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Molecular Biology > Replication > Flashcards

Replication Flashcards

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1
Q

Meselson and Stahl Experiment

A

Demonstrated the semi conservative nature of DNA replication

  • used centrifugation to distinguish between heavy and light nitrogen
  • grew cells in heavy nitrogen and moved to a medium of of light nitrogen
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2
Q

Replication Forks

A
  • origin of replication

- bidirectional replication

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3
Q

DNA Polymerase

A
  • catalyse replication
  • uses ssDNA as a template to add dNTPs to free 3’ OH group of a base paired nucleotide to synthesize a complementary strand
  • incoming nucleotides selected by Watson Crick base pairing
  • driven by energy of release of pyrophosphate and its subsequent hydrolysis to inorganic phosphate
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4
Q

Polymerase III

A

5’ - 3’ polymerase activity

3’ - 5’ exonuclease activity

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5
Q

Polymerase I

A

5’ - 3’ polymerase activity
5’ - 3’ exonuclease activity
- binds to dsDNA at ss nicks and cleaves beyond the nick to excise the DNA
- nick translation: replace nucleotides on 5’ side of ss nick, translating nick to 3’ end without changing molecule
3’ - 5’ exonuclease activity
- 3-5 activity activated by unpaired 3’ terminal nucleotide with free OH (nucleotide excised)

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6
Q

DNA helicase

A
  • unwinds duplex DNA
  • ATP hydrolysis used to unwind short sections of AT rich DNA: less energy needed
  • binds ssDNA and moves along the duplex prising DNA apart
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7
Q

Single Stranded Binding Proteins

A
  • prevent reannealing of DNA
  • cooperative protein binding straightens region of chain
  • stripped off for replication to occur
  • keeps strands apart
  • stop secondary structure formation
  • aligns strands
  • interacts with replication proteins
  • stimulates polymerases
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8
Q

Topoisomerase

A
  • supercoiling of DNA occurs ahead of helicase
  • tightening of helix will create intolerable strain and energy needed to unwind would be too great
  • nick in one of the strands phosphodiester bonds could allow swivel and relaxation

2 classes: type I makes ss breaks while type II makes staggered ds breaks

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9
Q

Type I topoisomerase

A
  • tyrosine in active site forms covalent phosphodiester link with top ss molecule of DNA
  • allows nick so rotation = relaxation
  • original phosphodiester bond energy conserved = spontaneous reversible reaction
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10
Q

Primase

A
  • synthesizes short RNA primer at replication origin so that DNAP can begin replication
  • forms primosome with helicase
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11
Q

Model of DNA replication

A
  • leading strand synthesized continously in 5-3’ direction
  • lagging strand synthesized discontinously in 3-5’ direction as a series of Okazaki fragments joined to make long nascent DNA chains
  • elongation of lagging strand in opposite direction to the direction of replication fork advance
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12
Q

E. Coli Replication Initiation

A
  • OriC origin of bidirectional replication fork
  • OriC bound by initiator protein DnaA that opens up a segment into single strands
  • DnaC binding permits helicase (DnaB) binding
  • primase activated by helicase
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13
Q

DNAP in E. Coli

A
  • versatile activity
  • 5 - 3 polymerase
  • 5- 3 exonuclease (removal of primers)
  • 3 - 5 exonuclease (proofreading)
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14
Q

Polymerase I

A
  • 3 -5 polymerase/exonuclease
  • 5 - 3 exonuclease
  • lower processivity and polymerisation rate
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15
Q

Polymerase III

A
  • holoenzyme required for survival

- can proofread (3-5 exonuclease) but not repair nicks (5-3 nick translation)

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16
Q

Structure of DNAP III

A
  • DnaB helicase
  • core (a, E, w subunits)
  • B clamp + clamp loader
  • y complex: DNA dependent ATPase
17
Q

B sliding clamp

A
  • clamp is a protein binding DNA pol and prevents enzyme dissociation from DNA (stabilizes molecule)
  • increases processivity (promoting factor in replication)
  • if ds region encountered the clamp releases it
  • clamp loader loads onto the DNA and releases it
18
Q

Accuracy of Polymerase

A
  • reliance on Watson Crick base pairing alone is not sufficient for perfect accuracy

Method 1:

  • correct nucleotide has higher affinity for polymerase as it correctly base pairs with template
  • phosphodiester bond formation involves conformational change in DNAP so that incorrectly bound nucleotide doesn’t fit in active site

Method 2:

  • 3-5 activity of DNAP
  • takes place immediately after incorrect addition to growing chain
  • polymerase can’t extend such a chain as 3-OH is needed
  • clips off mismatches at separate catalytic site
  • newly synthesized DNA transiently unpairs and polymerase has a conformational change to move this editing site into place
19
Q

Replisome

A
  • lagging strand template loops around bringing 2 DNA polymerases into a complex
  • brings 3’ end of a completed okazaki fragment close to the start site for the next fragment
  • trombone model
20
Q

Steps of Replication

A
  1. leading strand synthesised simultaneously and laggin strand begins later (unwinding is RDS)
  2. as DNA unwinds, 3’OH is continually replicated
  3. unwinding and synthesis is concommitant
  4. after enough ssDNA is made, lagging strand synthesiis occurs
    - fragment synthesis nears completion
    - primase binds to DnaB and synthesizes a new primer then dissociates
    - new B clmap loaded onto new primer by clamp loader
    - synthesis of new fragment completed on lag strand
    - lag strand core subunits transferred to the new template primer and its B clamp - old clamp discarded
    - next B clamp readied as fragment synthesis initiated
21
Q

RNA primer removal

A
  • DNAP III elongates chain and falls off
  • DNAP I binds and uses 5-3 exonuclease activity to remove RNA and replace it with DNA using 5-3 polymerase activity
  • DNA ligase links fragments
22
Q

Ligase

A
  • energy input from ATP

- 3-OH and 5-P joined and catalysed by ligase

23
Q

Uses of Replication Enzymes

A
  • recombinant gene technology
  • PCR
  • Sanger method of sequencing

Molecular Biology (11 decks)

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