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

Lecture 19: DNA Replication and Repair Flashcards

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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?

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A

relieves strain generated by unwinding

24
Q

DNA Replication: Initiation

What does SSB do?

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A

Binds to single-strand DNA
- helps stabalize

25
*DNA Replication: Initiation* What steps occur during Initiation?
1) DnaA binds at oriC 2) DUE region (part of origin) is denatured 3) DnaC-dependent loading of DnaB helicase
26
*DNA Replication: Elongation* What steps occur during Elongation (part I)?
● 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
*DNA Replication: Elongation* What is the difference between elongating the leading vs. lagging strand?
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
*DNA Replication: Elongation* What does the beta clamp do? Which strand uses the beta clamp the most?
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
*DNA Replication: Termination* What steps occur during termination? What kind of DNA topoisomerase is involved in this process?
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
*DNA Replication in Eukaryotes vs. Prokaryotes* Name the 3 major differences in DNA replication in Eukaryotes vs. Prokaryotes
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
*DNA Replication in Eukaryotes vs. Prokaryotes* Initiation in Eukaryotes requires 2 steps, what are they?
1) Formation of a pre-RC (replication complex) - CDK enzymes off (cyclin-dependent protein kinases) 2) Coordinate activation - CDK enzymes - Involves ATP hydrolysis
32
*Telomeres* What are telomeres, what do they do?
- 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
*Telomeres* What problem do telomeres help fix?
Problem with ends of linear chromosomes - Ends will not get replicated
34
*Telomeres* What does Telomerase do?
Synthesizes DNA from RNA template (reverse transcriptase) - Template is part of enzyme
35
*Reverse transcription* What is reverse transcription?
RNA-dependent DNA synthesis
36
*Reverse transcription* What are examples of Reverse Transcriptase?
1) HIV (retrovirus since it uses a reverse transcriptase) 3) LINE-1 (retrotransposon)
37
*DNA Damage* What are genetic mutations?
a permanent change in the DNA sequence
38
*DNA Damage* What are the different types of genetic mutations? Describe them
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
*DNA Damage* What are the different types of base substitutions?
- Transitions - Transversions - Base insertion - Base deletion
40
*DNA Damage* Describe a transition base substitution - What do they lead to?
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
*DNA Damage* Describe a transvertion base substitution - What do they lead to?
A transvertion occurs when - pur -> py or vice versa Can significantly change protein sequence, less common
42
*DNA Damage* What might cause a genetic mutation to occur?
- Deamination - Depurination/Alkylation - UV Radiation - Thymine Dimer
43
*DNA Damage* What is deamination? What specific deamination is frequent, what does this deamination lead to?
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
*DNA Damage* What might cause deamination at high levels?
Deaminating agents can induce conversions at high levels
45
*DNA Damage* What are examples of Deaminating agents
- Nitrosamine -Sodium nitrate - Sodium nitrate (nitrous acid precurors)
46
*DNA Damage* What is Depurination/Alkylation? How does this happen, and why? What is an example of this?
Alkylation can change base pairing properties Happens spontaneously, due to alkylating agents Example - O6-methylguanine will base pair with thymine instead of cytosine
47
*DNA Damage* What is the most common of type of modification that UV radiation causes?
kink creates block to replication and transcription! cross-linking (covalent bond) of cyclobutanes of neighboring pyrimidines: formation of thymine dimers
48
*DNA Damage* What do people that have impaired repair of UV damage inspired DNA lesions develop?
They develop a disease called xeroderma pigmentosum - extreme light sensitivity
49
*DNA Repair* What are the 4 types of DNA Repair Mechanisms?
1) Mismatch 2) Base Excision 3) Nucleotide Excision 4) Direct Repair
50
*DNA Repair* What type of errors does mismatch repair correct? How does it correct these errors?
Corrects replication errors through methylation
51
*DNA Repair* What is the mxn behind mismatch repair?
- 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
*DNA Repair* What type of damage does base excision repair? What enzymes does this repair use?
Repairs damages bases - example:uracil in DNA due to cytosine deamination Uses different glycolysase for each base lesion
53
*DNA Repair* What is the mxn behind base excision repair?
- 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
*DNA Repair* What damage does Nucleotide-excision repair fix? What enzyme does it employ?
Removes bulky lesions where more than one base is affected - Excinuclease
55
*DNA Repair* What is the mxn behind nucleotide-excision repair?
- 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
*DNA Repair* What does Direct Repair do?
Repairs defect directly - do nor remove any bases or nucleotides
57
*DNA Repair* What are the mxn of direct repair?
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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