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Molecules to Medicine Subblock 1 > DNA > Flashcards

DNA Flashcards

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Organic Chemistry 101 flashcards
1
Q

purines

A

Adenine and Guanine, 6 and 5 ring attached, have nitrogens at 1, 3, 7, and 9 positions. 9 attaches to sugar.

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2
Q

Pyrimadies

A

Thymine, cytosine, uracil. 6 ring, nitrogens at 1 and 3 positions. 1 attaches to sugar.

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3
Q

significance of 1’ position in ribose sugar

A

beta gylcosidic bond between the base and the sugar

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4
Q

Significance of 2’ position in ribose sugar

A

distinguishing position for DNA and RNA

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5
Q

Significance of 3’ position in ribose sugar

A

is involved with linking the chains of neucleotides together

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6
Q

Significance of 4’ position in ribose sugar

A

silent position, not much happens there

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7
Q

Significance of 5’ position in ribose sugar

A

phosphate linkage happens here

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8
Q

Nucleoside vs. nucleotide

A

Nucleoside is base and sugar, nucleotide has the addition of the phosphate(s)

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9
Q

relative solubility of bases and nucleotides/sides

A

nucleotides > nucleosides > pyrimadine > purine

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10
Q

How are nucleotide and deoxynucleotide residues linked together?

A

phosphodiester bonds, between 3’ carbon attached to phosphate, to the 5’ carbon.

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11
Q

What do ddl (dideoxyinosine) and AZT (azidothymadine) do?

A

inhibit DNA synthesis by reverse transcriptase. they look like nucleotides but they don’t have the 3’ OH.

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12
Q

experiments that lead to DNA being genetic material

A

Fred griffith: trasnformation of pneumococcus serotypes R and S (pathogenic). killed S + live R = pathogenic. Thought it was nucleic acid that was transforming it. Avery, McCloud, and McCarty proved that it was DNA that transformed it.

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13
Q

Chargoff’s rules

A

%G=%C, %A=%T. He reduced DNA to building blocks and measured GCAT content. Purines = pyrimadies ( G+A=C+T)

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14
Q

Experiments showing helical structure of DNA

A

X ray diffraction with moist DNA fragments. showed helical structure and 10 step rotation ( 3.4 mm)

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15
Q

Who proposed model for DNA?

A

Watson and Crick.

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16
Q

Basic properties of Normal (B form) DNA structure

A

2 antiparallel paired strands. Diameter of AT and GC pairs are the same, allowing it to keep a regular form. Sugar phosphate backbone with phosphates pointed out. There is a major and minor groove which is the product of how the bases are attached to their backbone. it is at a slight angle, or slightly set out of the plane. (complementary of bases suggests molecular mechanism for replication)

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17
Q

How many hydrogen bonds do the base pairs have?

A

GC -> 3

AT -> 2

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18
Q

Forces that keep DNA helix stable and together

A
  1. positively charged small molecules help to neutralize charge so that all the negatively charged phosphates on the DNA don’t repel eachother, disrupting stability
  2. hydrophobic interactions, stacking energies between bases
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19
Q

DNA melting, Tm, and affecting factors

A

DNA melting is separation of the two strands. This can happen at a specific temperature, Tm. Tm is affected by

  1. higher salt concentration (neutralizes the negative charges on the DNA to keep it from repelling itself
  2. extreme pH’s can alter the ionization states of the groups on bases that form H bonds and affect H bonding
  3. Increase in chain length increases Tm
  4. Increase in GC content will increase Tm
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20
Q

Methylation as a DNA modification

A

Cytosine methylation: at CpG. coordinates gene repression. NORMAL modification, done by DNA methylases.

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21
Q

Deamination

A

not favorable. can be caused by nitrous oxide, or nitrosamines (found in cigarette smoke) amino group, after reacting with water, turns into ammonia and is replaced by an oxygen that is double bonded to base. Note: deaminated methylated C looks like a T! In this case, the other strand will have a methylated C, so there are mechanisms that recognize and fix this

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22
Q

Depurination

A

water reacts to cause the purine to separate from the sugar. this is very susceptible to breakage.

