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MOL BIO Exam #1- Lo > Lec 03- DNA Replication, Repair, and Recombination 1 > Flashcards

Lec 03- DNA Replication, Repair, and Recombination 1 Flashcards

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1
Q

What is the error rate following DNA replication?

A

1 mistake in every 10^9 base pairs

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2
Q

How many base pairs are in the human genome?

A

3x10^9 base pairs

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3
Q

How many nucleotides are changed every cell division?

A

3 nucleotides

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4
Q

How are most DNA replication errors corrected?

A
  • proofreading

- DNA repair

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5
Q

How are DNA errors corrected post-replication?

A

by repair mechanisms

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6
Q

Multicellular organisms need high ___________

A

fidelity replication

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7
Q

What do germ cells have to have in order to maintain the species?

A

low mutation rates

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8
Q

Why do somatic cells need low mutation rates?

A

to avoid uncontrolled proliferation/cancer

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9
Q

How does DNA polymerase synthesize DNA?

A

by catalyzing the reaction:

(DNA)n residues + dNTP&raquo_space; (DNA)n+1 residues + P2O7

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10
Q

What does template directed DNA replication mean?

A

new chain is assembled in a preexisting DNA template that is complementary to the incoming bases

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11
Q

What does DNA replication require?

A

dATP
dGTP
dCTP
dTTP

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12
Q

What does DNA polymerase require?

A

Requires a primer with a free 3’OH to begin

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13
Q

Both strands of the replication fork are ___________ replicated

A

simultaneously

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14
Q

In what direction can DNA polymerase synthesize DNA?

A

5’&raquo_space; 3’

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15
Q

Which DNA strand is synthesized continuously?

A

leading strand

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16
Q

Which DNA strand is synthesized in segments?

A

lagging strand

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17
Q

What is the first step of proofreading?

A
  • just before a new nucleotide is added
  • enzyme must tighten its fingers around the active site
  • easier if the correct base is in place
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18
Q

When does exonucleolytic proofreading take place?

A

immediately after incorrect bases are added

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19
Q

What type of terminus does DNA polymerase require?

A

a perfectly paired 3’ terminus

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20
Q

What clips off unpaired residues at 3’ primer terminus?

A

3’&raquo_space; 5’ exonuclease

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21
Q

Why does 5’&raquo_space; 3’ replication allow for efficient error correction?

A

because it conserves energy when correcting mistakes

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22
Q

How is the lagging strand replicated?

A

through backstitching process

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23
Q

What are the steps of the backstitching process if the lagging strand?

A

1- DNA primase synthesizes a 10 nt long RNA primer (to prime DNA synthesis)
2- RNA primer is erased by RNAseH and replaced with DNA
3- DNA ligase joins the ends

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24
Q

Why can’t DNA initiate de novo synthesis in the lagging strand?

A

because it would increase the mutation rate

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25
Q

What does RNAseH do?

A

recognizes RNA/DNA hybrids

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26
Q

What does DNA helicase do?

A

unwinds DNA

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27
Q

What is DNA helicase?

A

a protein with 6 identical subunits that bind and hydrolyzes ATP

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28
Q

What happens when DNA helicase binds?

A

causes conformational change that propels it along the single stranded DNA

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29
Q

How fast can DNA helicase pry apart the helix?

A

1000 nucleotide pairs/second

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30
Q

What do single-stranded DNA binding proteins do?

A
  • bind tightly and cooperatively to exposed single stranded DNA
  • help stabilize unwound DNA
  • prevent formation of hairpins
  • DNA bases remain exposed
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31
Q

What does the sliding clamp do?

A

-keeps DNA polymerase on DNA when moving

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32
Q

When does the sliding clamp release?

A

when double stranded DNA is encountered

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33
Q

What does a clamp holder do?

A
  • required for assembly

- hydrolyzes ATP as it loads the clamp onto a primer-template junction

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34
Q

What happens to the clamp on the leading strand?

A

clamp remains associated to DNA polymerase for long stretches

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35
Q

What happens to the clamp on the lagging strand?

A

clamp loader stays close so it can assemble a new clamp at the start of each new Okazaki fragment

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36
Q

What is mismatch repair?

A
  • removal of almost all errors that are missed by proofreading
  • detects distortion caused by mispairing
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37
Q

How does mismatch repair know which strand is correct in E. Coli?

A

depends on methylation to distinguish new strand

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38
Q

How does mismatch repair know which strand is correct in humans?

A

depends on single strand breaks

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39
Q

Where are single strand breaks present in humans?

A

present on lagging strand before Okazaki fragments are ligated

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40
Q

What does MutS do?

A

binds to mismatch

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41
Q

What does MutL do?

A
  • scans for the nick

- triggers degradation of nicked strand

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42
Q

How many base pairs make one turn?

