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MOL BIO Exam #1- Lo > Lec 05- DNA Replication, Repair, and Recombination 3 > Flashcards

Lec 05- DNA Replication, Repair, and Recombination 3 Flashcards

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1
Q

What does homologous recombination mean?

A

to generate DNA molecules of novel sequence

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2
Q

What is a holliday junction?

A

DNA intermediate containing 4 DNA strands from 2 different helices

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3
Q

How are holliday junctions present?

A

transiently (not permanently)

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4
Q

What is resolution?

A

when strands of helices are cleaved by endonuclease (RuvC)

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5
Q

What enzyme cleaves strands of helices in holliday junctions?

A

endonuclease RuvC

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6
Q

What are the 2 outcomes of resolution?

A
  • crossing over

- gene conversion

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7
Q

How many crossing over events occur per chromosome?

A

only 2 crossover events per chromosome

rare event

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8
Q

What way are 90% of holliday junctions in humans resolved?

A

gene conversion

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9
Q

What does meiotic recombination occur with?

A

maternal and paternal homologous chromosomes

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10
Q

What does meiotic recombination repair usually occur between?

A

newly duplicated identical helices (sister chromatids)

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11
Q

What does meiotic recombination begin with?

A

a double strand break

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12
Q

What identifies the DNA damage during meiotic recombination?

A

Spo11 (yeast)

Mre11 complex

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13
Q

What follows double strand break in meiotic recombination?

A

strand invasion and double holliday junction formation

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14
Q

What occurs after strand invasion and double Holliday junction formation during meiotic recombination?

A

resolution

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15
Q

What happens if both strands in each HJ are cut in the same way?

A

they will separate with MINIMAL exchange of sequences

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16
Q

What happens if both strands in each HJ are cut in opposite directions?

A
  • portions of each chromosome upstream and downstream are swapped
  • yields crossing over
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17
Q

Crossing over and gene conversion can occur in the same __________ during homologous recombination

A

chromosome

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18
Q

Crossing over and gene conversion in the same chromosome leads to __________________

A

multiple opportunities for genetic reassoortment

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19
Q

What does recombination result in?

A

regions of heteroduplex DNA

region where a strand from the maternal homolog is base-paired with a strand from the paternal homolog

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20
Q

In meiosis, each parent should make ________________ to the genetic material of the offspring

A

an equal contribution

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21
Q

What did analysis of all 4 haploid gametes reveal?

A

rare cases where equal contribution from each parent did not occur

  • 3 maternal alleles
  • 1 paternal allele
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22
Q

What causes divergence from the expected distribution of alleles during meiosis?

A

gene conversion

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23
Q

What are some of the ways gene conversion problems occur?

A
  • DNA synthesis during homologous recombination

- Repair of mismatches in regions of heteroduplex DNA

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24
Q

As a consequence of repair of mismatches in regions of heteroduplex DNA, what happens to the alleles?

A

1 allele is lost

the other allele is duplicated resulting in “conversion” of one to the other

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25
Q

Why must HR be regulated?

A

to prevent inappropriate cross-overs

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26
Q

What happens if recombination occurs between repeated sequences?

A

it could scramble the genome

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27
Q

What happens when the mismatch repair pathway interrupts HR between matched sequences?

A

prevents recombination events

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28
Q

What is another way of preserving speciation?

A

blocking recombination between closely related species

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29
Q

What are 3 types of transpositional recombination?

A
  • DNA-only transposons
  • Retroviral-like retrotransposons
  • Nonretroviral retrotransposons
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30
Q

What is a type of conservative site-specific recombination?

A

bacteriophage lambda

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31
Q

What are transposons?

A

specialized segments of DNA that move from one position in the genome to another

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32
Q

What are 3 other names for transposons?

A
  • transposable elements
  • selfish DNA
  • jumping genes
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33
Q

What is the range in size for transposons?

A

hundreds to thousands of nucleotide pairs

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34
Q

What does each transposon have?

A

a unique set of genes

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35
Q

What do the 100-1000 nucleotide pairs encode for the transposon?

A

enzyme that catalyzes the movement of the transposon

contain the enzyme that they repair

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36
Q

How can transposons provide benefits to the cell?

A

antibiotic resistance in bacteria

gives the cell an advantage because it can survive antibiotic treatment

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37
Q

How can transposons produce genetic variation?

A
  • occasionally rearrange neighboring DNA of host

- can induce spontaneous mutations

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38
Q

Is sequence homology required for transposons?

A

NO

-can insert anywhere in the genome (host can go anywhere)

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39
Q

How often do transposons move?

A

infrequently

one in every 10^5 cell divisions in bacteria

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40
Q

What is transposase?

A

an enzyme encoded by the transposon itself

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41
Q

What is the function of transposase?

A

allows insertion into a target DNA site

doesn’t require specific sequence on the host

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42
Q

Where does the transposase act?

