Ch. 22: DNA Replication, Repair, & Mutagenesis Flashcards Preview

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Flashcards in Ch. 22: DNA Replication, Repair, & Mutagenesis Deck (94):
1

What are the 3 general features of DNA replication?

1) DNA replication is semiconservative
2) Replication is bi-directional
3) Replication is semi-discontinuous

2

What were the 3 proposed models of DNA replication? Describe each.

1) Conservative Replication: parent molecule directs synthesis of new double-stranded molecule; after 1 round of rep ➡️ one molecule conserved as 2 old strands

2) Semi-conservative Replication: 2 parent strands separate, each makes copy of itself; after 1 round of rep ➡️ 2 daughter molecules both have 1 new & 1 old strand

3) Dispersive Replication: Random distribution of parent material between 2 daughter molecules

4

What are the 3 active sites on E. coli DNA polymerase I? What is the function of each site?

Which one is the smaller fragment? Which two are part of the larger fragment?

1) 3' to 5' exonuclease activity: proofreading & editing
2) 5' to 3' exonuclease activity: DNA repair
3) 5' to 3' polymerase activity: nick translation (nick & replace old strand)

Smaller fragment: 5' exonuclease
Larger fragment (Klenow fragment): polymerase & 3' exonuclease

5

What is the chief DNA replicating enzyme of E. coli?

Which subunits have polymerase function? Which subunits have proofreading ability (3' to 5' exonuclease)?

E. coli DNA polymerase III

Alpha subunits: polymerase function
Epsilon subunits: proofreading ability

6

E. coli DNA polymerase I & III: Molecular weight of each?

Polymerase I: 105,000

Polymerase III: 130,000

7

E. coli DNA polymerase I & III: How many molecules/cell for each?

Polymerase I: ~400

Polymerase III: ~10

8

E. coli DNA polymerase I & III: How many nucleotides/second for each?

Polymerase I: ~20

Polymerase III: ~1000

9

E. coli DNA polymerase I & III: which has 3' Exonuclease activity?

Polymerase I: yes

Polymerase III: no

10

E. coli DNA polymerase I & III: which has 5' Exonuclease activity?

Polymerase I: yes

Polymerase III: no

11

E. coli DNA polymerase I & III: what is the biological function of each?

Polymerase I: RNA primer excision, DNA repair

Polymerase III: Replicase

12

What experiment showed DNA replication is semiconservative?

Meselson and Stahl experiment

13

Where in the cell is eukaryotic DNA polymerase alpha located?

What is its biological activity?

Nucleus

Replication (primase, replication initiator)

14

Where in the cell is eukaryotic DNA polymerase beta located?

What is its biological activity?

Nucleus

DNA repair (base excision repair)

15

Where in the cell is eukaryotic DNA polymerase gamma located?

What is its biological activity?

Mitochondria

Mitochondrial DNA replication

16

Where in the cell is eukaryotic DNA polymerase delta located?

What is its biological activity?

Nucleus

Replication (main polymerase at leading & lagging strand)

17

Where in the cell is eukaryotic DNA polymerase epsilon located?

What is its biological activity?

Nucleus

Replication (leading & lagging strand)

18

What are the main eukaryotic DNA polymerases?

DNA polymerases alpha and delta

19

What are the 3 enzymatic activities of reverse transcriptase?

1) RNA-directed DNA polymerase activity

2) RNase H activity: exonuclease that specifically degrades RNA chains in DNA:RNA hybrids

3) DNA-directed DNA polymerase activity: replicates ssDNA, forms dsDNA

20

What is an example of a reverse transcriptase?

What drug inhibits this enzyme?

HIV reverse transcriptase

AZT (3'-azido-2',3'-dideoxythymidine): ➖ DNA synthesis

21

What enzymes are used to unwind the DNA helix?

DNA gyrase: ATP dependent negative supercoiling

Helicase: ATP dependent unwinding of DNA double helix

22

What enzyme, as an RNA polymerase, synthesizes DNA primers?

Primase

23

What enzyme seals nicks in dsDNA?

DNA ligase: ATP dependent, joins Okazaki fragments together

24

Where does initiation of DNA replication occur in E. coli?