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23
Q

UV mediated linkage

A

mostly of pyrimidines, especially thymines. this can happen in two different confirmations. cyclobutane, or a 6’ - 4’ phosphoproduct. they can affect DNA structure (put kink in) and can affect DNA metabolism mechanisms.

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24
Q

alkylation, benzopyrene example

A

nucleophilic groups, on pyrimidies especially are very susceptible to alkylation. this is deleterious to DNA. for example, benzopyrene is a alkylating agent from chimney soot, and chimney sweeps would get cancer at a much higher rate. Other environmental carcinogens, and synthetic, like nitrogen mustard,

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25
Q

What does topoisomerase do?

A

relaxes super coiling of DNA, allowing for proper replication, (also txn?) Many cancer drugs target this as well. when DNA is too supercoiled, p53 is called in to kill the cell.

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26
Q

Define “semiconservative” as it applies to DNA replication

A

a DNA molecule serves as its own template, so both of the new molecules have one parent strand and one daughter strand.

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27
Q

Define “bidirectional” as it applies to DNA

A

DNA replication proceeds in both directions from each replication fork

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28
Q

Define “okazaki fragments” as it applies to DNA

A

These 100-1000 bp fragments make up the lagging strand. DNA can be synthesized only in 3’ - 5’ direction, so these fragments are used to replicate the 5’-3’ strand at each fork. These small fragments are synthesized backwards and then joined

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29
Q

What does DNA pol need to proceed with replication (3)

A

A template ssDNA, a free 3’ OH from the proceeding nucleotide (a primer), and free dNTPs

30
Q

Define “semidiscontinuous” as it applies to DNA replication

A

At each replication fork, there is a leading (continuous strand) and a lagging (discontinuous strand)

31
Q

Define “origin” and “fork” as it applies to DNA replication

A

Origin is where a replication bubble begins. The replication forks are on either side of the bubble, where the two parent strands separate.

32
Q

Function of origin binding proteins

A

recognize and binds to the replication origin

33
Q

Function of helicase

A

Melts and unwinds DNA

34
Q

function of single strand binding proteins

A

Protects ssDNA from attacks by a nuclease, keeping it single stranded
Also important for sensing DNA damage and for repair. It serves as a scaffold for other proteins that come in to deal with repair or detect damage.

In humans, RFA (or RPA) (a complex, with one subunit that looks like SSB from ecoli)

35
Q

function of primase

A

Synthesizes the RNA primer.

36
Q

function of DNA pol I

A

5’ to 3’ polymerase activity and 5’ to 3’ exonuclease activity, to remove the primer and then fill in the gap. DNA pol I is distributive, low processivity

37
Q

Function of DNA pol III

A

5’ - 3’ polymerase activity, to elongate the new strand from the primer

38
Q

Function of DNA ligase

A

Using ATP, it ligates the two replicated DNA fragments

39
Q

Function of sliding clamp

A

Holds the DNA pol III to the strand to keep it from falling off. In humans this is called PCNA.

40
Q

function of topoisomerase/gyrase

A

Release of tension caused by torsional strain of unwinding the DNA. Type II cuts double strands and moves the uncut strand across the gap and reseals it. Type I cuts a single strand, and moves the intact strand around the gap until the stress is relieved and then seals it.

41
Q

Function of telomerase

A

Telomerase provides RNA template for finishing replication of the lagging strand in linear DNA. It is a specialized reverse transcriptase. It adds a run of CA or TGs (telomeres) to the end of DNA strands.

42
Q

function of reverse transcriptase

A

makes DNA from RNA.

43
Q

List the 8 steps of DNA replication and the enzymes involved in each step.

A
  1. recognition of replication origin by ORIGIN BINDING PROTEINS (example DnaA)
  2. Melting and Unwinding DNA by DNA HELICASE
  3. relaxation of torsional stress ahead of the fork by TOPOISOMERASE/GYRASE
  4. Protection of ssDNA by SSB (single stranded binding proteins, like RFA or RPA in humans)
  5. Synthesis of RNA primer by PRIMASE
  6. Elongation of DNA from primer, DNA POL III
  7. Removal of primer and copying into DNA by DNA POL I
  8. Ligation of DNA fragments by DNA LIGASE
44
Q

Describe a DNA replication origin (in Bacteria)

A
  1. Unique DNA segments with multiple short repeats
  2. recognized by origin binding proteins
  3. rich in AT pairs
45
Q

How can DNA pol confirm accuracy?

