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Biochem BLOCK 3 > DNA Replication > Flashcards

DNA Replication Flashcards

1
Q

What two things are DNA replication considered?

A

Bidirectional

Semiconservative

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2
Q

Bidirectional DNA replication

A

Replication begins in the interior of a DNA molecule and proceeds in both directions

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3
Q

Semiconservative DNA replication

A

Each copy of the DNA molecule, after replication, contains one strand from the original template and one newly synthesized strand

Old strand plus a new strand

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4
Q

Origin of replication in prokaryotes

A

One origin

Circular DNA

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5
Q

Origin of replication in eukaryotes

A
  • bidirectional

- multiple origins of replication, in order to replicate in a reasonable amount of time

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6
Q

Separation of the two complementary DNA strands in prokaryotes

A
  • origin of replication needs to be melted (site where 2 strands separate
  • origin of replication sequences are usually almost exclusively composed of A-T bases
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7
Q

Formation of the replication fork in prokaryotic DNA

A
  • single stranded binding proteins (SSBs: bind to single strands to prevent reannealing and protect DNA from nuclease degradation
  • DNA helicases then move toward the double stranded region and force the strands apart
  • SSBs bind the newly separated strands
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8
Q

SSBs

A

Bind to single strands to prevent reannealing and protect DNA from nuclease degradation

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9
Q

DNA helicases

A

Forces apart the strands at the replication fork

-use ATP

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10
Q

Supercoiling

A

DNA is a helix, so when helicases separate the strands of DNA, supercoiling ahead of the replication fork will occur

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11
Q

Topoisomerases

A

Alleviate supercoiling ahead of the replication fork

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12
Q

Type I topoisomerase

A

Creates a nick in ONE strand which allows the DNA to swivel around the intact strand, then seals the nicked strand

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13
Q

Type II topoisomerases

A

Cut BOTH strands to relieve the supercoil, then re-legates the two strands

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14
Q

DNA gyrase

A
  • special type II topoisomerase
  • introduces negative supercoils
  • also important for the separation of circular chromosomes after replication
  • important for replication, packaging of the chromosome, and separation of replicated, circular chromosomes
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15
Q

What do quinolones inhibit??

A

DNA gyrase

-inhibits it so prokaryotes cannot replicate DNA

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16
Q

Too much quinolone

A

Can be toxic because it can inhibit mitochondrial DNA in eukaryotes

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17
Q

Direction of DNA replication

A

All plymerases that synthesize nuclei acids only catalyze synthesis in the 5’ to 3’ direction

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18
Q

What direction does the DNA template strand read?

A

3’ to 5’

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19
Q

LEading strand

A

At each replication fork, one strand of DAN fragment can be replicated continuously as the replication fork advances

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20
Q

Lagging strand

A
  • synthesized discontinuously
  • as the replication fork advances, small fragments of DNA are synthesized 5’ to 3’ away from the replication fork
  • the fragments are called Okazaki fragments
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21
Q

Okazaki fragments

A

The discontinuously synthesized fragments.
Later joined to become a continuous segment of DNA
-combo of DNA and RNA

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22
Q

DNA polymerase

A

Require free 3’ OH group to being synthesis

-begin synthesis from the free 3’ OH group from the RNA primer after primase copies the first 10 nucleotide

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23
Q

Primase

A

an RNA polymerase that copies the first ~10 nucleotides to “prime” synthesis
-does not require free 3’ OH

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24
Q

RNA primer

A

Each new DNA fragment on the lagging strand begins with the action of primase laying down an RNA primer

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25
Q

DNA polymerase catalyze..

A

A reaction between the 3’ OH group of the strand being synthesized, and the 5’-triphsophate of an incoming nucleotide specified by the template being copied

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26
Q

Net reaction of DNA polymerase

A

Addition of a nucleotide to a growing DNA strand and the release of pyrophosphate

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27
Q

What makes the reaction of DNA polymerase irreversible?

A

Pyrophosphate
-it is further cleaved to inorganic phosphate to make the reaction irreversible and drive th reaction in the forward direction

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28
Q

What drivers the reaction that DNA catalyzes in the forward reaction?

A

Pyrophosphate

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29
Q

What does pyrophosphate do to the reaction catalyzes by DNA polymerase?

A

Coupled irreversible reaction

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30
Q

Coupled irreversible reaction

A

A common theme in many condensation reactions in biochemistry
-two high energy bonds are cleaved for each added nucleotide in a growing DNA chain

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31
Q

DNA polymerase III (pol III)

A

Enzyme in prokaryotes that elongates the leading and lagging strand

32
Q

what elongates both the leading and lagging strand in prokaryotes?

A

DNA polymerase IIII

33
Q

Proofreading

A

DNA replication needs to as accurate as possible, a single nucleotide mutation can have devastating consequences

34
Q

What DNA polymerase has proofreading ability?

A

POL III (as well as many other DNA polymerases)

35
Q

Pol III proofreading

A

Checks each added nucleotide to make sure it is correctly base-paired with the template strand
-exonuclease in reverse direction, 3’-5’ activity

36
Q

What happens when Pol III detects a mistake?