What occurs? (What binds to start initiation?)

Occurs at origin (special site, rich in A-T ➡️ easily separated)

dnaA protein binds at origin ➡️ local denaturation of DNA ➡️ 2 replisomes assemble at this site ➡️ bidirectional replication

25

Where does termination of DNA replication occur in E. coli?

What occurs to cause termination?

Special site opposite origin

Terminator utilization substance (tus) binds to DNA ➡️ ➖ helicase & prevents replisome from passing through

26

Elongation of DNA replication:

What is the leading strand synthesis?

1st RNA primer synthesized by primase ➡️ DNA polymerase III synthesized DNA progressively until reaches terminus

27

Elongation of DNA replication:

What is the lagging strand synthesis?

Each Okazaki fragment made & joined to those made previously:

RNA primers w/each Okazaki fragment removed by 5' to 3' exonuclease activity of DNA polymerase I (replaces RNA w/DNA) ➡️ nick between Okazaki fragments sealed by DNA ligase

28

What is the function of single stranded DNA binding proteins in DNA replication?

(After DNA helicase unwinds dsDNA): Keeps DNA strands from coming back together

29

Fidelity of DNA replication: How many errors are made per # of bases?

1 error/10^8 bases

30

What is rolling circle replication used for?

Phage DNA replication

Bacterial mating process

31

What are 4 important characteristics of rolling circle replication?

1) primer synthesis not necessary
2) leading strand covalently linked to template
3) Replication can continue for many rounds ➡️ generates concatameric branches
4) leading strand template never separates from circular part of molecule

32

How is DNA replication in eukaryotes initiated?

via extracellular signal (from other cells, usually paracrine)

33

In eukaryotes, what is required for progression of the cell cycle?

Timed activation of cell cycle via cyclin/cdk complexes (via signal transduction)

34

What 3 "special considerations" occur in eukaryote DNA replication (not in prokaryotes)?

1) multiple initiation sites (due to huge chromosome)
2) Histone complexes
3) telomere structures

35

What are the phases of the cell cycle?

G0: senescence ➡️ metabolism done, cell eventually dies (can't return to cell cycle)
G1 (gap): external signal to replicate is sent to nucleus & protein synthesis enables passage to S phase
S: DNA synthesis
G2 (gap): ⬆️ cell size
M: mitosis (chromosomes & cells divide)

36

What molecules drive the cell cycle?

What molecules inhibit the cell cycle?

➕: oncoproteins

➖: tumor suppressor proteins

37

Purpose of G1➡️S checkpoint of cell cycle?

Check for DNA damage ➡️ if damage detected, cell arrested in G1 and DNA repaired

38

What protein is important at G1➡️S checkpoint ("guardian of the genome")?

What are the functions of this protein?

p53 (tumor suppressor)

cell cycle arrest
DNA repair
Apoptosis (if DNA damage extensive)
Senescence (if DNA damage extensive)
Autophagy
Metabolic reprogramming

39

What is metabolic reprogramming? (In relation to p53 & tumor suppression)

p53 inhibits glycolysis (needed by cancer cells) ➡️ alpha-ketoglutarate important in hydroxylation reactions

40

Purpose of G2➡️M checkpoint in cell cycle?

Check for complete DNA replication ➡️ if not, cell arrested in G2 & DNA replication completed

41

How many replication forks are there in average human chromosome? In Drosophila embryogenesis?

Humans: 100-200

Drosophila: 6000

42

Compare histones for the leading versus lagging strand during DNA replication.

Histones retained by leading strand barely dissociate

After replication, lagging strand bare (as new histones made & assembled)

43

What type of DNA do histones have ⬆️ affinity for?

dsDNA

44

Which is bigger: DNA or histone proteins?

DNA much bigger than histones proteins

45

Describe steps of what occurs with nucleosomes during DNA replication.

1) nucleosome (histone octamer) unwinds & 2 halves divide
2) Replication fork passes & new DNA strands synthesized
3) both halves of nucleosome attached to same daughter strand (leading) ➡️ recombine
4) new histone octamer will be quickly made and added to other daughter strand (lagging)

46

Describe structure & function of telomeres.