A

10 bp

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43
Q

What is DNA topoisomerase?

A

a reversible enzme

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44
Q

What does DNA topoisomerase do?

A
  • breaks a phosphodiester bond to change superhelicity

- relieves supercoiling

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45
Q

What does type 1 topoisomerase do?

A

catalyzes the reaction of supercoiled DNA

thermodynamically favorable process

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46
Q

How does type 1 topoisomerase work?

A
  • creates transient single strand break in DNA
  • allows DNA on either side of the nick to rotate freely relative to each other
  • use other phosphodiester bond as a swivel point
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47
Q

Why doesn’t DNA resealing require any energy after Type I topoisomerases?

A
  • rapid

- energy is stored in the phosphotyrosine linkage

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48
Q

What are Type II Topoisomerases?

A

-enzymes that make a transient double-stranaded break in the DNA

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49
Q

Where are Type II Topoisomerases activated?

A

at sites on chromosome where 2 double-stranded helices cross each other

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50
Q

How do Type II Topoisomerases use ATP?

A

1) Break 1 double-stranded helix reversibly to crease “gate”
2) Cause 2nd strand to pass through
3) Reseals break and dissociates

51
Q

What are the 2 functions of Type II Topoisomerase?

A
  • separate “decatenate” two interlocked DNA circles

- prevent severe tangling problems that would arise during DNA replication

52
Q

What are replication origins?

A

A-T rich regions where sequence attacks initiator proteins to pry open DNA

(similar process for PRO and EUK)

53
Q

Why is initiation highly regulated for E. coli replication?

A

Because it is the only point of control for E. coli

54
Q

Initiation of E. coli replication proceeds only when __________.

A

sufficient nutrients are present

55
Q

What is the refractory period for the initiation of E. coli replication?

A

a delay in replication until the new strand is methylated

56
Q

How long does it take for bacterial genomes to replicate using 2 replication forks?

A

40 minutes

57
Q

How long does it take for EUK genomes to replicate using a single ORI?

A

800 hours for an average chromosome with a single ORI

traveling 50 nt/sec

58
Q

What proteins bind to specific sites in ORI to form a complex?

A

initiator proteins

59
Q

What does the initiator protein complex attract?

A
  • DNA helicase

- helicase loader

60
Q

What is placed around a single-stranded DNA that is exposed by the initiator complex?

A

helicase

61
Q

What remains engaged until the helicase is properly loaded?

A

the helicase loader

62
Q

Why does helicase unwind the DNA?

A

so the primes can make the RNA primer on the Leading strand

63
Q

What do the remaining proteins do after helicase unwinds the DNA?

A

remaining proteins assemble to create 2 replication forks with complexes moving in opposite direction with respect to the ORI

64
Q

During which phase does EUK DNA replication occur?

A

during DNA synthesis (S) phase

65
Q

How long does EUK S-phase last?

A

8 hours

66
Q

Why are chromosomes replicated?

A

to produce 2 complete copies

67
Q

Until which phase are the chromosomes joined at centromeres?

A

until M phase

68
Q

Replication is activated in ___________ consisting of 20-80 origins.

A

clusters/replication units

69
Q

Depending on chromatin structure, different regions of each chromosome are replicated in a reproducible order during _______ phase.

A

S phase

70
Q

With timing related to the packing of DNA in chromatin, what is late-replicating?

A

heterochromatin

71
Q

X chromosomes of females:
Almost all of inactive X is condensed into heterochromatin and is replicated late in S-phase. The active homolog is __________ and replicates through ______ phase

A
  • less condensed

- S-phase

72
Q

Which regions of a genome replicate first?

A

regions with less condensed chromatin

73
Q

What are the 3 minimum requirements for a sequence to be ORI (yeast)?

A
  • binding site for ORC (origin recognition complex)
  • A-T rich stretch for easy unwinding
  • binding site for proteins (Abf1) that help attract ORC
74
Q

ORC interaction with ______ persists throughout the cell cycle.

A

ORI

75
Q

What proteins bind to ORC to form a pre-replicative complex and regulate origin activity?

A
  • helicase

- helicase loading proteins (Cdc6 and Cdt1)

76
Q

What 4 things does activated Cdks lead to in S-phase?

A

1) dissociation of helicase loading proteins
2) activation of helicase
3) unwinding of DNA
4) loading of DNA polymerase, etc..

77
Q

Assembly of new ORC is prevented until ___________

A

next M-phase resets the cycle

78
Q

When is the single chance for ORC to form in G1?

A

when Cdk activity is low

79
Q

What is the second window for pre-replicative complex to be activated and disassembled in S-phase?

A

when CDk activity is high

80
Q

Specific human sequences have been identified that can serve as ORIs. They are _______ nucleotide pairs in length.

