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Biochem Exam 2 > DNA damage and repair > Flashcards

DNA damage and repair Flashcards

1
Q

two sources of DNA damage/lesions

A

Environmental sources
Endogenous (internal) sources

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2
Q

DNA damage can lead to…

A

genomic instability, apoptosis, senescence
loss of genomic integrity predisposes the organism to…
Immunodeficiency
Neurological disorders
Cancer

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3
Q

how does oxidation and hydroxyl radicals (OH-) cause DNA damage?

A

Hydroxyl radicals (highly reactive species that can be formed from various sources (environmental radiation, ionizing radiation, and certain chemical reactions) oxidize guanine to 8-oxoguanine which base-pairs with A instead of C (aka a G-C pair is replaced by A-T pair)

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4
Q

Depurination

A

a spontaneous reaction (hydrolysis of N-glycosidic bond that links the purine base to the sugar backbone of the DNA
) that removes a purine base ( A or G) from a nucleotide/DNA, and leaves behind an apurinic site (AP site) –> if not repaired before DNA replication, DNA polymerase may insert an inccorect nucleotide opposite of the missing base or insert nothing at all –> leads to a point mutation

More common under conditions of cellular stress, exposure to mutagens, or in environments with increased levels of reactive oxygen species (ROS)

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5
Q

deamination

A

the spontaneous removal of an amino group (NH2) from nucleotide bases
the major type of deamination reaction: converts cytosine to uracil –> C-G pair replaced by U-A pair

additional reactions:
Adenine deaminated to form hypoxanthine: hypoxanthine pairs with C instead of T → leads to a A-T to G-C mutation
Guanine deaminated to form xanthine: xanthine still pairs with cytosine like guanine does → has less drastic effects/mutations

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6
Q

examples of agents that can cause DNA damage?

A

Aflatoxin, psoralen, UV radiation, high energy electromagnetic radiation (ie. X-rays)

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7
Q

aflatoxin

A

the most potent and dangerous groups of mycotoxins (toxic chemical compounds produced by certain species of fungi or mold that can contaminate food products)

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8
Q

aflatoxin B1

A

the major toxin of aflatoxins and is produced by molds that grow on peanuts

it is activated by cytochrome P450 to form a highly reactive species that modifies bases: Aflatoxin B1-8-9-epoxide binds to guanine –> results in a C-G pair replaced by A-T pair

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9
Q

psoralen

A

a product of a chinese herb that intercalates into DNA and when exposed to UV light, it forms covalent bonds with thymine T bases in DNA → creates interstrand crosslinks that prevent the two DNA strands from separating → blocks transcription and replication of DNA

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10
Q

high energy electromagnetic radiation

A

radiation has sufficient energy to ionize molecules and break chemical bonds → causes single-strand or double-strand breaks in DNA → leads to chromosomal rearrangements, deletions, or translocations if not repaired

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11
Q

Xeroderma pigmentosum (XP)

A

disorder in individuals that have a nucleotide excision repair (NER) mutation that causes failure to repair thymine dimers –> eventually leads to lesions and skin cancer

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11
Q

UV radiation (thymine dimers)

A

Promotes COVALENT linkages between 2 ADJACENT pyrimidine bases on the SAME DNA strand → distorts the DNA helix and blocks normal DNA replication and transcription

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12
Q

what happens if DNA damage is left unrepaired?

A

leads to either the substitution of one nucleotide pair for another as a result of incorrect base-pairing during replication (deamination) or to deletion of one or more nucleotide pairs in the daughter DNA strand after DNA replication (depurination)

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13
Q

what are the five DNA damage repair systems?

A
  1. DNA polymerase proofreading
  2. Mismatch repair (MMR)
  3. Base excision repair (BER)
  4. Nucleotide excision repair (NER)
  5. double-strand DNA break repair (DSBR)
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14
Q

what types of DNA damage do each of the repair systems fix?

A

DNA polymerase proofreading: mismatches in newly synthesized DNA strand
Mismatch repair: mismatches and small indels that were not fixed by DNA polymerase
base excision repair: deamination and depurination (small non-helix-distorting base lesions)
nucleotide excision repair: UV radiation (thymine dimers); bulking helix-distoring lesions
double-strand DNA break repair: when both DNA strands are broken

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15
Q

nonhomologous DNA end joining (NHEJ)

A

quick but error-prone repair mechanism that does NOT require a homologous template –> DNA is lost
used primarily during the G1 phase where no sister chromatids are available
1. Nuclease: processes the broken ends and makes them into clean breaks
2. DNA ligase: joins the ends together

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16
Q

homologous recombination (HR)

A

a more accurate repair mechanism that uses a sister chromatid as a template to repair double-strand breaks; primarily active in the S and G2 phases of cell cycle when sister chromatids are available
1. Nuclease digests the 5’ ends of the two broken strands at the break to make them a clean break
2. Strand invasion by complementary base-pairing
3. Repair polymerase synthesizes DNA using the undamaged complementary DNA as a template
4. Invading strand is released and the complementary base-pairing allows broken helix to form
5. DNA synthesis continues using complementary strands from damaged DNA as a template
6. DNA ligase seals the nicks

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17
Q

What are the two regions in DNA polymerase?

