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BIOC 302 Part 2 > DNA Replication 1 > Flashcards

DNA Replication 1 Flashcards

1
Q

central dogma of biology (3)

A
  • genetic information moves to proteins
  • once information is transferred to protein, it cannot be transferred back to RNA/DNA
  • RNA and DNA can pass information back and forth and replicate
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2
Q

what are the challenges that need to be resolved to replicate DNA? (5)

A
  1. DNA copy must be accurate
  2. must be able to free DNA strands from protein and from each other
  3. must be repair/proof-reading mechanisms
  4. must be fast
  5. eukaryotes only: issue with replicating telomeres
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3
Q

telomeres

A
  • the ends of linear DNA
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4
Q

semi-conservative

A
  • describes how DNA molecules contain one strand from original parent DNA molecule and one from newly synthesized strand after replication
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5
Q

DNA polymerase (2)

  • function
  • what does it require
A
  • enzyme that uses DNA as a template to make new DNA copies

- requires primer and magnesium 2+

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6
Q

DNA polymerase primer

A
  • short strand oligonucleotide with free 3’-OH
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7
Q

what is magnesium’s function in DNA replication? (2)

A
  • acts as a co-factor for DNA polymerase by coordinating dNTP in the active site
  • aspartate forms ionic bond with magnesium in the active site
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8
Q

what direction does DNA polymerase catalyze the synthesis of new DNA?

A
  • ONLY in the 5’ -> 3’ direction
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9
Q

fidelity

A
  • the degree of exactness with which something is copied or reproduced
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10
Q

how does DNA polymerase achieve high fidelity (3)

A
  1. Mg2+ ensures proper orientation of dNTP
  2. geometry of active site ensures only correct base pairing can occur
  3. alpha-helix “lid” closes on active site when correct base pairing forms to trap dNTP so catalyses can occur
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11
Q

how much does magnesium, active site geometry and alpha-helix allow mistakes in DNA?

A
  • allows a mistake every 10^4 - 10^5 nucleotides
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12
Q

how does DNA polymerase correct mistakes that do occur?

A
  • 3’ -> 5’ exonuclease activity removes nucleotides one by one from DNA strand
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13
Q

what are the steps involved in DNA polymerase’s exonuclease activity? (3)

A
  1. if mismatch base is incorporated, DNA polymerase will shift to 3’ -> 5’ exonuclease site
  2. mismatch is removed
  3. DNA polymerase will switch back to 5’ -> 3’ active site and continue polymerase activity
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14
Q

what is a downside of DNA polymerase exonuclease activity and why is it still used anyway? (2)

A
  • it is wasteful as it can remove pairings that are not mismatched
  • still used because it does ensure high fidelity
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15
Q

how much does DNA polymerase exonuclease activity allow mistakes in DNA?

A
  • allows a mistake every 10^6 - 10^8 nucleotides
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16
Q

what happens if the exonuclease activity fails to detect a mismatch?

A
  • DNA repair enzymes can correct error after replication
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17
Q

what is the final error rate considering all of the mechanism used to ensure high fidelity in DNA replication?

A
  • a mistake every 10^10 nucleotides
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18
Q

describe the E.coli genome

A
  • circular genome with one oriC
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19
Q

oriC definiton

A
  • origin of replication
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20
Q

oriC structure (2)

A
  • 4-5 repeats of a sequence recognized by DnaA protein

- DNA unwinding elements (DUE)

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21
Q

DNA unwinding elements (DUE) (2)

A
  • 3 tandem repeats (~13 nucleotides each) rich in A and T

- where first strand separation occurs due to weaker H-bonding (only 2 bonds) between A-T

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22
Q

replication fork

A
  • point where DNA is being synthesized

- replication occurs bidirectionally: 2 replication forks per circular genome

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23
Q

does DnaA require ATP (2)

A
  • yes, it required ATP

- ATP is a co-factor for DnaA because it is not hydrolyzed

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24
Q

DnaB (2)

A
  • helicase: separates the DNA helix

- one DnaB per replication fork, so there are 2 DnaB proteins total during DNA replication

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25
Q

DnaC

A
  • ATPase: it hydrolyzes 1 ATP to load DnaB
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26
Q

how does DnaB bind to the replication fork?

A
  • with the help of DnaC, which hydrolyzes an ATP
27
Q

what Dna proteins stay/leave the DNA helix? (2)

A
  • DnaB stays in front of DNA polymerase in each replication fork to separate strands
  • DnaC and DnaA dissociate from DNA after initiation
28
Q

what enzyme is located in front of DnaB during DNA replication? (2)
- function

A
  • topoisomerase, specially DNA gyrase

- remove supercoils formed by DNA unwinding the helicase/DnaB

29
Q

ss binding proteins

A
  • bind to ssDNA during DNA replication to prevent DNA renaturation
30
Q

leading strand (2)

A
  • synthesized continuously

- synthesized in direction that DNA unwinds

31
Q

lagging strand (2)

A
  • synthesized discontinuously in short stretches

- synthesized in the direction opposite to DNA unwinding

32
Q

okazaki fragments

A
  • short stretches of DNA synthesized on lagging strand
33
Q

DNA polymerase I (3)

  • where is it found
  • different activities
  • main function
A
  • DNA polymerase found in E. coli
  • has 3 activities: polymerase, proof-reading (3’ -> 5’ exonuclease), and 5’ -> 3’ exonuclease
  • nick translation: using 5’ -> 3’ exonuclease it removes RNA primers and substitutes them with DNA
34
Q

what does each synthesized DNA segment start with?

