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Flashcards in DNA Modification And Repair Deck (36):
1

Form of normal DNA modification

Methylation

2

What are the major methylated bases in prokaryotes?

Adenine and Cytosine

-happens after replication

N6 methyladenine

N4 methylcytosine

3

What is the role of methylation in bacteria?

Methylation in bacteria occurs at specific sites

Protects the bacteria's DNA from cleavage by restriction endonucleases

4

What is the functional importance of methylation of adenine in E.coli?

Methylation of adenine residues in the sequence GATC is involved in mismatch error correction

Can be on either A or C or both

5

Cytosine in Eukaryotes

The only normal base modification in eukaryotes

5-methylcytosine

3-5% cytosine content of most DNA

6

Where is the methylation present in Eukaryotes?

5-methylcytosine is usually found in C residues 5' to G

When a C in one strand is methylated, the C in the complementary strand is also methylated

7

What does it mean for methylation in eukaryotes to be heritable?

1) Sites of new methylation can be selected during gametogenesis by a de novo methylase

2) Not all C's are methylated

3) fertilization and replication

4) Methylation is carried out by a maintenance methylase after replication

8

How does eukaryotic methylation leave room for error recognition?

Daughter strands not immediately methylated after replication --> so you can do error repair earlier on

9

How does methylation control gene expression?

Unmethylated promoters expressed to produce the protein

Methylated promoters are inactive and are not expressed

10

What is 5-azacytidine?

Inactivity of methylated genes reversed by treatment of 5-azacytidine

5-azacytidine is a cytosine analog that can be metabolized into dCTP and incorporated into DNA

11

How does 5-azacytidine work as a potential treatment for beta-thalassemia?

1) a normal methylated DNA goes through replication in 5-azacytidine

2) The cystosine in the daughter strands are actually 5-azacytidine

3) Replication of these new DNA leads to methylation after

4) one daughter DNA is normally methylated (due to the original methylation on that strand)

5) one daughter DNA has a loss of methylation leading to gene expression

12

Factors that cause DNA mutation

-Mistakes during replication

-Reactive oxygen species (ROS)

-Chemical damage

-Radiation

-ionizing agents

-Deamination of a cytosine or 5-methylcytosine

13

What base does a deamination of 5-methylcytosine (5mC) lead to?

It become thymine (Bc of that CH3 on C5)

Would become uracil if not methylated cytosine

14

How does 5-methylcytosine cause a transition mutation?

1) Deamination of methylated cytosine

2) Changed to a thymine

3) Replication of this leads to a daughter DNA which makes an originally what should be a GC base pair become an AT base pair

4) one daughter DNA is normal

15

Why is Deamination of 5-methylcytosine seen as dangerous if not repaired?

Thymine is not seen as an abnormal base by repair mechanisms but it's legit changing the gene sequence that the DNA should be encoding

16

What is a transition point mutation?

Purine-pyramidine base pair changed into a different purine-pyramidine base pair


Ie: CG --> TA

17

What is a transverse on point mutation?

A purine-pyramidine base pair is changed into a pyrimidine-purine base pair

Ie: AT ---> TA

18

Deletion or insertion mutations- causes and examples

Addition or removal of one or more base pairs

Caused by intercalating agents that fit in between adjacent base pairs of the double helix
Ie: ethidium bromide used in labs or doxorubicin used to treat lymphoma

Can also be caused by transposable elects, errors in replication, and repeating elements such as the triplet repeats

19

How does an insertion/deletion mutation work?

DNA is a 3 base code in which 3 bases code for 1 amino acid

Insertion/deletion of 1 nucleotide into a gene shifts the reading frame

20

What does photodimerization result in?

Results in infra strand dimerization of adjacent thymines

Catalyzed by UV light

Usually dimerizes pyrimidines: TT, CC, CT

21

What are the 2 sorts of dimers that can result?

1) cyclobutane thymine dimer (bonds are 2 vertical lines between C6-C6 and C5-5 on adjacent thymines

2) 6-4 Photoproduct (bond is diagonal between C6-C4 on adjacent thymines)

22

What is the main target for base damage/modification?

Primarily Purines

Can be lethal if not treated

23

How does O6-alkylguanine work?

Has a high probability of being base paired with thymine during replication

This leads to a GC to AT transition if not repaired

24

Repair of O6-alkylguanine? - prokaryotes and eukaryotes

O6-alkylguanine [or methylguanine] DNA methyl transferase (MGMT) transfers the methyl from O6- methylguanine to itself

MGMT self alkylates and restores the guanine

*if you can change the guanine before replication, you can change the base pairing back to C*

25

Excision repair- basic idea

Damaged DNA is recognized, removed, and then replaced by DNA polymerase

26

Nucleotide excision repair (NER)

Repairs intra strand thymine dimers caused by UV irradiation

1) UvrAB complex scans DNA 3' --> 5'

2) Damange DNA site causes UvrAB complex to stop

3) UvrAB complex bends DNA

4)Dissociation of UVrA and binding of UvrC to UvrB

5) UvrBC complex endonuclease activity cuts both sides of the dimer

6) Helicase unwinds damaged DNA

7) Polymerase 1 fills in the gap

8) backbone rejoined by DNA ligase

27

Function of DNA-N-glycosylases

They remove incorrect bases in DNA

Ie: Uracil that can appear as a result of Deamination of cytosine Deamination --> can cause a GC to AT transition mutation

28

Base Excision Repair of mismatched uracil by Uracil DNA N-glycosylase (prokaryotes)

1) Recognition of damage

2) Base removed from back bone by Uracil-DNA N-glycosylase

3) Endonuclease cute backbone 5' to the damage

4) Nick translation by DNA polymerase I--> DNA pol I ends up replacing more than 1 base because of nick translation

5) Nick in backbone sealed by DNA ligase

29

What is a apyrimidinic site?

A site without a pyrimidine base Bc it easy removed as seen in base excision repair for mismatched uracil

30

2 BER pathways for eukaryotes

Short patch BER similar mechanism to prokaryotes

Long patch BER takes over if 5' to abasic site is refractive to cleavage by DNA pol epsilon

31

How does mismatch repair work?

During replication DNA proofread for mistakes by DNA polymerase III epsilon subunit

Nacent DNA also scanned for errors- mismatched bases and single base insertions or deletions

If found corrected by mismatch repair.

32

Mismatch repair- MutHLS system

1) Recognition of damage by MutS

2) Using ATP hydrolysis for energy, MutS pulls DNA in from both directions until reaching a GATC sequence, the sequence in E.coli where A is methylated

3) MutH Endonuclease cleaves the unmethylated DNA strand 5' to the G in the GATC sequence

4) DNA Helicase II unwinds the DNA back past the mismatch

5) An exonuclease then removes the damaged DNA

6) DNA polymerase III and DNA ligase then fill the gap and seal the nick

33

MutS, MutL, MutH

MutS- DNA mismatch/damage recognition

MutL- molecular matchmaker; Endonuclease; termination of mismatch-provoked excision

MutH- strand discrimination

34

CMMRD

Constitutional mismatch repair deficiency

Rare autosomal recessive syndrome caused by homozygous mutations in mismatch repair genes

35

Nucleotide excision repair in eukaryotes- Global genome

Probes the genome for helix-distorting lesions

36

Nucleotide excision repair in eukaryotes- transcription coupled

Removes transcription blocking lesions to permit unperturbed gene expression