A

Polymerization site or polymeryzing activity region (P site) and Exonuclease site or editing activity region (E site)

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18
Q

describe how DNA polymerase proofreading works

A

proofreading occurs simultaneously with DNA synthesis but in 2 different regions.
Recognition: Before DNA adds the next nucleotide, it checks the previous one to see if it is correctly matched
Excision: If it adds an incorrect nucleotide, the newly synthesized DNA strand transiently unpairs from the template strand and DNA polymerase shifts the 3’ end of the synthesized DNA strand into the editing site (E site: exonuclease active site domain) of DNA polymerase and the mispaired base is removed
DNA polymerase shifts the synthesized DNA strand back into the P site and elongation continues

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19
Q

what is the error rate of DNA replication with proofreading

A

one mistake per 10^7 nucleotides copied

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20
Q

what is the basic mechanism of DNA repair?

A
  1. excision
  2. resynthesis: repair DNA polymerase
  3. ligation: DNA ligase
21
Q

mismatch repair (MMR) in E. Coli

A

important proteins: MutS, MutL, and MutH
1. MutS recognizes and binds to mismatch and then recruits MutL
2. MutH distinguishes between the new DNA and parent strands and cleaves the backbone in the vicinity of the mismatch
3. Exonuclease I removes a segment of the DNA strand containing the erroneous T
4. DNA polymerase III synthesizes a new DNA

two proteins are required for mismatch repair: one to recognize the
error and one to recruit an endonuclease to cleave the DNA

22
Q

how does MMR recognize which DNA strand contains the error?

A

newly synthesized DNA strands are temporarily unmethylated; parent/template DNA strands are methylated (MutH protein recognizes the difference)

22
Q

base excision repair (BER) steps

A
  1. AlkA enzyme recognizes and removes the alkylated bases, leaving behind an AP site (spot in DNA where a base has been removed)
  2. AP endonuclease: cleaves the DNA backbone at the AP site, creating a single-strand break
  3. ligation: DNA polymerase fills the gap and DNA ligase seals the backbone nick
23
Q

nucleotide-excision repair (NER) in bacteria

A

UvrA-UvrB complex: recognizes and binds to the thymine dimer
UvrC excinuclease: makes two cuts (one on 5’ side and one on 3’ side of lesion) → excising the damaged DNA fragment
DNA polymerase I: synthesizes the correct sequence using the undamaged DNA strand as a template to feel the gap; and then DNA ligase follows behind to seal the nick and complete the repair

24
Q

what is the structure of AlkA enzyme and flipping mechanism?

A

AlkA binds tightly to the damaged DNA → forms a complex that allows precise interaction between AlkA and the AP site
Flipping mechanism: when AlkA encounters an AP site, the sugar-phosphate backbone at the AP site FLIPS OUT from the regular structure of the DNA double helix (undergoes a conformational change) and INTO the active site of the enzyme

25
Q

Why is thymine used in DNA instead of uracil? give reasoning in terms of cytosine deamination

A

Presence of T instead of U in DNA permits the repair of deaminated cytosine → preserves the integrity of genetic information

  • C in DNA can spontaneously deaminate to become U → would lead to an C-G replaced by A-U pair after the next round of replications
  • If U was a natural base in DNA, the repair machinery would not be able to distinguish between a legit U and a U formed by C deamination (that should not be there and causes damage)
    –> THUS the use of T allows the DETECTION of deamination of cytosine: to prevent this confusion, DNA uses T instead of U such that when C deaminates to U, it is easily recognized and replaces the U with C
  • If U IS detected in DNA it is removed by uracil DNA glycosylase and the site is repaired with the insertion of C
26
Q

sickle cell anemia

A

reduced ability to carry oxygen and their tendency to block blood flow in small vessels
caused by a single nucleotide change (A–>T) aka a point and missense mutation –> normal Hb –> abnormal HbS

27
Q

what are 3 examples of the consequences of failure to repair DNA damage

A

a. xeroderma pigmentosum (failure to repair thymine dimers)
b. sickle cell anemia (recessive hereditary; point and missense mutation)
c. cancer

28
Q

how do mutations cause cancer?

A

Gradual accumulation of changes in DNA (DNA damaged) in somatic cells leads to cancer
Defects in DNA repair systems: mutation frequency increases → likelihood of cancer-causing mutations increase
Age is the LARGEST factor of cancer incidence

29
Q

Leob Lab experiment design and results?

A

tested how mutation rates influenced the evolutionary success of bacterial populations by generating 66 DNA polymerase I mutants in E. coli with comparable growth properties but with differing DNA replication fidelities (spanning 10^3 fold higher and lower than that of wild type E. coli)
- all E. coli strains were mixed and cultured for 31 days

findings: The bacterial strains that DOMINATED at the end have MODERATELY enhanced mutation rate of DNA polymerase
- extreme mutators did not fare any better in competition: two mutators with rates of around 150- and 110-fold greater than that of wild type failed to survive to the end of the competition
- Moderate mutators evolve MORE efficiently than wild type in pairwise competition

30
Q

2015 Nobel prize in chemistry?

A

Tomas Lindahl: base excision repair
Paul Modrich: mismatch repair
Aziz Sancar: nucleotide excision repair

They delineated the mechanisms of BER, MMR, and NER
significance: challenged and dismissed the early view that the DNA molecule was very stable
→ paved the way for the discovery of human hereditary diseases associated with distinct DNA repair deficiencies and a susceptibility to cancer
- brought a deeper understanding of cancer and neurodegenerative or neurological diseases → led to novel strategies to treat cancer

31
Q

What are translesion or error-prone polymerases?

A

special DNA polymerases that can replicate across the damage and generate a rough draft of the damaged sequence

32
Q

What are examples of environmental sources of DNA damage?

A

Ultraviolet light (UV), toxic chemicals, ionizing radiation (IR)

33
Q

What are examples of endogenous sources of DNA damage?

A

Cell’s own metabolites, reactive oxygen species (ROS), hydrolysis, oxidations, alkylation

34
Q

what are the 4 main types of aflatoxins?

A

B1, B2, G1, G2

35
Q

How do alkylating agents cause mutations?

A

Alkylating agents can modify bases, often by transferring an alkyl group (a carbon-containing group) to the nitrogen or oxygen atoms of the base
ie. Guanine can be alkylated to form O6-methylguanine, which pairs with thymine instead of cytosine, leading to G→A transitions
- crosslinking, base mispairing, strand breaks

36
Q

Depurination and deamination

A

they are the MOST FREQUENT spontaneous chemical reactions known to produce endogenous mutations and serious DNA damage in cells
Both take place on the double-helical DNA and neither break the phosphodiester backbone

37
Q

what are the clinical effects of aflatoxins?

A

causes death, liver cancer, reproductive problems, anemia, immune system suppression, and jaundice

38
Q

what is cytosine deamination and what does it result in?

A

cytosine deamination (removal of NH2 group) results in uracil
–> C-G base pair becomes a U –> A base pair instead

39
Q

What is 5-methylcytosine deamination and what does it result in?

A

5-methylsytosine deamination results in thymine
–> C-G base pair becomes a T–> A base pair instead

40
Q

what does it mean for something to be methylated?

A

the molecule gains a CH3 group (ie. cytosine vs 5-methylcytosine)

41
Q

What is the difference between amino acid changes and DNA sequence changes?

A

A point mutation in the context of PROTEINS would be a change in amino acid sequence (ie. sequence of amino acids: arginine-lysine-histidine –> arginine-lysine-lysine)
a point mutation in the context of DNA are describing SINGLE NUCLEOTIDE changes (ie. ATGC –> AGGC)

a point mutation in DNA does NOT always result in a point mutation in the encoded protein: ie. a DNA point mutation may result in an mRNA sequence change of AUU –> AUC, but in this case, both codons code for for the same amino acid, so you won’t get a point mutation in the protein

BUT some point mutations changes BOTH the DNA sequence and the amino acid sequence: ie. the point mutation in the DNA sequence from AUU –> UUU ALSO is an amino acid point mutation since they code for two different amino acids

42
Q

what is the error rate for DNA replication WITHOUT proofreading?

A

one mistake per 10^5 nucleotides

43
Q

what is the error rate for DNA replication with proofreading AND MMR?

A

one mistake per 10^9 nucleotides copied
- to put into perspective, the human entire genome is around 3x10^9 base pairs of DNA

44
Q

What is the structural difference between thymine and uracil?

A

thymine has a methyl group CH3 in its C5 position
uracil has an H group in its C5 position
(aka U is just T methylated)

45
Q

what is uridine repair, when is it used, and what are the key steps?

A

Uridine bases in DNA, formed by the deamination of
cytidine, are excised and replaced by cytidine

  1. Uracil DNA glycosylase: cleaves the N-glycosidic bond between the U base and the sugar-phosphate backbone to leave an AP site
  2. AP endonuclease: recognizes the AP site and cuts the DNA backbone at this location which creates a nick in the DNA strand near the lesion
    → a phosphodiesterase enzyme: removes the deoxyribose sugar and phosphate group at the AP site, leaving a small gap in the DNA
  3. DNA polymerase I: fills the gap with the correct base; DNA ligase: seals the nick by forming a phosphodiester bond between the new nucleotide and the existing DNA strand
46
Q

How does the defective repair of DNA cause many cancers?

A

the gradual ACCUMULATION of DNA mutations/damage in somatic cells leads to cancer: aka the defects in DNA repair systems increase mutation frequency, which increases the likelihood of cancer-causing mutations

  • age is the largest factor of cancer incidence
47
Q

direct repair definition

A

forms of repair that correct mistakes WITHOUT having to
remove any fragments of DNA

48
Q

what is an example of direct repair?

A

DNA photolyase: uses light energy to cleave pyrimidine dimers

49
Q

How does the nick direct mismatch repair machinery?

A

the nick in NDA could be a signal of the newly synthesized DNA strand (another signal other than methylation)
the nick directs MMR machinery to correct DNA strand

Biochem Exam 2 (6 decks)

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