A

Shifts backward one nucleotide and excises the misincorporated nucleotide
-exonuclease activity 3’-5’

37
Q

3’-5’ exonuclease activity

A

Pol III shifts backward one nucleotide and excises the misincorporated nucleotide

38
Q

What must happen to complete replication of circular DNA and the joining of Okazaki fragments?

A

The RNA primer must be removed and replaced with dNTPs (via Pol I)

39
Q

DNA polymerase I (pol I)

A
  • 5’-3’ polymerase activity

- 3’-5’ AND 5’-3’ exonuclease activity

40
Q

What has BOTH 3’-5’ and 5’-3’ exonuclease activity?

A

DNA polymerase I

41
Q

What all does Pol I do?

A
  • removes RNA primer (5’ to 3’ exonuclease)
  • replaces the rNTPs with the correct dNTP (5’-3’ polymerase)
  • proof reads and corrects misincorporated nucleotides (3’-5’ exonuclease)
42
Q

What can Pol I’s 5’-3’ exonuclease activity do?

A

Actually remove incorrectly base paired nucleotides (in this case the RNA primer)
-also important for another pol I function, DNA repair

43
Q

What are the two important functions of Pol I 5’-3’ exonuclease activity?

A
  1. Replication of DNA

2. Repair of DNA

44
Q

DNA ligase

A

Seals the nick that remains after the RNA primer is removed and replaced with dNTPs

45
Q

DnaA protein

A

Initial strand separation

High A-T content

46
Q

What prevents reannealing?

A

SSB proteins

47
Q

G1 phase

A

Most variable in terms of time; growth and metabolism

48
Q

G0 phase

A

Semipermanent G1 phase, appropriate signals can cause a re-entry into the cell cycle

49
Q

S Phase

A

Replication of DNA (synthesis)

50
Q

M phase

A

The stage of cell division (mitosis)

51
Q

Pol alpha

A

Contains primase + DNA polymerase (begins strand synthesis)

52
Q

Pol delta

A

DNA polymerase + proofreading (extends strands)

53
Q

Pol beta and Pol epsilon

A

DNA repair enzymes

54
Q

Pol gamma

A

Mitochondrial DNA polymerase

55
Q

What happens to the lagging strand on the eukaryotic linear chromosome?

A

Will have a gap once the primer is removed

-overhang that is susceptible to degradation and has enzymes to properly manage them

56
Q

Telomerase

A

Extends the ends of linear chromosomes (at the overhang)

57
Q

Telomeres

A

The ends of eukaryotic chromosomes contain repeated sequences called this
-6-nucleotide repeats (>1000 6-nucleotide repeats)

58
Q

What does telomerase contain?

A
  • Segment of RNA that is complimentary to the telomere repeat and extends beyond the repeat, the extension acts a template
  • also contains reverse transcriptase
59
Q

Telomerase reverse transcriptase

A

Copies its own template (RNA) into DNA extending the 3’ overhang on the chromosome

  • repeated many times
  • keeps nucleases from chopping into important coding regions on end
60
Q

After telomerase extends the repeat many times…

A

The overhang is filled in by the action of primase and DNA polymerase
-however, there will always be a section of DNA left that is single stranded

61
Q

Section of DNA left that is single stranded

A

Assumes a special structure with the dsDNA and certain proteins to protect the end of the DNA

62
Q

Is telomerase expressed in every cell?

A

No

63
Q

Where is telomerase expressed?

A

In cells that continually divide and are not terminally differentiated

64
Q

Cells that do not express telomerase

A

Have their chromosomes shortened at each cell division-have a finite number of cell divisions, cellular equivalent to aging

65
Q

What causes cellular aging?

A

Cells that do not have telomerase have their chromosomes shorten every time there is replication, there is a finite number of cell divisions

66
Q

Molecular reason behind cancer

A

Some cells can activate telomerase

67
Q

What polymerases can copy RNA into DNA?

A
  • Reverse transcriptase
  • RNA dependent DNA polymerase
  • telomerase

Common strategy in many viruses (HIV)
Lacks proofreading ability , high mutation rate

68
Q

Strand-directed mismatch repair

A

Corrects errors made during replication

69
Q

Damage repair

A

Similar process as strand-directed mismatch

70
Q

DNA repair proteins

A
  • With endonuclease activity recognize misincorporated bases or damaged bases and nick the damaged strand
  • some of these proteins also remove the damaged region
71
Q

DNA Pol I in DNA repair

A

-Can remove the damaged region (exo- or endonuclease)
-then fills in the previously damaged region
=-DNA ligase seals the final nick

72
Q

HNPCC (hereditary nonpolposis colorectal cancer)

A

A defect in mismatch repair is responsible for this

One of the most common inherited cancers

73
Q

DNA mutations

A

Can be causes by other mechanisms besides replication errors

74
Q

Spontaneous mutations

A

Exposure to chemicals or radiation

-cigs

75
Q

UV light

A

Causes pyrimidine dimers

-usually thymine dimers

76
Q

UV light damaged DNA repair

A

UV specific endonuclease

-cuts DNA on both sides of damage and removes it, gap filled in by repair DNA polymerase (Pol I in prokaryotes)

77
Q

Xeroderma pigmentosum

A

Results from deficiency in excision endonuclease

Biochem BLOCK 3 (12 decks)

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