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Flashcards in DNA Replication Deck (77):
1

What two things are DNA replication considered?

Bidirectional
Semiconservative

2

Bidirectional DNA replication

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

3

Semiconservative DNA replication

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

4

Origin of replication in prokaryotes

One origin
Circular DNA

5

Origin of replication in eukaryotes

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

6

Separation of the two complementary DNA strands in prokaryotes

-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

7

Formation of the replication fork in prokaryotic DNA

-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

8

SSBs

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

9

DNA helicases

Forces apart the strands at the replication fork
-use ATP

10

Supercoiling

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

11

Topoisomerases

Alleviate supercoiling ahead of the replication fork

12

Type I topoisomerase

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

13

Type II topoisomerases

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

14

DNA gyrase

-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

15

What do quinolones inhibit??

DNA gyrase
-inhibits it so prokaryotes cannot replicate DNA

16

Too much quinolone

Can be toxic because it can inhibit mitochondrial DNA in eukaryotes

17

Direction of DNA replication

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

18

What direction does the DNA template strand read?

3' to 5'

19

LEading strand

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

20

Lagging strand

-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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Okazaki fragments

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

22

DNA polymerase

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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Primase

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

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RNA primer

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

25

DNA polymerase catalyze..

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

26

Net reaction of DNA polymerase

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

27

What makes the reaction of DNA polymerase irreversible?

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

28

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

Pyrophosphate

29

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

Coupled irreversible reaction

30

Coupled irreversible reaction

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

31

DNA polymerase III (pol III)

Enzyme in prokaryotes that elongates the leading and lagging strand

32

what elongates both the leading and lagging strand in prokaryotes?

DNA polymerase IIII

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Proofreading

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

34

What DNA polymerase has proofreading ability?

POL III (as well as many other DNA polymerases)

35

Pol III proofreading

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

36

What happens when Pol III detects a mistake?

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

37

3'-5' exonuclease activity

Pol III shifts backward one nucleotide and excises the misincorporated nucleotide

38

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

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

39

DNA polymerase I (pol I)

-5'-3' polymerase activity
-3'-5' AND 5'-3' exonuclease activity

40

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

DNA polymerase I

41

What all does Pol I do?

-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

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

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

43

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

1. Replication of DNA
2. Repair of DNA

44

DNA ligase

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

45

DnaA protein

Initial strand separation
High A-T content

46

What prevents reannealing?

SSB proteins

47

G1 phase

Most variable in terms of time; growth and metabolism

48

G0 phase

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

49

S Phase

Replication of DNA (synthesis)

50

M phase

The stage of cell division (mitosis)

51

Pol alpha

Contains primase + DNA polymerase (begins strand synthesis)

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Pol delta

DNA polymerase + proofreading (extends strands)

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Pol beta and Pol epsilon

DNA repair enzymes

54

Pol gamma

Mitochondrial DNA polymerase

55

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

Will have a gap once the primer is removed
-overhang that is susceptible to degradation and has enzymes to properly manage them

56

Telomerase

Extends the ends of linear chromosomes (at the overhang)

57

Telomeres

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

58

What does telomerase contain?

-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

Telomerase reverse transcriptase

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

After telomerase extends the repeat many times...

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

Section of DNA left that is single stranded

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

62

Is telomerase expressed in every cell?

No

63

Where is telomerase expressed?

In cells that continually divide and are not terminally differentiated

64

Cells that do not express telomerase

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

65

What causes cellular aging?

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

66

Molecular reason behind cancer

Some cells can activate telomerase

67

What polymerases can copy RNA into DNA?

-Reverse transcriptase
-RNA dependent DNA polymerase
-telomerase

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

68

Strand-directed mismatch repair

Corrects errors made during replication

69

Damage repair

Similar process as strand-directed mismatch

70

DNA repair proteins

-With endonuclease activity recognize misincorporated bases or damaged bases and nick the damaged strand
-some of these proteins also remove the damaged region

71

DNA Pol I in DNA repair

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

72

HNPCC (hereditary nonpolposis colorectal cancer)

A defect in mismatch repair is responsible for this
One of the most common inherited cancers

73

DNA mutations

Can be causes by other mechanisms besides replication errors

74

Spontaneous mutations

Exposure to chemicals or radiation
-cigs

75

UV light

Causes pyrimidine dimers
-usually thymine dimers

76

UV light damaged DNA repair

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

Xeroderma pigmentosum

Results from deficiency in excision endonuclease