Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

LS3 > The Mutability and Repair of DNA > Flashcards

The Mutability and Repair of DNA Flashcards

1
Q

What are some sources of mutations/damage to DNA?

A
  • inaccuracy in DNA replication
  • spontaneous damage
  • exogenous damage: radiation, chemicals, by-products of cellular metabolism
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

What are 2 major consequences of DNA damage?

A
  • mutations (gene or regulatory sequences)

- lesions or structural changes to the DNA can prevent its use as a template for replication or transcription

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

What are point mutations and what are the two types that exist?

A

They alter a single nucleotide 1. transitions: purine to purine or pyrimidine to pyrimidine
A - G or T - C
2. transversions: purine to pyrimidine or vice versa

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

What are 4 different mutations that can occur in coding regions?

A
  1. silent
  2. missense
  3. nonsense
  4. frameshift
    - base addition
    - base deletion
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

silent mutation

A

doesn’t change the amino acid

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

missense mutation

A

a change in the amino acid

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

nonsense mutation

A

stops protein synthesis because it turns into a stop codon

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

frameshift mutation

A

base is added or deleted to change the animo acid sequence from that point on

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

A major limit to DNA replication accuracy is___.

A

The occasional flickering of the bases into the wrong tautomeric form
amino –> imino
keto–> enol

The alternate tautomer for each base changes its base-pairing specificity. It allows the incorrect bp to be correctly positioned for catalysis during DNA replication and incorporated into the daughter DNA.
1 in 10^5 nts

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

DNA polymerase has _____ proofreading activity.

A

3’–> 5’ exonuclease activity

this is a separate active site on the same polypeptide as the polymerase active site; it detects and removes mistakenly added nucleotides.

decreases the error rate of DNA replication by a factor of 100

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

The 3 stages that define the accuracy of DNA replication.

A
  1. DNA polymerase incoporation “kinetic proofreading”
  2. 3’ exonuclease activity of DNA polymerase
  3. postreplication mismatch repair
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

Mismatch Repair

A

A second chance to repair replication errors that escape proofreading

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

What are the two challenges of mismatch repair?

A
  1. find mismatches in the genome quickly. They are transient and after a second round of replication the mismatch is eliminated and leads to a mutation that won’t be detected
  2. repair the mismatch accurately; choose the correct nucleotide to replace
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

How does Mismatch repair occur in E.coli?

A
  1. MutS (dimer) scans the dsDNA and detects mismatches based on the distortion to the DNA structure. This forms the MutS-DNA complex where MutS binding to a mismatched bp induces a kink in the DNA and a conformational change in the MutS protein.
  2. MutL is then recruited and activates MutH. MutH is an endonuclease that creates a single stranded nick in the DNA near the mismatch. It binds to the hemimethylated GATC sites following replication, but its endonucelase activity is only activated when MutS/MutL detects a mismatch nearby. Following activation, MutH selectively nicks the unmethylated DNA strand
  3. an Exonuclease digests the DNA from the nick past the mismatched nucleotide
  4. single strand gap is filled in by DNA polymerase and sealed by DNA ligase
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

How do you identify the parental template from the newly synthesized strand?

A

(in E.coli only)
Dam methylase: methylates “A” residues at 5’ GATC 3’ sequences on parental strands; hemimethylated
-newly synthesized daughter strands are initially unmethylated

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

T/F. Mismatch repair can require either 5’ or 3’ exonuclease activity.

A

true

17
Q

How does mismatch repair work in Eukaryotic cells?

A

Eukaryotic mismatch repair machinery includes MutS, MutL homologs (MSH and MLH)

  • mutations in MSH and MLH genes leads to a genetic predisposition to colon cancer
  • no MutH homolog; hemimethlation is not used to tag the parental strands; okazaki fragments?
18
Q

Hydrolysis

Spontaneous DNA damage

A
  • unnatural bases/ alterations
    1. spontaneous deamination of cytosine generates uracil
    2. spontaneous depurination produces an abasic/ apurinic site in the DNA where the deoxyribose sugar lacks a purine base
  • natural base/alteration
    3. deamination of 5-methylcytosine generates thymine. this will not be recognized as an abnormal base. It can lead to a transition mutation if unprepared
19
Q

Radiation- UV light

A

thymine dimer is formed where it is incapable of base pairing and interferes with DNA replication and transcription

20
Q

O6-methylguanine

DNA damage

A

methylation of guanine to O6-methylguanine will mispair with thymine. after additional DNA replication this will lead to a GC to AT transition mutation

21
Q

Intercalating Agents

DNA damage

A

cause the deletion or addition of a base pair or a few; can lead to frameshift mutations

i.e- ethidium, proflavin, acridine orange

22
Q

What is the most cytotoxic DNA damage that can occur?