A

acts on specific DNA sequence on each end of the transposon

43
Q

What is the structure of DNA-only transposons?

A

short inverted repeats at each end

44
Q

What is the structure of Retroviral-like retrotransposons?

A

directly repeated long terminal repeats (LTRs) at each end

45
Q

What is the structure of Nonretroviral retrotransposons?

A

poly A at 3’ end of RNA transcript

5’ end is often truncated

46
Q

What specialized enzymes are required for movement for DNA-only transposons?

A

transposase

47
Q

What specialized enzymes are required for movement for Retroviral-like retrotransposons?

A
  • reverse transcriptase

- integrase

48
Q

What specialized enzymes are required for movement for Nonretroviral retrotransposons?

A
  • reverse transcriptase
  • endonuclease

requires enzymes but doesn’t code for their enzymes

49
Q

What is the mode of movement for DNA-only transposons?

A

moves as DNA either by:

  • cut and paste
  • replicative pathways
50
Q

What is the mode of movement for Retroviral-like retrotransposons?

A

moves via an RNA intermediate produced by a promoter in the long terminal repeats (LTRs)

51
Q

What is the mode of movement for Nonretroviral retrotransposons?

A

moves via an RNA intermediate that is often produced from a neighboring promoter

52
Q

What are some examples of DNA-only transposons?

A
  • P element (Drosophila)
  • Ac-Ds (maize)
  • Tn3 and Tn10 (E. coli)
  • Tam3 (snapdragon)
53
Q

What are some examples of Retroviral-like transposons?

A
  • Copia (Drosophila)
  • Ty1 (yeast)
  • THE1 (human)
  • Bs1 (maize)
54
Q

What are some examples of Nonretroviral retrotransposons?

A
  • F element (Drosophila)
  • L1 (human)
  • Cin4 (maize)
55
Q

Which transposon type exists ONLY as DNA in their movement?

A

DNA-only transposons

56
Q

What organism does DNA-only transposons predominate in?

A

bacteria

57
Q

DNA-only transposons is largely responsible for the spread of _____________________

A

antibiotic resistance

58
Q

What do DNA-only transposons contain?

A
  • gene encoding transposase

- sequences recognized by the enzyme necessary for movement (short inverted repeat sequences)

59
Q

How does the cut and paste transposition work for DNA-only transposons?

A

1) Sequences on each end of element bind transposase
2) Two transposase molecules come together forming a transposome
3) Transposase introduces cuts at the base of the loop
4) Element removed
5) Central intermediate forms

60
Q

What is a transposome?

A

A loop juxtaposing two ends of an element created when 2 transposases come together

61
Q

What does the central intermediate of DNA-only transposons do?

A

catalyze direct attack on random site of target DNA

62
Q

What does the attack on random site of target DNA create?

A

creates a staggered break between 2 phosphodiester bonds

creates new phosphodiester bonds as it joins the DNA together

63
Q

What repairs the gaps created by staggered breaks in the target DNA?

A

DNA pol and DNA ligase

64
Q

What is the result of the repair of the staggered breaks/gaps in DNA created by central intermediate?

A

results in duplication of the target’s insertion site

65
Q

What is the “hole” in the donor chromosome repaired by?

A
  • Double-stranded break repair (if chromosome has just been replicated and there is an identical copy)
  • Homologous recombination (using homologous chromosome)
  • Nonhomologous end joining
66
Q

What will double-stranded break repair do to the DNA-only transposons?

A

will restore the transposon

67
Q

What will homologous recombination repair do to the DNA-only transposons?

A

will NOT restore the transposon

68
Q

What till non homologous end joining do at the break site of the DNA-only transposons?

A

will produce a mutation at the break site

69
Q

Some viruses are considered ___________

A

mobile elements

70
Q

How do retroviruses integrate into the host genome?

A

use transposition

71
Q

How are retroviruses different from transposons?

A

they encode proteins that package their genetic information into virus particles that can infect other cells

(HIV)

72
Q

What do retroviruses consist of?

A

SS RNA genome packed into protein caspid with a virus-encoded reverse transcriptase (RT) enzyme

73
Q

What does reverse transcriptase do?

A

synthesizes a DNA copy from RNA

74
Q

What happens during the infection process for retroviruses?

A

1) viral RNA enters the cell
2) converted to ds DNA molecule by reverse transcriptase activity
3) viral encoded integrase creates 3’-OH ends on the DNA that attacks the host DNA
4) new viral RNAs are synthesized by host’s RNA pol

75
Q

How are Retrovirus-like transposons different from Retroviruses?