Repeated terminal sequences (T & G), w/# of telomere repeats ranging from 20-100 (single celled eukaryotes) to >1500 in mammals

Help stabilize chromosome

47

Why would chromosomes become shorter with each round of replication without telomerase?

RNA primers at 5' removed by polymerase alpha ➡️ cannot fill gap because no 3' OH group present

48

What is the short RNA sequence which telomerase carries?

AUC-CCA-AUC

49

Compare telomerase activity of normal somatic cells versus germ-line (stem) cells.

Somatic: lack telomerase activity ➡️ chromosome shortening to senescence/cell death

Germ-line: telomerase extends 5' end of lagging strand ➡️ cells have long life span/immortal

50

What disease does telomerase contribute to? How may this potentially impact treatment of this disease?

Contributes to immortality of cancer cells

Anti-telomerase drugs being investigated to treat cancer

51

What is responsible for this action in (1) prokaryotes versus (2) eukaryotes during DNA replication?

Inducing (➖) supercoils ahead of replication fork

1) pro: gyrase

2) euk: nucleosome unwinding

52

What is responsible for this action in (1) prokaryotes versus (2) eukaryotes during DNA replication?

Unwinds dsDNA ➕supercoils

Both pro & euk: helicase

53

What is responsible for this action in (1) prokaryotes versus (2) eukaryotes during DNA replication?

Adds RNA primers at start of Okazaki fragments at 5' end

1) pro: primase

2) euk: primase subunit of DNA polymerase alpha

54

What is responsible for this action in (1) prokaryotes versus (2) eukaryotes during DNA replication?

Polymerizes leading & lagging strands 5' ➡️ 3'

1) pro: DNA polymerase III

2) euk: DNA polymerase delta, DNA polymerase alpha

55

What is responsible for this action in (1) prokaryotes versus (2) eukaryotes during DNA replication?

Exonuclease removes primers & fills in DNA gaps

1) pro: DNA polymerase I

2) euk: DNA polymerase beta, assisted by DNA polymerase alpha

56

What is responsible for this action in (1) prokaryotes versus (2) eukaryotes during DNA replication?

Links Okazaki fragments

Both pro & euk: Ligase

57

What is responsible for this action in (1) prokaryotes versus (2) eukaryotes during DNA replication?

Extends 3' ends of DNA strands
Fills in complementary strand of telomere

1) pro: not necessary (circular DNA)

2) euk: telomerase, DNA polymerase alpha

58

What are the 3 main classes of inhibitors of DNA replication?

1) prevent/reduce synthesis of precursors (bases, nucleotides)
2) affect either template or priming ability of growing strand
3) act directly on polymerases or other enzymes needed for replication

59

What do intercalating agents do to DNA?

1) Induce unwinding, lengthening, & stiffening of DNA double helix
2) Inhibits binding of enzymes (DNA/RNA polymerases, topoisomerase)
3) induce mutations during DNA replication ➡️ ➖ cell division

60

Examples of intercalating agents?

Acridines
Ethidium bromide
Actinomycin D
Anthracyclines (daunomycin, doxorubicin, epirubicin, idarubicin)

61

What agents covalently bind to DNA ➡️ cause chain breakage?

Bleomycin
Zinostatin

62

What agents cause cross-linking of DNA strands?

Alkyl sulphonates
Anthramycin
Mitomycin
Nitrogen mustards

63

What compounds bind to guanine ➡️ ➖ DNA expression?

Platinum & gold coordination compounds

64

What agents prevent extension of growing DNA chain?

2',3'-dideoxyribonucleosides
cordycepin

65

agents which act on & inhibit DNA polymerases:

Acyclovir: specific inhibition?

inhibits DNA polymerase of HSV

66

agents which act on & inhibit DNA polymerases:

Aphidicolin: specific inhibition?

Inhibits DNA polymerase alpha & delta

67

agents which act on & inhibit DNA polymerases:

2'-dideoxyazidocytidine: specific inhibition?

Inhibits bacterial primase

68

agents which act on & inhibit DNA polymerases:

Which agents inhibit DNA gyrase in bacteria?

Coumermycin
Novobiocin
Oxolinic acid
Nalidixic acid

69

What is the function of topoisomerase I?