A

Ionizing radiation like X-rays cause double stranded breaks in the DNA backbone!

23
Q

What are two direct reversal mechanisms of DNA damage?

A
  1. photo reactivation: damaged bases are directly repaired: DNA photolyase breaks the covalent bonds linking adjacent pyrimidines like thymine dimers, using energy from light
  2. methyl group removal from O6-methylguanine
24
Q

What is Base excision repair?

A
  1. DNA glycosylase removes damaged bases by hydrolyzing the glycosidic bond between a damaged base and its deoxyribose sugar. This generates an basic (OH) residue in the backbone. DNA glycosylases are specific. There are 11 in human cells.
  2. Abasic residue is excised and replaced. How? The basic residue gets excised from the DNA backbone via endonuclease/exonuclease activity, leaving both a free 3’OH group and a 5’phosphate. A repair DNA polymerase fills in the gap using the undamaged strand as template and DNA ligase seals the nick.
    Endonucleolytic cleavage also removes abasic residues that arise from spontaneous hydrolysis like depurination of G residue
25
Q

What about the T:G mismatch that can arise from spontaneous deamination of 5-methylcytosine?

A

There is no ‘damaged’ base, just a mismatch.

Thymine DNA glycosylase (G/T mismatch-specific thymine glycolsylase) selectively removes a T opposite a G, by ‘assuming’ that the T arose from deamination of 5-methylcytosine (which occurs frequently in vertebrate DNA)

26
Q

How do DNA glycosylases detect damaged bases if they are buried in the helix?

A

Structure of DNA-Glycosylase complex. Base flipping of the damaged base places it in the catalytic center of the glycosylase

27
Q

Nucleotide Excision Repair (NER)

A

•Recognizes and removes bulky lesions that distort the shape of the double helix (e.g., a thymine dimer or a bulky chemical group on a base)
•Similar repair pathways found in all organisms, from bacteria to humans
•Mutations in genes involved in nucleotide excision repair lead to UV light sensitivity
•Basic Mechanism:
–DNA lesion is identified
–DNA is melted around the lesion (ss)
–The damaged DNA strand is cleaved on either side of the lesion by an endonuclease and a ss fragment is removed
–The ss gap is filled in by DNA polymerase and ligase, using the undamaged strand as template

28
Q

Explain NER in E.coli.

A
  1. UvrA-UvrB complex scans the DNA to detect distortions to the helix
  2. UvrB melts the DNA around the lesion, making a ssDNA bubble
  3. UvrC cleaves the damaged DNA strand on either side of the lesion (5’ and 3’)
  4. UvrD – a DNA helicase – removes the damaged DNA fragment (ss)
  5. DNA Polymerase and DNA ligase fill and seal the gap
29
Q

Xeroderma Pigmentosum

A
  • a genetic disease that makes individuals extremely sensitive to sunlight; gives rise to skin lesions and a high frequency of skin cancer
  • can develop from mutations in 7 different genes in humans that are involved in NER (‘XP’ genes)
  • Cells have limited ability to repair UV-induced DNA damage, causing increased mutagenesis and cell death
30
Q

Transcription-Coupled Repair

A

RNA Polymerase can serve as another damage sensing protein in the cell:

1) RNAP stalls at a lesion in the DNA template and transcription stops
2) NER proteins are recruited and repair the lesion

31
Q

DSB Repair

A
  1. Recombination-based DSB repair
    •retrieves sequence information from the sister chromosome (after DNA replication)
    •Error-free process – precise restoration of the original sequence across the site of the break
    •predominant mechanism to repair breaks in yeast cells
    •What about early in the cell cycle before two sister chromosomes have been generated by DNA replication? It won’t work.
    **polymerase and ligase is required
  2. Non-homologous end joining (NHEJ):
    •The predominant mechanism of DSB repair in multicellular eukaryotic organisms
    •Mutagenic/error-prone repair of DSBs
    –The NHEJ enzymes process the free ends of a DSB and DNA sequence can be lost before the two strands are ligated together
    –Still less cytotoxic than leaving the DSB unmended!
    •Ku70 and Ku80 bind the broken DNA ends and recruit the kinase, DNA-PKcs
    •DNA-PKcs recruits Artemis, an enzyme that processes the broken ends and prepares them for ligation
    •Ligation of the broken ends occurs via Ligase
32
Q

Translesion DNA Synthesis ‘Tolerating’ DNA Damage

A
  • Translesion DNA synthesis may occur if a DNA lesion (e.g., thymine dimer or apurinic site) is not fixed prior to DNA replication
  • Considered a last resort – DNA replication may proceed but the process is highly error-prone
  • Translesion DNA polymerases (2 in E. coli; 5 in humans)
  • Nucleotides are incorporated in a way that does not depend on base pairing with the template

LS3 (7 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions