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ML2001: Introductory Molecular Biology > DNA Metabolism - Replication > Flashcards

DNA Metabolism - Replication Flashcards

1
Q

What is the function of Polymerases?

A

Synthesis DNA

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2
Q

What is the function of Nucleases?

A

Digest DNA
(endo and exonucleases)

Endo - cleave in middle; Exo - cleave at end

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3
Q

What is the function of Ligases?

A

Ligate (join) DNA fragments

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4
Q

When does DNA replication occur?

A

During cell division

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5
Q

When does DNA synthesis occur?

A

During S phase of cell cycle

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6
Q

What are the SIX fundamental rules for DNA replication?

A
  1. Semi-conservative
  2. Starts at origins
  3. Always synthesised 5’ to 3’
  4. Semi-discontinuous
  5. Synthesised by DNA Polymerases
  6. Very accurate
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7
Q

How many origins of replication do Eukaryotic cells have?

A

Multiple

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8
Q

How many replication forks does E. coli have?

A

2

(Bidirectional)

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9
Q

What end are nucleotides added to during DNA synthesis?

A

3’ end of new strand

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10
Q

What direction are template strands read in?

A

3’ to 5’ end

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11
Q

Which strand of DNA is synthesised continuously?

A

Leading strand

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12
Q

Which strand of DNA is synthesised in fragments?

A

Lagging strand

(Ozaki fragments)

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13
Q

Which DNA Polymerase is involved in adding nucleotides to the 3’ end of DNA?

A

DNA Pol I

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14
Q

What is required for DNA Polymerase to synthesise DNA?

A
  • Template strand
  • Free 3’ OH
  • Primer (RNA)
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15
Q

Why do mistakes in DNA synthesis only occur 1 in 10^9 to 10^10 bases?

A

Base Pairing:
Only correct base pairs fit in enzyme active sites

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16
Q

What improves the accuracy of DNA synthesis?

A

Intrinsic 3’ to 5’ exonuclease activity in DNA Polymerase
(Proofreading)

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17
Q

What is the specialised function of DNA Pol I?

A

5’ to 3’ exonuclease activity

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18
Q

What is the function of Klenow?

A

Proof reading & Polymerisation

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19
Q

What part of Pol I does the Insertion and Postinsertion sites sit?

A

Palm

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20
Q

How does the polymerase proofread in DNA replication?

A
  • Forward movement is impeded
  • Polymerase repositions to remove and replace mis-paired nucleotide
20
Q

How does the polymerase proofread in DNA replication?

A
  • Forward movement is impeded
  • Polymerase repositions to remove and replace mis-paired nucleotide
21
Q

How many enzymes and proteins are involved in DNA replication in E. coli?

A

20 plus DNA Pol III

22
Q

What is the DNA synthesis complex known as in E. coli?

A

Replisome

23
Q

What is the function of Helicases?

A

Separate parent DNA strands (ATP)

24
Q

What is the function of Topoisomerases?

A

Control separation stress and twisting

25
Q

What is the function of Single Strand DNA Binding Proteins?

A

Stabilise strands

26
Q

What is the function of Primases?

A

Lay down RNA primer

27
Q

What is the function of DNA Pol I?

A

Replaces RNA primer with DNA

28
Q

What is the function of DNA Ligases?

A

Close nicks

29
Q

What are the THREE stages of DNA Replication?

A
  1. Initiation
  2. Elongation
  3. Termination
30
Q

What is the Initiation phase of DNA replication?

A

Regulated process so that DNA replication occurs only one in every cell division

31
Q

Where does initiation occur in E. coli?

A

At Ori C - origin of replication site

32
Q

How many base pairs does the replication of origin, Ori C, contain in E. coli?

A

245 bp

33
Q

What is the DNA Unwinding Element (DUE) on the E. coli replication of origin site?

A

Three 13bp A-T rich repeats

34
Q

Where does the key initiator protein DnaA bind to on the E. coli origin of replication site?

A

Five 9bp repeats
(R1 - R5)

35
Q

What are the steps for the Initiation Phase of DNA replication in E. coli?

A
  1. DnaA (ATPase) binds to R1-R5 sites
  2. Denatures DNA at A-T rich DNA Unwinding Element (DUE)
  3. DnaB (helicase) binds unwound DNA
  4. Unwinds DNA in both directions
  5. SSB proteins and Topoisomerase II stabilise DNA strands
  6. Thousands of bases become unwound
36
Q

What are the steps for LEADING strand synthesis in the Elongation phase of DNA replication?

A
  1. Primase synthesises 10-60 nucleotide RNA primer at origin
  2. Nucleotides added successively by DNA Pol III
  3. Synthesis proceeds continuously
  4. Keeps pace with replication fork
37
Q

What is the name of the protein complex on the lagging strand in the Elongation phase of DNA replication?

A

Primosome

38
Q

What are the steps for LAGGING strand synthesis in the Elongation phase of DNA replication?

A
  1. Primer synthesised at intervals
  2. Each primer extended by Pol III
  3. Synthesis extends as far as previous primer
  4. RNA primers removed
  5. Replaced with DNA by Pol I
  6. Nicks closed with DNA ligase
39
Q

Why does DNA Pol III holoenzyme has three sets of core subunits?

A

So one or two Okazaki fragments can be synthesised simultaneously, along with the leading strand

40
Q

Where do the replication forks meet in the Termination phase of DNA replication in E. coli?

A

At the terminus region:
Ter sequences

41
Q

What complex arrests replication in the Termination phase of DNA replication in E. coli?

A

Ter-Tus complex

42
Q

What enzyme is involved in the separation of circles in the Termination phase of DNA replication in E. coli?

A

Topoisomerase IV

43
Q

How many times does DNA replicate per cell division cycle in Eukaryotes?

A

Only once

44
Q

What controls the replication of DNA in Eukaryotes?

A

Cyclins & Cyclin-dependent kinases

45
Q

How many polymerases are involved in DNA replication in Eukaryotes?

A

Five or more

46
Q

What is the function of DNA Pol a?

A

Primer synthesis

47
Q

What is the function of DNA Pol d?

A

Leading and lagging strand synthesis (PCNA)

Acts like Pol III

48
Q

What is the function of DNA Pol e?

A

DNA repair

ML2001: Introductory Molecular Biology (18 decks)

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