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FTM Molecular Biology Group > L02 DNA Replication > Flashcards

L02 DNA Replication Flashcards

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1
Q

__________________________, received the Nobel prize for his research studying the enzymes responsible for DNA replication - most importantly - the identification of the enzyme

A

Arthur Kornberg

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2
Q

________________________ produced evidence that DNA replicates in a semi-conservative fashion.

A

Meselson and Stahl produced evidence that DNA replicates in a semi-conservative fashion.

They used heavy Nitrogen (15N)

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3
Q

_________________________ are usually circular and contain a single ORI (origin of replication)

A

Prokaryote genomes: are usually circular and contain a single ORI (origin of replication)

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4
Q

Required for DNA polymerase activity (cofactor)

A 
Magnesium ions (Mg2+)
-required for DNA polymerase activity
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5
Q

_______________________ has many replication origins and no replication termini.

A

Eukaryotic chromosomes: have many replication origins and no replication termini

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6
Q

OriC, the origin of replication in E.coli has a length of ________________ bp.

A

OriC, the origin of replication in E.coli has a length of 245.

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7
Q

OriC, the origin of replication in E.coli has a length of 245 bp and contains two important sequences:

What are they?

A
  • 13-nucleotide sequences (three in tandem array)

* 9-nucleotide sequences (four are present).

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8
Q

The _____________________ at the Ori are A-T rich. Bonding between A-T is weaker than G-C. This facilitates easier melting and strand separation of the DNA molecule at this region.

A

13-mer tandem sequences

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9
Q

At the ___________________, DnaA initiator proteins bind to begin the process of replication.

A

9-nucleotide regions

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10
Q

Initiator proteins that bind to ORI and breaks hydrogen bonds between bases.

A

DnaA

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11
Q

What is Dna C

A

Helicase inhibitor, delivers helices to DNA template

(acts as the controller)

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12
Q

What is Dna B

A

Helicase, opens helix and binds primase to form primosome protein complex

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13
Q

DNA Primase

A

RNA Polymerase which is required for DNA polymerase to begin elongation of DNA daughter strands (leading and lagging strand).

Provides DNA Polymerase III with free 3’-OH group

(makes RNA primer)

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14
Q

Function of DNA polymerase III:

A

Multi-subunit protein for elongation of DNA daughter strands, DNA synthesis of leading and lagging strands.

(elongates DNA and needs free 3’ OH)

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15
Q

Function of DNA polymerase I:

A

removes and replaces RNA primer with DNA

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16
Q

Function of DNA ligase:

A

ligates Okazaki fragments together creating phosphodiester bonds

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17
Q

RNA primer

A

4-15 nucleotides long providing free 3’ -OH group

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18
Q

___________________________ catalyzes the formation of a phosphodiester bond between the 3’ OH group of the deoxyribose on the last nucleotide and the 5’-phosphate of the dNTP precursor.

A

DNA polymerase III

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19
Q

____________________ is released as a result of DNA polymerase III incorporating a single base and elongating the new DNA strand.

A

Pyrophosphate

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20
Q

Tautomeric Shift

A

shift of C to normal cytosine (C), correction of misincorporation of C pairing with A.

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21
Q

Short DNA molecules on the lagging strand

A 
Okazaki fragments 
(in bacteria, 1000 - 2000 bp long)
22
Q

___________________ removes the RNA primer in prokaryotes.

A

DNA polymerase I

23
Q

___________________ removes the RNA primer in eukaryotes.

A

RNAseH and FEN-1 proteins

24
Q

___________________ joins the DNA fragments together by forming a phosphodiester bond between the two fragments

A

DNA ligase

25
Q

DNA Polymerase I: (pick all that apply)

  1. implicated in DNA repair
  2. involved in repairing damaged DNA
  3. the main polymerase in bacteria
  4. 5’ to 3’ exonuclease activity (RNA primer removal)
  5. 5’ to 3’ polymerase activity (replace primer with DNA)
  6. 3’ to 5’ exonuclease activity (proofreading)
  7. 5’ to 3’ (responsible for elongation of leading and lagging strands)
A

Pol I: implicated in DNA repair; has

5’ to 3’ exonuclease activity (RNA primer removal)
5’ to 3’ polymerase activity(replace primer with DNA)
3’ to 5’ exonuclease activity (proofreading)

26
Q

DNA Polymerase II: (pick all that apply)

  1. implicated in DNA repair
  2. involved in repairing damaged DNA
  3. the main polymerase in bacteria
  4. 5’ to 3’ exonuclease activity (RNA primer removal)
  5. 5’ to 3’ polymerase activity (replace primer with DNA)
  6. 3’ to 5’ exonuclease activity (proofreading)
  7. 5’ to 3’ (responsible for elongation of leading and lagging strands)
A

Pol II: involved in repairing damaged DNA

has 3’ to 5’ exonuclease activity

27
Q

DNA Polymerase III: (pick all that apply)

  1. implicated in DNA repair
  2. involved in repairing damaged DNA
  3. the main polymerase in bacteria
  4. 5’ to 3’ exonuclease activity (RNA primer removal)
  5. 5’ to 3’ polymerase activity (replace primer with DNA)
  6. 3’ to 5’ exonuclease activity (proofreading)
  7. 5’ to 3’ (responsible for elongation of leading and lagging strands)
A

Pol III: the main polymerase in bacteria

5’ to 3’ (responsible for elongation of leading and lagging strands)

has 3’ to 5’ exonuclease activity (proofreading)

28
Q

Single-stranded binding proteins (SSB proteins)

A

prevents reannealing, formation of secondary structure, and straightens out DNA template for DNA polymerization.