A
  1. It can sense proper vs. improper hydrogen bonds
  2. It can sense proper vs. improper geometry of base pairing
  3. both DNA pols have 3’ - 5’ exonuclease activity to remove the improper base pair
46
Q

Differences between DNA pol I and II

A
  1. DNA pol I has 5’ - 3’ exonuclease activity (to remove primer)
  2. DNA pol III is associated with the sliding clamp, so it is very processive.
  3. Pol I has slow replication rate, Pol II is faster.
47
Q

Tell me about the two DNA pols for Eukaryotes!

A

DNA pol epslion is for leading strand, and DNA pol gamma is for lagging strand.

48
Q

Tell me about replication at the end of a chormosome

A

In the lagging strand, there is a primer at the very end of the chromosome, and when the primer is removed, there is a gap. Telomerase binds to the last few ssBP, by an RNA template it carries around with it. It uses this template to extend the strand further, though multiple rounds of extension, using the ss 3’ end of 5’ to 3’ parent strand as a primer. Then a primase and DNA pol synthesize DNA to complement the sequences added by the DNA pol. The last bit of single stranded DNA synthesized by the telomerase loops back around to form a t-loop, and is stablized and bound by Telomere binding proteins like TRF1 and TRF2.

49
Q

Describe the relationship between mutations, DNA repair, and cancer

A

Cancer may be caused by mutations in DNA, which occur when damage is not recognized and fixed by DNA repair mechanisms (including apoptosis)

50
Q

Examples of heritable human diseases that are caused by defective repair mechanisms

A

Cancer, XP, TTD, cockayne’s, HNPCC (hereditary non-polyposis colorectal cancer, aka Lynch Syndrome)

51
Q

What are some sources of DNA damage

A
  1. Endogenous: UV, radiation, chemicals

2. Exogenous: Reactive oxidative species, hydrolysis, deamination, or alkylation of DNA.

52
Q

What are some types of DNA damage

A
  1. Change of base: depurination, deamination, deamination, alkylation, oxidation, thymidine dimers,uricil misincorperation,
  2. Change of structure: insertion, deletion, crosslinks, mismatch, strand breaks especially ds, bulky adducts
53
Q

What are some consequences of unrepaired DNA damage, and some examples for deamination, alkylation, strand breaks and base adduct

A

cell may be signaled to undergo apoptosis, or become cancerous.

Base deamination : of methylated C, so CG to TA mutation

Base alkylation: Methlated G can pair with T, so you can have GC to AT mutation

Strand breaks : can lead to crosslinks that affect Txn and replication

Base adduct: of benzopyrine can cause a GC-TA mutation

54
Q

What are the 4 types of DNA repair?

A
  1. direct reversal
  2. excision repair
  3. tolerance/bypass
  4. strand break repair
55
Q

Tell me about direct reversal, give me a few examples

A
  1. DNA ligase can fix a single strand break
  2. photolyase in plants can fix UV damage
  3. O6 methylguanine methyl transferase (MGMT) removes methyl on the methylated guanine.
56
Q

Name the 3 types of excision repair

A
  1. Base excision repair (base damage that does not affect DNA duplex structure
  2. nucleotide excision repair (damage that distorts structure and blocks polymerase)
  3. mismatch repair (mistakes made during replication)
57
Q

What are the steps common to all forms of excision repair

A
  1. recognition of damage/mismatch
  2. exonuclease cleaves phosphodiester bonds around the damaged area
  3. nuclease removal of damaged fragment
  4. DNA pol synthesizes missing sequence by complementing the intact strand.
  5. ligase seals the nick between original and synthesized strands
58
Q

Describe the process of mismatch repair.

