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BIOL 0280 UNIT 4 > Lecture 19: DNA Replication and Repair > Flashcards

Lecture 19: DNA Replication and Repair Flashcards

1
Q

DNA Synthesis

What does DNA polymerase require?

A
  • Template (copy)
  • RNA primer with 3’ OH
  • dNTP substrates
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2
Q

DNA Synthesis

What does DNA polymerases catalyze?

A

-Nucleophilic attack by 3’ OH
- Phosphodiester bond
- 5’ → 3’ synthesis

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3
Q

DNA Synthesis

What is a SNUG active site?

A

A SNUG active site can only accommodate “correct” base pairs

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4
Q

DNA Synthesis

In what direction does DNA always occur?

A

DNA synthesis is always 5’ -> 3’

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5
Q

Possible Models For Replication

Describe Semiconservative replication?

A

Hybrid of old and new strands

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6
Q

Possible Models For Replication

Describe Conservative replication?

A

Duplex of only old OR only new

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7
Q

Possible Models For Replication

Describe Dispersive replication?

A

Alternative new/old strand pieces throughout duplex
- Complementarity allows replication with high fidelity

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8
Q

DNA Polymerases

What do DNA polymerases do?

A

Accurately and efficiently replicate the genome
- They join individual nucleotides to produce a new strand of DNA

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9
Q

DNA Polymerases

What does DNA Polymerase III do?

What is a advantageous quality does it have?

A

Replicates the genome

Highest processivity

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10
Q

DNA Polymerases

What is processivity?

A

The number of nucleotides incorporated by a polymerase in a single binding event

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11
Q

DNA Polymerases

What are the two types of exonuclease activity that occur in DNA replication?

A

3’ -> 5’ exonuclease activity
and
5’ -> 3’ exonuclease activity

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12
Q

DNA Polymerases

What does 3’ -> 5’ exonuclease activity involve?

A

Proofreading
- allows for the removal of one base at a time

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13
Q

DNA Polymerases

What does 5’ -> 3’ exonuclease activity involve?

A

Removing primers
- Allows for the removal of many bases at a time

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14
Q

DNA Polymerases

What direction do polymerases work in?

A

All have 3’ -> 5’ exonucleocase activity for proofreading

  • This allows for removal of one base at a time
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15
Q

DNA Polymerases

How often is an incorrect nucleotide inserted?
By how much do DNA Polymerases improve accuracy?

A
  • Inserted approx every 10^4 to 10^5 bps
  • Improves accuracy 10-100x
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16
Q

DNA Replication

What does DNA Polymerase I do?

A

Has 5’ -> 3’ exonuclease activity
- Removing primers
- Allows for the removal of many bases at a time

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17
Q

DNA Replication

What are the three major steps in DNA replication?

A

1) Initiation
2) Elongation
3) Termination

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18
Q

DNA Replication

Where does DNA replication occur in Eukaryotic cells?

A

In the nucleus

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19
Q

DNA Replication: Initiation

What are the the 5 specific sequences and proteins involved in initiation of DNA replication?

A

1) DUE (DNA Unwinding Element)
2) DnaA
3) DnaB
4) DNA gyrase/ topoisomerase II
5) SSB

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20
Q

DNA Replication: Initiation

What does DUE do?

A

DUE = DNA Unwinding Element
- Makes AT rich easier to melt
- It can be unzipped with less energy

AT-rich region is exactly the site where a replication complex is formed and where the DNA synthesis is initiated

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21
Q

DNA Replication: Initiation

What does DnaA do?

A

recognizes oriC sequence (where
duplex splits)

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22
Q

DNA Replication: Initiation

What does DnaB do?

A

Helicase: Unwinds DNA

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23
Q

DNA Replication: Initiation

What does DNA gyrase/topoisomerase II do?

A

relieves strain generated by unwinding

24
Q

DNA Replication: Initiation

What does SSB do?

A

Binds to single-strand DNA
- helps stabalize

25
Q

DNA Replication: Initiation

What steps occur during Initiation?

A

1) DnaA binds at oriC
2) DUE region (part of origin) is
denatured
3) DnaC-dependent loading of DnaB helicase

26
Q

DNA Replication: Elongation

What steps occur during Elongation (part I)?

A

● Starts at the origin of replication (circular DNA)
● Primase lays down RNA primers
○ At origin for leading strand
○ Before each Okazaki fragment in lagging strand
● DnaB helicase unwinds DNA
● DNA polymerase III adds nucleotides
● SSB stabilizes strands
● DNA gyrase relieves strain by unwinding

27
Q

DNA Replication: Elongation

What is the difference between elongating the leading vs. lagging strand?

A

DNA polymerase III can only works from 5’ to 3’

Leading strand synthesis is continuous

Lagging strand synthesis is discontinuous
- Leads to formation of Okazaki fragments
- Primer is laid before each one

28
Q

DNA Replication: Elongation

What does the beta clamp do?
Which strand uses the beta clamp the most?

A

Intended to make sure DNA polymerase does not fall off the DNA strands

  • The lagging strand requires constant loading and unloading of beta clamp, which requires ATP hydrolysis
29
Q

DNA Replication: Termination

What steps occur during termination?

What kind of DNA topoisomerase is involved in this process?

A

1) Replication forks stop in terminus region
2) After termination, the two duplicated chromosomes are catenated (entangled)
3) DNA topoisomerase IV seperates the two chromosomes

30
Q

DNA Replication in Eukaryotes vs. Prokaryotes

Name the 3 major differences in DNA replication in Eukaryotes vs. Prokaryotes

A

Replication in Eukaryotes is…
1) A LOT slower and more complex
2) Chromosomes in eukaryotes are long and linear
3) Initiation requires 2 steps

31
Q

DNA Replication in Eukaryotes vs. Prokaryotes

Initiation in Eukaryotes requires 2 steps, what are they?

