on lagging strand , the flap that forms is degraded by exonucleases (substitutes function of Pol 1 in prokaryotes)

Pol epsilon (elongates leading strand, same exonuclease activity) if dysfunctional pol delta CAN SUBSTITUTE.

Question 15

What happens in nucleosomes as fork advances?

Answer 15

Histones will remain loosely associated to a parental strand, new histones are synthesized simultaneously with DNA replication and nucleosomes reform behind the replication fork

Question 16

Describe telomeres. What do t-loops do?

Answer 16

They are at the end of linear chromosomes.

Telomerase contains an RNA code and reverse transcribes to extend to 3’ ends. The primer will be inserted on the 3’ end to synthesize from 5’ to 3’. Once replication is over, the primer is removed and there is a 3’ overhang which will get digested. This is so only nonsense DNA is lost.

Telomeres are short noncoding G-rich sequences (TTAGGG) and they form t-loops.

Protect the ends from degradation, prevents recombination, and end to end fusion.

Question 17

Describe the role of telomeres in aging and cancer

Answer 17

telomerase is active in all cells before birth and remain active in stem cells and germ cells.

When telomeres get too short that end to end fusion can occur, DNA damage sensors arrest cells (p53), and serves as a mitotic clock by monitoring telomere length.

Telomerase is usually active and p53 is deactivated in cancers. Telomerase inhibitors.

Question 18

What are the effects of Dyskeratosis congenita (reduced telomerase activity)

Answer 18

affects precursor cells in proliferative tissues :

Hair = baldness,

Intestinal crypt = gut disorders

Basal layer of epidermis = abnormal skin pigmentation.

Bone marrow progenitor cells. (fail to produce new blood cells so patients usually die from bone marrow failure.

Question 19

Hutchinson-Gilford progeria

cause and effect

Answer 19

accelerated telomere shortening,

aged skin, alopecia, short stature, early death by MI.

Question 20

What kind of repair mechanism is best for each of the following?

damage to a single base?
replication errors
bulky adducts 
oxidative damage - single strand break
ionizing radiation, oxidizing agents.

Answer 20

damage to a single base = BER

replication errors = MMR

bulky adduct = GG NER, TC NER

single strand break repair

doulble strand break = homologous, non homologous.

Question 21

Describe MMR

Answer 21

mismatch, no nucleotide damage.

MSH2/6 recognizes mismatch while MSH 2/3 recognizes insertions and deletions. They will recruit MLH-1/PMS2 which are endonucleases.

Prokaryotes recognize parental strand by methylation and eukaryotes perhaps recognize the nicks on lagging strand.

The nucleases nick on either side of the mismatch. The helicase unwinds so it is singlestrand and exonuclease digests it. DNA pol III and ligase fill the gap.

Question 22

Defects in MMR

Answer 22

Lynch syndrome: significantly increased risk of colorectal, endometrial, ovarian, skin, gastric, renal cancers.

Question 23

BER

Answer 23

ROS (reactive oxygen specifies can damage our bases) example cytosine deaminated into uracil.

First the damaged base is recognized by base specific glycosylase, uracil DNA glycosylase. Which cleaves the N-glycosidic bond and removes the base.

AP endonuclease cleaves the sugar phosphate backbone.

Deoxyribose phosphate lyase removes the residue. DNA pol I fills the gap

Question 24

Defects in BER

Answer 24

mutation in DNA glycosylase leads to very high risk for colon cancer - adenomatous colorectal polyposis syndrome.

Werners syndrome: for some fucking reason helicase isn’t mentioned for BER, however WRN helicase is necessary for BER…

autosomal recessive disorder ft. premature aging, skin atrophy, hair loss and cancer disposition.

Wrn is also involved in processing telomeric DNA, activating DNA damage responses.

Question 25

NER

Answer 25

Helicase releases the strand because it is unfavorable to digest it with that large ass thing on it.

GG-NER (inactive region) - XPE, XPC (repair proteins that recognize the Helix distortions and recruit the repair complex. Endonucleases come to make incisions (XPF, XPG) common pathway Helicases unwind DNA to release damaged portion (XPB-XPD) also common.

TC-NER (transcriptionally active) (CSA- CSB) proteins recognize the stalled RNA pol II And can ubiquitinate it. Ribosome leaves and then the common proteins are recruited and do the same.

both pathways, XPA, XPF, XPG, XPB, XPD

The ribosome will stall at the site where there is a bulky DNA adduct. This recruits the proteins for repair.

Question 26

NER defects

Answer 26

Vital for what?
Cigarette smoke contains carcinogens that once oxidized will covalently bind to residues in the DNA of lung cells. Without NER, it will interrupt hydrogen bonding, distort helix, cause frameshifts and lung cancers.

UV light- induces formation of dimers between pyrimidines, distorts DNA and causes frameshifts, leads to skin cancers.

GG-NER - typically cancer.

also XP - due to mutations in XPC, XPE (GG-NER exclusives) or XPD, XPA (shared), high risk of melonoma, extreme solar sensitivity, high risk of internal cancers.

TC-NER - no cancer, typically neurological defects.

Cockayne Syndrome
Mutations in CSA, or CSB (recognize stalled ribosome) , mental retardation, sun sensitive but not increased skin cancer (transcription does not resume once damage is sensed, stalled transcription in active cells likely leads to apoptosis.

this has been a way to cure cancer by causing cancer cells to form intrastrand adducts and die.

If common pathway is affected, both CNS and cancer.

Question 27

Single strand break repair

Answer 27

Usually the loss of a nucleotide and the 3’ OH and 5’ phosphate.

SSB is recognized by PARP-1. Which recruits the complex.

Aprataxin reestablishes the ends. DNA polymerase and ligation.

Question 28

Defects in SSB.

Answer 28

Ataxia Oculomotor Apraxia

uncoordinated movement and gait, limited eye movement, involuntary movements, neurological issues

but there is no non-neurologic features like immune deficiency.

Caused by mutation in aprataxin.

Question 29

DSB repair

Answer 29

Non-homologous end joining: the major pathway, just rejoins what remains of two broken ends, frayed ends are cleaved and joined. Error prone.

Ku 70/80 heterodimer senses broken DNA. Recruits DNA, PKCs: Artemis
Artemis - digests frayed ends. DNA polymerase extends ends using overhangs and the ligase joins the two together.

Homologous repair - aligns highly homologous DNA molecules and it is non-mutagenic. Only occurs in S and G2 cycles of cell cycle when sister chromosomes are available.

RAD 52 binds the DNA ends
RAD 51 searches for sequence homology and aligns the homologous DNA.
BRCA1 and BRCA2 regulate RAD51.
Nuclease and helicase liberates a single strand
RAD51 (using ATP) mediates single strand invasion into the homologous DNA molecule. It replicates, then returns and fixes the other damaged strand.

Question 30

Defects in DSB

Answer 30

BRCA1 or BRCA2 leads to higher chance of developing breast cancer because victims are more sensitive to radiation. Some drugs induce DSBs for killing cancer but there is severe side effects

ATaxia telangiectasia - caused by mutation in the ATM protein.
ATM: activated by DSBS, singals the cell cyle checkpoint to slow down, signals repair machinery to start DSB repair. Especially in B and T cells.

They are hypersensitive to Radiation, propensity of lymphoid cancer, because they have fucked up B and T cells.

Question 31

How are primers removed in eukaryotes

Answer 31

DNA polymerase delta, PCNA and Flap exonuclease