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Nucleic Acid Biochem I > DNA Repair and Mutations > Flashcards

DNA Repair and Mutations Flashcards

Readings: 409 - 437 447 - 453

1
Q

What does a second generation mutated molecule of DNA arise from? What percent of second generation molecules will be effected?

A

A first generation replication error or parent strand with altered nucleotide

Only 25% (1/4) DNA molecules will be effected by ONE replication error

50% (2/4) second generation DNA molecules will be effected by a parent strand with an altered nucleotide

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2
Q

How are mismatch repairs carried out?

A
  1. DNA polymerase adds a wrong nucleotide, creating a mismatch. The newly synthesized GATC site is humimethylated
  2. MutS binds the mismatch and forms a complex with MutL
  3. MutS-MutL scan DNA bidirectionally, forming a loop
  4. MutS-MutL finds the nearest GATC site and recruits MutH to cleave the newly synthesized unmethylated GATC sequence
  5. Helicase II and an exonuclease unwind and degrade the newly replicated DNA strand past the mismatch
  6. Pol III fills the gap and ligase seals the DNA
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3
Q

What six proteins are needed to repair mismatched DNA?

A
  • MutS
  • MutL
  • MutH
  • DNA helicase II
  • Pol III
  • Ligase
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4
Q

What is a GATC sequence used for in DNA repair?

A

The complementary sequence, CTAG, is methylated, meaning GATC is hemimethylated. When there is a mismatch during DNA replication, GATC is searched for down the line by MutS and MutL. When found, MutH hydrolyzes everything from MutS-MutL to GATC. This allows Pol III to come in and put in correct nucleotides and ligase to seal the nick

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5
Q

What are base-excisions?

A

Abnormal bases (eg. uracil, hypoxanthine or xanthine), alkylated bases and in some other organisms, pyrimidine dimers being put in.

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6
Q

What 4 proteins are involved in the base-excision repair?

A
  • DNA glycosylases
  • AP endonucleases
  • DNA polymerase I
  • DNA ligase
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7
Q

What is the consequence of base deamination by deaminases?

A

If the amine is taken off bases, they become analogs. Eg. cytosine could become uracil or adenine could become hypoxanthine. This could lead to base-excisions

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8
Q

How are base-excisions and damaged bases repaired in DNA?

A
  1. DNA glycosylase removes the damaged base
  2. Ap endonuclease breaks the phosphate sugar backbone near the damaged base
  3. DNA polymerase I incorporates correct NTPs as new DNA, creating a nick
  4. DNA ligase repairs the nick
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9
Q

What are nucleotide-excisions?

A

DNA lesions that cause large structural changes (eg. pyrimidine dimers)

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10
Q

What proteins are involved in repair of nucleotide-excisions (such as a thymine primer) in prokaryotes?

A
  • UvrA Recognizes lesion
  • Topoisomerase (UvrB)
  • ABS excinuclease (Uvr C)
  • Helicase (UvrD)
  • DNA polymerase I
  • DNA ligase
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11
Q

How are nucleotide-excisions (lesions) repaired in prokaryotes? What is the difference in humans?

A
  1. ABS exinuclease makes two nicks in the sugar phosphate backbone surrounding the DNA lesion
  2. DNA helicase detaches DNA strand containing nick (13 mer)
  3. DNA polymerase I synthesizes new DNA strand
  4. DNA ligase seals nick

In humans the lesion strand is 29 mer and DNA polymerase ε is used to synthesize new DNA

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12
Q

Explain the repair of a thymine dimer in prokaryotes.

A
  1. 2 UvrA recognize the excision and attach to it
  2. a UvrB unwinds the DNA at the excision and UvrAs detach
  3. UvrC creates a 3’ and 5’ nick surrounding excision (about 12’13 nucleotides apart
  4. UvrD detaches the strand thats been nicked (helicase activity)
  5. Pol I and ligase repair the strand
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13
Q

What enzyme directly repairs pyrimidine dimers?

A

DNA photolyases

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14
Q

What part of DNA sequence is particularly vulnerable to UV light?

A

Two adjacent thymines, which can bind to make a pyrimidine dimer

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15
Q

What is the maximum UV absorbance of DNA?

A

260 nm

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16
Q

How are pyrimidine dimers directly repaired?

A
  1. The chromophore in DNA photolyase absorbs a photon of visible light and passes the excitation energy to FADH-
  2. FADH- donates an electron to catalyze bond rearrangements in the pyrimidine dimer
  3. Electron rearrangement restores pyrimidines and is transferred back to FADH to regenerate FADH-
17
Q

What enzyme repairs O6-methylguanine damage?

A

DNA methyltransferase

18
Q

What is O6-methylguanine damage?

A

When guanine is replaced by O6-methylguanine, it has a methyl attached to the 6’ O, which prevents triple bonding with cytosine (like guanine normally would), so O6-methylguanine incorrectly pairs with thymine.

This can arise from methylation of guanine

19
Q

How is O6-methylguanine damage repaired?

A

A methyltransferase takes the methyl group and is then degraded, leaving the guanine to base pair correctly

20
Q

What happens if the DNA replication fork encounters an unrepaired lesion or strand break? How is this repaired?

A

Replication usually halts and the fork may collapse. This is because there is no correct template to direct accurate repair.

Two ways for repair: Recombinational DNA repair and error-prone repair where novel DNA polymerase V can replicate inaccureately over many types of lesions

21
Q

What is done to GATC sequences in strands of DNA?

A

The adenosine nucleoside is methylated in both parental strands, when replication occurs, it takes a while for a daughter strand to me methylated, which is what makes it detectable.

22
Q

Which protein recognizes mismatches?

A

MutS

23
Q

Which enzyme has helicase-like threading capabilities and can make loops up to a GATC sequence in mismatch repair?

A

MutL

24
Q

Which enzyme cleaves unmethylated GATC sequences?

A

MutH

25
Q

What does a deaminated cystine molecule become?

A

Uracil

26
Q

What does DNA glycosylase do to repair damaged bases?

A

Cuts glycosidic bonds between the damaged base and its phosphate.

27
Q

What are triplet expansion diseases?

A

Human diseases caused by the insertion of triplet sequences

Normally this is expressed as repeated sequences going over a certain threshold for repeats. Most commonly, the CAG repeated codon.

28
Q

What proteins are needed to regress a replication fork in order to provide a template for a leading strand that has a lesioned template?

A

Recombinases (RecA)

Pol I

29
Q

When there is a nick in DNA during replication, DNA replication can’t complete, what can fill the nick to allow replication to complete?

A

crossing over with Holliday intermediate junctions can fill the gap. Recombinase proteins are required.

30
Q

When is DNA polymerase V used in E. coli?

A

During its SOS response, E. coli will recruit polymerase V to guess at base pairs for lesioned template DNA. This is an inaccurate process, may cause mutations, but is better than apoptosis.

Nucleic Acid Biochem I (5 decks)

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