A

Retrovirus-like transposons lack a coat

Move in and out of chromosomes the same way but Retrovirus-like transposons are unable to leave the resident cell (can’t infect another cell)

76
Q

Transposition steps

A

1) Entire transposon transcribed by host (RNA transcript contains reverse transcriptase enzyme translated by host cell)
2) Reverse transcriptase makes ds DNA copy of RNA molecule via hybrid DNA/RNA intermediate
3) ds DNA integrates into site on chromosome

77
Q

What enzyme is used to integrate the ds DNA into the site on the chromosome?

A

integrase

encoded by the Retrovirus-like transposons

78
Q

What does integrase cut?

A

one strand at each end of the viral sequence

79
Q

Each exposed 3’ OH end attacks a _____________ of the target DNA

A

phosphodiester bond

80
Q

What process allows viral DNA to insert into the target?

A

each exposed 3’ OH end attacking a phosphodiester bond of the target DNA

81
Q

What happens to the DNA once the viral DNA attacks the target?

A

it leaves gaps to be filled/ligated

82
Q

What happens after the gaps are filled by DNA repair and the viral DNA has fully integrated into the target?

A

leaves short repeats on each side of the integrated DNA segment

83
Q

What type of transposons comprise a large portion of our genome?

A

Nonretroviral transposons

84
Q

What are mutated and truncated Nonretroviral transposons?

A

repeated sequences in the genome

85
Q

Which transposons are mostly immobile but few retain the ability to move?

A

Nonretroviral transposons

L1 element (LINE)

86
Q

What 2 enzymes are required for Nonretroviral transposons to move?

A
  • endonuclease

- reverse transcriptase

87
Q

Do Nonretroviral transposons encode for their required enzymes?

A

NO

They use enzymes from other transposons

88
Q

What % of the human genome is made of Nonretroviral transposons?

A

40%

89
Q

Steps of transposition by Nonretroviral transposons

A

1) Endonuclease and reverse transcriptase bind to L1 RNA
2) Endonuclease nicks the target DNA at insertion point (Releases 3’ OH to serve as primer in reverse transcription step)
3) ss DNA copy of L1 directly linked to target DNA
4) Insertion of ds DNA copy of L1 at target site

90
Q

What is the function of conservative site specific recombination?

A
  • mediates rearrangements of other types of mobile DNA elements
  • breaks and joins 2 DNA double helixes on each molecule
91
Q

Depending on positions and relative orientations of recombination sites, the breakage and joining of 2 DNA double helices can lead to __________, _________, or _________ in conservative site specific recombination

A
  • DNA integration
  • DNA excision
  • DNA inversion
92
Q

How is conservative site specific recombination different from transcription?

A
  • Need special sites on EACH DNA that serve as recognition sites for recombinase
  • Only the transposon sequence is required for transposition
  • Form transient high energy covalent bonds and use this energy to complete DNA rearrangement
  • NO covalent protein/DNA intermediate in transposition
  • Gaps filled by DNA pol and ligase
93
Q

Does transposition use a covalent protein/DNA intermediate?

A

NO

94
Q

What is different about the outcome of conservative site specific recombination?

A

the relative orientation of the DNA sites

95
Q

What moves in and out of the host genome by the mechanism of conservative site specific recombination?

A

many bacterial viruses

bacteriophage lambda

96
Q

If the DNA sites are in the same orientation what happens to the DNA sequence? > >

A

DNA sequence can be INTEGRATED or EXCISED

97
Q

If the DNA sites are inverted in orientation, what happens to the DNA sequence? > <

A

DNA sequence is INVERTED instead of excised

98
Q

What is the first mobile element to be understood in biochemical detail?

A

bacteriophage lambda

99
Q

Steps of the lifecycle of bacteriophage lambda

A

1) Host chromosome + bacterial cell attachment
2) Injection of lambda DNA
3) Lambda DNA circularizes
4) Integration of lambda DNA into host chromosome
5) Lytic or Prophage pathway

100
Q

What happens in the Prophage pathway of the bactriophage lambda lifecycle?

A

After integration of lambda DNA into host chromosome:

1) Cell division
2) Integrated lambda DNA replicates along with host chromosome

101
Q

What happens in the Lytic pathway of the bacteriophage lambda lifecycle?

A

After integration of lambda DNA into host chromosome:

1) Synthesis of viral proteins needed for formation of new viruses
2) Rapid replication of lambda DNA and its packaging into complete viruses
3) Cell lysis releases large number of new viruses

102
Q

Steps of lambda phage insertion into bacterial chromosome

A

1) Circular chromosome of bacteriophage lambda and bacterial chromosome bind at attachment site sequences
2) Integrase binds to the lambda protein complex
3) Catalysis of double strand breakage and rejoining
4) Integrase dissociates
5) Heteroduplex joints form and bacteriophage DNA integrated into bacterial chromosome

103
Q

What can site-specific recombination be used for?

A

to turn genes on and off

tool for knocking out gene function in specific tissues in mice

MOL BIO Exam #1- Lo (8 decks)

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