29
Q

_________________ tightly holds the DNA polymerase onto the template for synthesis of long templates, and releases DNA polymerase when it stalls at a region of double stranded DNA.

A

Clamp protein

30
Q

___________________ transiently forms a single covalent bond with DNA and breaking a phosphodiester bonds. This allows free rotation of the DNA around the covalent backbone bonds.

A

Topoisomerase I

31
Q

__________________ is similar to reverse transcriptase in that it synthesizes DNA using an RNA template covalently bound to the protein in eukaryotes

A

Telomerase

32
Q

___________________ loads DNA Polymerase III onto single-stranded DNA template. Offloads DNA Polymerase once it encounters double-stranded DNA

A

clamp proteins

33
Q

Name 11 Summary steps of DNA Replication

A

1) Initiator Proteins binds at ORI (DnaA)
2) Helicase inhibitor (Dna C)
3) DNA helicase (Dna B)
4) SSB proteins
5) DNA primase
6) Clamp proteins
7) DNA polymerase III synthesizes DNA
8) DNA polymerase I removes primer
9) DNA ligase joins DNA strands
10) Topoisomerase cleaves phosphodiester bind on one parent strand
11) EUKARYOTES ONLY - Telomerase, adds RNA to parent strand, allowing for DNA Pol III to synthesize complementary DNA, where primer was.

34
Q

Without Telomerase, what problem would we encounter during DNA replication?

A

A lose of genetic information due to gap of RNA primer not being replaced.

35
Q

What direction does polymerization and proofreading by DNA Polymerase III occur in?

a) both 5’ –> 3’
b) both 3’ –> 5’
c) 5’ –> 3’ ; 3’ –> 5’
d) 3’ –> 5’; 5’ –> 3’

A

C)

36
Q

Which of these is true? From the ORI,

a) one parent strand is leading, while other lagging
b) both parent strand leading
c) both parent strands have leading & lagging portions
d) both strand lagging

A

C

37
Q

What is the purpose of DNA primase?

A

to synthesize the primer

Note: DNA primase is not the primer itself, it makes the primer

38
Q

Compare the genomes of Prokaryotes and Eukaryotes.

A

Prokaryotes: circular DNA; one ORI; once began, must finish entire genome Eukaryotes: linear DNA; many ORI, can start & stop for portions of DNA needed

39
Q

Where on E.coli does DnaA bind?

A

the 9 mers at ORI

40
Q

Where does strand separation occur in E. coli?

A

the tandem 13 mers

41
Q

What has the ability to release or inhibit helicase?

A

DnaC (helicase inhibitor)

42
Q

T/FDNA polymerase III binds to single parent strand to begin synthesis of DNA

A

false. DNA polymerase III needs double stranded region to bind to. It binds to the RNA/DNA primer.

43
Q

Why is a primer needed for DNA synthesis?

A

provides free 3’ -OH

44
Q

What did one band after 20 min incubation vs two bands after 40 minutes in E.coli indicate in the Meselson & Stahl experiment?

A

E. coli replicates every 20 minutes.On band at 20 min = 1 replication; 1 band indicated that the two new DNA helices were the same.two bands at 40 min = 2 replications; 2 bands indicate that there were two different kinds of DNA found.This indicated that DNA replicated in semi- conservative form.

45
Q

What did kornberg discover?

A

DNA polymerase is enzyme responsible for DNA replication

46
Q

Once ORI is opened, what is required for prokaryotic elongation?

A

1) DNA primase
2) DNA polymerase III
3) DNA polymerase I
4) DNA ligase

47
Q

What mechanism enables DNA polymerase III to correct base misincorporation?

A

3’ –> 5’ proofreading exonuclease

48
Q

Why can Cytosine sometimes pair with Adenine?

A

Cytosine has a rare tautomeric form that can present at Thymine, allowing it to be paired with Adenine, but once bond returns to its original form.

49
Q

T/F. All forms of DNA polymerase have 3’ –> 5’ exonuclease activity.

A

TRUE

50
Q

What is the main polymerase in bacteria?

A

Polymerase III

51
Q

Where does Telomerase add new RNA material?

A

to the 3’ end of parent strand that is being replicated

FTM Molecular Biology Group (8 decks)

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