A

1) Formation of a pre-RC (replication complex)
- CDK enzymes off (cyclin-dependent protein kinases)

2) Coordinate activation
- CDK enzymes
- Involves ATP hydrolysis

32
Q

Telomeres

What are telomeres, what do they do?

A
  • Repetitive nucleotide sequence at end of chromosome
  • Shorten at end of each replication cycle
  • Don’t care if we lose them (they don’t encode for anything!)
33
Q

Telomeres

What problem do telomeres help fix?

A

Problem with ends of linear chromosomes
- Ends will not get replicated

34
Q

Telomeres

What does Telomerase do?

A

Synthesizes DNA from RNA template (reverse transcriptase)
- Template is part of enzyme

35
Q

Reverse transcription

What is reverse transcription?

A

RNA-dependent DNA synthesis

36
Q

Reverse transcription

What are examples of Reverse Transcriptase?

A

1) HIV (retrovirus since it uses a reverse transcriptase)

3) LINE-1 (retrotransposon)

37
Q

DNA Damage

What are genetic mutations?

A

a permanent change in the DNA sequence

38
Q

DNA Damage

What are the different types of genetic mutations?

Describe them

A

Silent: No effect on Gene function
- No change in AA sequence

Deleterious: impaires gene function

Advantageous: enhances gene function
- Will be selected for and maintained

39
Q

DNA Damage

What are the different types of base substitutions?

A
  • Transitions
  • Transversions
  • Base insertion
  • Base deletion
40
Q

DNA Damage

Describe a transition base substitution
- What do they lead to?

A

A transition occurs when
- pur -> pur
- py -> py

Very frequently leads to synonymous changes in code (so do not change AA sequence in protein)

41
Q

DNA Damage

Describe a transvertion base substitution
- What do they lead to?

A

A transvertion occurs when
- pur -> py or vice versa

Can significantly change protein sequence, less common

42
Q

DNA Damage

What might cause a genetic mutation to occur?

A
  • Deamination
  • Depurination/Alkylation
  • UV Radiation - Thymine Dimer
43
Q

DNA Damage

What is deamination?

What specific deamination is frequent, what does this deamination lead to?

A

It is…
- Removal of amino acid group
- NH2 to a carbonyl

Frequent..
- Deamination of of cytosine is very frequent
- How you can get a Uracil in a DNA molecule

44
Q

DNA Damage

What might cause deamination at high levels?

A

Deaminating agents can induce conversions at high levels

45
Q

DNA Damage

What are examples of Deaminating agents

A
  • Nitrosamine
    -Sodium nitrate
  • Sodium nitrate (nitrous acid precurors)
46
Q

DNA Damage

What is Depurination/Alkylation?

How does this happen, and why?

What is an example of this?

A

Alkylation can change base pairing properties

Happens spontaneously, due to alkylating agents

Example
- O6-methylguanine will
base pair with thymine instead of cytosine

47
Q

DNA Damage

What is the most common of type of modification that UV radiation causes?

A

kink creates block to replication and transcription!

cross-linking (covalent bond) of cyclobutanes of neighboring pyrimidines: formation of thymine dimers

48
Q

DNA Damage

What do people that have impaired repair of UV damage inspired DNA lesions develop?

A

They develop a disease called xeroderma pigmentosum
- extreme light sensitivity

49
Q

DNA Repair

What are the 4 types of DNA Repair Mechanisms?

A

1) Mismatch
2) Base Excision
3) Nucleotide Excision
4) Direct Repair

50
Q

DNA Repair

What type of errors does mismatch repair correct?

How does it correct these errors?

A

Corrects replication errors through methylation

51
Q

DNA Repair

What is the mxn behind mismatch repair?

A
  • MutL-MutS binds to mismatch
  • MutL-MutS + MutH find Me site
  • MutH cleaves unmodified strand
  • Exonuclease activity degreades DNA from the Me past mismatch pair
  • DNA pol II replaces the degraded DNA by copying methylated parent strand
52
Q

DNA Repair

What type of damage does base excision repair?

What enzymes does this repair use?

A

Repairs damages bases
- example:uracil in DNA due to cytosine deamination

Uses different glycolysase for each base lesion

53
Q

DNA Repair

What is the mxn behind base excision repair?

A
  • DNA glycolysase cleaves N-glycosyl bond
  • AP endonuclease removes sugar
  • DNA pol I removes the nick and adds a new base
  • DNA ligase seals nick
54
Q

DNA Repair

What damage does Nucleotide-excision repair fix?

What enzyme does it employ?

A

Removes bulky lesions where more than one base is affected
- Excinuclease

55
Q

DNA Repair

What is the mxn behind nucleotide-excision repair?

A
  • Exinuclease makes two cuts around the affected area to remove damaged DNA
  • DNA helicase removes the damaged DNA
  • DNA pol I adds new bases
  • DNA ligase seals new fragments with old DNA
56
Q

DNA Repair

What does Direct Repair do?

A

Repairs defect directly
- do nor remove any bases or nucleotides

57
Q

DNA Repair

What are the mxn of direct repair?

A

Metabolically expensive
- Uses a “suicide enzyme”
- Cost: one protein per repair (protein CANNOT be used again)

BIOL 0280 UNIT 4 (6 decks)

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