Relieves torsional stress in DNA by inducing reversible single strand break (no energy required) ➡️ essential in DNA replication & cell growth

70

What do topoisomerase inhibitors do?

Example?

Produce double strand breaks in DNA that are irreversible ➡️ leads to cell death

Camptothecins

71

What two things contribute to genetic stability?

1) highly accurate DNA replication system

2) DNA repair system when DNA damaged

72

What is a mutation?

Any change in genetic material or base sequence of DNA

73

Compare somatic mutations to germ-line mutations.

Somatic: cell changes = mostly deleterious for affected individual

Germ-line: heritable or stable changes ➡️ lead to evolution, new species

74

At what rate (rate/cell cycle) do mutations continuously occur?

1 mutation / 10^9 base pairs

75

Small scale mutations: single/a few base changes

Types?

1) Base substitution: transition, transversion (based on base change); silent, missense, nonsense (based on consequence)
2) Base deletion
3) Base insertion

76

Large scale mutations: chromosomal mutations

Translocations?

Interchange of large segments of DNA

77

Large scale mutations: chromosomal mutations

Inversions?

Region of DNA flips its orientation w/respect to rest of chromosome

78

Large scale mutations: chromosomal mutations

Deletion?

Loss of important genes

79

Large scale mutations: chromosomal mutations

Nondisjunction?

Lost track of where they are supposed to go in cell division

80

What base changes = transition?

What base changes = transversion?

Transition = purine ➡️ purine (A ↔️ G) or pyrimidine ➡️ pyrimidine (C ↔️ T)

Transversion = purine ↔️ pyrimidine (A ↔️ C , A ↔️ T, G ↔️C, G ↔️ T)

81

What occurs in silent mutations? Missense? Nonsense?

1) Silent: no amino acid change
2) Missense: amino acid change
3) Nonsense: introduces new stop codon ➡️ termination of protein synthesis

82

Insertions & deletions may cause what?

Codon frame shifts: changes reading frame of base sequence of gene ➡️ synthesis of completely different protein

3 base insertions or deletions do not change reading frame but may cause diseases: fragile X syndrome (CGG repeat ➡️ mental retardation), Huntington's disease (CAG repeat ➡️ chorea, dementia, death)

83

What is a mutagen?

Physical/chemical agent that causes mutations

84

What is mutagenesis?

Process of producing a mutation (induced or spontaneous)

85

What mutations do chemical mutagens cause?

Modification of bases (alkylation)

Insertion between bases

86

What mutations does UV & ionizing radiation cause?

Cross-linking of base pairs
Ring opening
DNA strand breaks

87

What is the Ames test?

Determines if a chemical is a mutagen

Assumption: any substance that is mutagenic may also be a carcinogen (cause cancer)

88

Types of DNA damage:

Base loss?

Base lost but sugar-P backbone intact ➡️ strand can't replicate if not repaired

89

Types of DNA damage:

Base modification?

1) Deamination of cytosine ➡️ uracil
2) chemical modification: ROS and environmental chemicals can modify bases via alkylation
3) photodamage (UV) ➡️ thymine dimers

90

Types of DNA damage:

Replication errors?

During DNA replication: mismatch, insertion, deletion

91

Types of DNA damage:

Inter-strand cross links?

Crosslinks formed via bifunctional alkylating agents, UV, ionizing radiation

92

Types of DNA damage:

DNA-protein crosslinks?

Crosslinks between DNA strands & protein molecules formed via bifunctional alkylating agents, UV, ionizing radiation

93

Types of DNA damage:

Strand breaks?

ss nicks and ds breaks via ionizing radiation

94

Types of DNA repair:

Direct reversal of damage: 3 examples?

1) Light induced (300-600 nm) enzymatic cleavage of T, C, and CT dimers via photolyase (does not occur in placental mammals)

2) O6-methyl-guanine-DNA-methyltransferase: O6-methyl group transferred from DNA ➡️ Cys of enzyme

3) DNA ligase seals nicks

95

Types of DNA repair:

Excision repair + replacement with new DNA: 3 types? (In both pro & euk)

1) mismatch repair
2) base excision repair
3) nucleotide excision repair