A
  • RNA primer
35
Q

what must be done before Okazaki fragments can be stitched together

A
  • RNA primers must be removed
36
Q

how are RNA primers removed form Okazaki fragments (2)

A
  • DNA polymerase I 5’ -> 3’ exonuclease activity removes nucleotides in front of moving enzyme
  • results in nick translation
37
Q

nick translation

A
  • due to removal of RNA primer by DNA polymerase I, ss break in DNA will be moved further down the strand
38
Q

function of DNA polymerase I 5’ -> 3’ exonuclease activity (2)

A
  • removal of RNA primers from Okazaki fragments

- DNA repair

39
Q

DNA polymerase III (4)

  • where it is found
  • physical characteristics
  • function
  • physical features and characteristics
A
  • DNA polymerase found in E. coli
  • large protein with many polypeptide chains
  • one part of enzyme synthesizes leading strand while another part synthesizes lagging strand at the same time (2 active subunits at the same time) using RNA primers as a starting point
  • has high fidelity and processivity due to beta-clamp and clamp-loading complex
40
Q

how does DNA polymerase III have high fidelity and processivity

A
  • it can catalyze many reactions without releasing the substrate (DNA)
41
Q

beta-clamp (2)

A
  • made of 2 beta-subunits that can close on DNA to allow DNA polymerase III to have high processivity
  • 1000 nt/s vs 10 nt/s in DNA polymerase I
42
Q

clamp-loading complex

A
  • δ-loader that can open beta-clamp and thread DNA through
43
Q

what DNA polymerases are used in E. coli

A
  • DNA polymerase I and III
44
Q

what primase is used in E. coli for DNA replication

A
  • DnaG
45
Q

DnaG (2)

A
  • primase used for DNA replication in E. coli

- synthesizes RNA primers (4-5 nt long) on both leading and lagging strand

46
Q

describe discontinuous synthesis in E. coli DNA replication

A
  • every 1000-2000 bp, DNA polymerase III releases lagging strand of DNA and binds it further down the replication fork where primase has synthesized a new primer
47
Q

does clamp loading require ATP?

A
  • yes, it requires 3 ATP molecules
48
Q

DNA ligase (2)

A
  • connects ss breaks and seals nicks between Okazaki fragments
  • requires 1 ATP (2 ATP equiv) molecule per nick
49
Q

ter sites

A
  • special sequences in E. coli that are located opposite to the oriC
50
Q

Tus protein

A
  • bind to ter site sequences and “slow down” the replication fork
  • 2 sets of Tus-ter complexes
51
Q

role of topoisomerase in E. coli DNA replication

A
  • topoisomerase IV resolves catenated DNAs after termination
52
Q

what is happening during the same time as DNA replication in prokaryotes

A
  • cell division
53
Q

how does replication in eukaryotes differ from replication in prokaryotes (9)

A
  1. more complex and not fully understood
  2. larger genomes
  3. linear chromosomes
  4. movement of replication fork is slower
  5. presence of histones
  6. multiple origins of replication per chromosome
  7. okazaki fragments are shorter
  8. different main polymerase enzyme used
  9. primers made and removed by different enzymes
54
Q

how much slower is the movement of the replication fork between eukaryotes and prokaryotes (2)
- why is it slower in eukaryotes

A
  • 50 nt/s vs 1000 nt/s

- mistakes are more detrimental in eukaryotes

55
Q

how much shorter are Okazaki fragments between eukaryotes and prokaryotes

A
  • 100-200 nt vs 1000-2000 nt
56
Q

what is the main enzyme of replication in eukaryotes?

A
  • polymerase δ
57
Q

what makes RNA primers in eukaryotes

A
  • primase: polymerase alpha
58
Q

what removes RNA primers from Okazaki fragments in eukaryotes?

A
  • combination of RNase H and MF1
59
Q

RNase H

A
  • degrades RNA-DNA duplexes
60
Q

MF1

A
  • has 5’ -> 3’ exonuclease activity
61
Q

what problem does DNA polymerase δ have with replicating telomeres

A
  • DNA polymerase δ can only synthesize in 5’ -> 3’ direction, so DNA telomeres are left unreplicated after primer is removed
  • ss overhang is degraded so DNA gets shortened
62
Q

telomere

A
  • linear chromosome ends
63
Q

what is the solution to replicating telomeres?

A
  • telomerase enzyme uses reverse transcription
64
Q

telomerase (2)

A
  • special enzyme in eukaryotes capable of reverse transcription
  • can extend DNA ends by using RNA as a template

BIOC 302 Part 2 (7 decks)

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