A

1000

81
Q

How can specific human sequences still function if they are moved to a different locus?

A

must be placed where chromatin is uncondensed

82
Q

What does ORI function depend on?

A

distant sequences

83
Q

ORI dependence on distant sequences also affects _______ and has a global effect of _________________.

A
  • affects transcription

- decondensing chromatin structure

84
Q

What else is required for replication besides DNA replication?

A

synthesis and assembly of new proteins

85
Q

EUK have ________ copies of genes for each histone

A

multiple

86
Q

In what phase are histone proteins mainly synthesized?

A

S-phase

87
Q

How many histone proteins are synthesized in S-phase?

A

amount made = highly regulated to meet requirements

88
Q

What is needed for efficient replication?

A

chromatin remodeling proteins

89
Q

What do chromatin remodeling proteins do?

A

destabilize DNA-histone interface

90
Q

What does the histone octamer break into as the replication fork passes through chromatin?

A
  • (1) H3-H4 tetramer

- (2) H2A-H2B dimers

91
Q

From where are the (2) H2A-H2B dimers released as the replication fork passes through chromatin and the histone octamer breaks?

A

released from the DNA

92
Q

How is the H3-H4 tetramer distributed?

A

distributed randomly to daughter duplexes

93
Q

What does freshly made H3-H4 do in the reassembly process?

A

fills in the spaces

94
Q

What % of the H2A-H2B dimers are new and old?

A

50% new

50% old

95
Q

What is required for the orderly reassembly of chromatin?

A

histone chaperones (chromatin assembly factors)

96
Q

What directs the histone chaperones to the DNA?

A

PCNA (sliding clamp)

97
Q

Some daughter nucleosomes contain only ________ histones or only _____ histones, but most are hybrids of old and new.

A
  • parental histones

- new histones

98
Q

How are parental patterns of histone modification spread?

A

through reader-writer complexes

99
Q

What may be responsible for some types of epigenetic inheritance?

A

Patterns of histone modification

100
Q

What happens when there is an end replication problem on the lagging strand?

A

there is no place for the RNA primer

101
Q

What kind of genome does bacteria have?

A

circular genome

102
Q

_______ have telomeres

A

Eukaryotes

103
Q

What is the special sequence that is at the end of each chromosome and repeated x1000?

A

GGGTTA

104
Q

What enzyme replenishes the special sequence at the end of the chromosome?

A

telomerase

105
Q

How does the telomerase replenish the end of the chromosome?

A

by elongating parental strand in the 5’–>3’ direction using an RNA template on the enzyme

106
Q

After extension of parental strand by telomerase, replication of lagging strand can be completed by ______________, using the extension as a template.

A

DNA polymerase

107
Q

What mechanism & nuclease ensure the 3’ end is longer, leaving a protruding single stranded end that loops back and tucks into the repeat?

A

telomerase replication mechanism using DNA pol and 5’ nuclease

108
Q

What are T-loops?

A
  • structures that protect chromosome ends

- distinguishes chromosome ends from broken ends that need to be repaired

109
Q

Our somatic cells are born with ___________.

A

full complement of telomere repeats

110
Q

What can retain full telomerase activity?

A

stem cells

111
Q

Each chromosome end in a given cell contains ______________ depending on age.

A

variable number of telomere repeats

112
Q

What causes loss of telomere repeats?

A

insufficient telomerase activity

113
Q

What are the characteristics of daughter cells after many generations?

A
  • have defective chromosomes

- stop dividing

114
Q

What is replicative senescence?

A
  • the way the cell’s lifetime is regulated to guard against cancer
  • daughter cells will have defective chromosomes and will stop dividing after many generations
115
Q

How many times do normal human fibroblasts divide before undergoing replicative senescence?

A

60 times

116
Q

What happens to human fibroblasts after 60 divisions when telomerase is provided experimentally?

A

cells will continue dividing and not undergo replicative senescence

117
Q

What is responsible for aging in animals?

A

replicative senescence

118
Q

What is the result of transgenic mice that lack telomerase?

A

mice develop progressive defects in highly proliferative tissues

119
Q

What are 2 of the consequences of progressive defects in highly proliferative tissues which are a result of a lack of telomerase in transgenic mice?

A
  • premature aging

- prone to cancer

120
Q

What is dyskeratosis congenita?

A

a disease in humans in which they carry a mutant telomerase RNA gene

121
Q

What does dyskeratosis congenita do to the telomeres?

A

develop prematurely shortened telomeres

122
Q

How do humans with dyskeratosis congenita die?

A

progressive bone marrow failure

123
Q

Why is it risky for an organism to control cell proliferation using replicative senescence?

A
  • not all cells stop dividing

- gives rise to variant cells that lead to cancer

MOL BIO Exam #1- Lo (8 decks)

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