A
  1. recognition of mismatch by MSH, and MLH
    (or MutS and MutL in bacteria). newly synthesized strand identified by lack of methylation (in eukaryotes, in Lagging strand it can recognze the nicks, in leading strand, it can recognize nicks left by an enzyme that removes the accidental rNTPs that DNA pol accidently puts in.)
  2. Endonuclease cleaves backbone of new strand
  3. exonuclease chews away a segement including the mismatch. helicase helps by unwinding.
  4. DNA pol repairs the single strand gap
  5. DNA ligase seals the backbone1
59
Q

Describe Base excision repair

A
  1. recognition of altered base by a specific glycosylase.
  2. glycosylase removed base by hydrolyzing N-glycosidic bond, forming apurinic or apyramidic (AP site)
  3. AP specific endonuclease and AP lyase cut backbone (5’ and 3’ respectively)
  4. gap is filled by DNA pol, nick is fixed by DNA ligase
60
Q

Describe Nucleotide excision repair

A
  1. recognition and binding by a complex, either globalgenome NER or txn-coupled NER
  2. local unwinding by helicases (in TFIIH complex)
  3. double incision by two endonucleases, removed of ~30bp oligonucleotide with the lesion,
  4. gap filled by DNA pol, nick fixed by DNA ligse
61
Q

describe double strand break repair by homologous recombination (HR)

A

With a homologous DNA molecule, repair mechanism uses the homologous molecule as a template for the patching up. It needs sequence homology and is very accurate

62
Q

Describe double strand break repair by NHEJ

A

Non homologous end joining does not need sequence homology between the two broken ends. It ligates them together. can lead to insertions or deletions. (choice between NHEJ and HR can be dependent of cell type and stage in cell cycle.

63
Q

What repair mechanism fixes DNA damage due to DNA pol mistakes

A

Mis match repair

64
Q

What repair mechanism fixes DNA damage due to deamination, cysteine methylation or other damages that done affect DNA duplex structure

A

Base excision repair

65
Q

What repair mechanism fixes DNA damage that does cause DNA structure defects, such as thymidine dimers or bulky adducts

A

Nucleotide excision repair

66
Q

What repair mechanism fixes DNA damage due to O6 methylated guanine

A

direct reversal, by O6methylated guanine methyl transferase

67
Q

What is the DNA damage checkpoint and its role in maintaining genome stability

A

A cellular surveillance mechanism via signaling, that halts cell cycle progression
when sensors detect DNA damage, signal the signal transducers (kinases like ATM & ATR, very important to detection system) which signal effectors.
to initiate the recruitment and activation of downstream proteins. ATM and ATR
become activated in human cells undergoing earlier stages of tumorigenesis to delay or prevent
cancer. Mutations disrupting this checkpoint lead to genomic instability, and malignant conversion

68
Q

Describe the process of tolerance/bypass and the possible consequences

A

If excision repair systems cannot fix all the DNA damage that is blocking replication, cells use “loosened” or “error prone” DNA pols to continue through the damaged strand. These DNA pols lack proof reading and 3’-5’ exonuclease activity. example: Pol eta recognizes TT dimers, and Adds A (one or 2), BUT when you have a guanine adduct, it still adds A! This can lead to a lot of mutations.

69
Q

Name five sources of DNA double-strand breaks.

A

Endogenous:

  1. immune system rearrangements
  2. single-strand breaks during DNA replication
  3. meiosis

Exogenous

  1. ionizing radiation (cosmic rays and soils)
  2. medical imaging and treatments
70
Q

Distinguish between the two mechanisms used to repair DNA doublestrand breaks.

A

Non homologous end joining: imperfect, loss of few nucleotides, occurs throughout the cell cycle

Homologous recombination: perfect repair, requires sister chromatid, limited to S and G2 of cell cycle

71
Q

Explain why defects in either of these two modes of double-strand break repair can increase cancer risk.

A

Defects in DNA repair of any kind, including DSB repair allows for accumulation of mutations at a greater than average rate. Thus your chance of getting a mutation that promotes cancer is also increased.

72
Q

Describe three classes of proteins with distinct functions that, when mutated, lead to failure to repair DNA double-strand breaks

A

sensors, transducers, and effectors

Molecules to Medicine Subblock 1 (11 decks)

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