Lec 05- DNA Replication, Repair, and Recombination 3 Flashcards
1
Q
What does homologous recombination mean?
A
to generate DNA molecules of novel sequence
2
Q
What is a holliday junction?
A
DNA intermediate containing 4 DNA strands from 2 different helices
3
Q
How are holliday junctions present?
A
transiently (not permanently)
4
Q
What is resolution?
A
when strands of helices are cleaved by endonuclease (RuvC)
5
Q
What enzyme cleaves strands of helices in holliday junctions?
A
endonuclease RuvC
6
Q
What are the 2 outcomes of resolution?
A
- crossing over
- gene conversion
7
Q
How many crossing over events occur per chromosome?
A
only 2 crossover events per chromosome
rare event
8
Q
What way are 90% of holliday junctions in humans resolved?
A
gene conversion
9
Q
What does meiotic recombination occur with?
A
maternal and paternal homologous chromosomes
10
Q
What does meiotic recombination repair usually occur between?
A
newly duplicated identical helices (sister chromatids)
11
Q
What does meiotic recombination begin with?
A
a double strand break
12
Q
What identifies the DNA damage during meiotic recombination?
A
Spo11 (yeast)
Mre11 complex
13
Q
What follows double strand break in meiotic recombination?
A
strand invasion and double holliday junction formation
14
Q
What occurs after strand invasion and double Holliday junction formation during meiotic recombination?
A
resolution
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
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
17
Q
Crossing over and gene conversion can occur in the same __________ during homologous recombination
A
chromosome
18
Q
Crossing over and gene conversion in the same chromosome leads to __________________
A
multiple opportunities for genetic reassoortment
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
20
Q
In meiosis, each parent should make ________________ to the genetic material of the offspring
A
an equal contribution
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
22
Q
What causes divergence from the expected distribution of alleles during meiosis?
A
gene conversion
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
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
25
Why must HR be regulated?
to prevent inappropriate cross-overs
26
What happens if recombination occurs between repeated sequences?
it could scramble the genome
27
What happens when the mismatch repair pathway interrupts HR between matched sequences?
prevents recombination events
28
What is another way of preserving speciation?
blocking recombination between closely related species
29
What are 3 types of transpositional recombination?
- DNA-only transposons
- Retroviral-like retrotransposons
- Nonretroviral retrotransposons
30
What is a type of conservative site-specific recombination?
bacteriophage lambda
31
What are transposons?
specialized segments of DNA that move from one position in the genome to another
32
What are 3 other names for transposons?
- transposable elements
- selfish DNA
- jumping genes
33
What is the range in size for transposons?
hundreds to thousands of nucleotide pairs
34
What does each transposon have?
a unique set of genes
35
What do the 100-1000 nucleotide pairs encode for the transposon?
enzyme that catalyzes the movement of the transposon
contain the enzyme that they repair
36
How can transposons provide benefits to the cell?
antibiotic resistance in bacteria
gives the cell an advantage because it can survive antibiotic treatment
37
How can transposons produce genetic variation?
- occasionally rearrange neighboring DNA of host
| - can induce spontaneous mutations
38
Is sequence homology required for transposons?
NO
-can insert anywhere in the genome (host can go anywhere)
39
How often do transposons move?
infrequently
one in every 10^5 cell divisions in bacteria
40
What is transposase?
an enzyme encoded by the transposon itself
41
What is the function of transposase?
allows insertion into a target DNA site
doesn't require specific sequence on the host
42
Where does the transposase act?
acts on specific DNA sequence on each end of the transposon
43
What is the structure of DNA-only transposons?
short inverted repeats at each end
44
What is the structure of Retroviral-like retrotransposons?
directly repeated long terminal repeats (LTRs) at each end
45
What is the structure of Nonretroviral retrotransposons?
poly A at 3' end of RNA transcript
5' end is often truncated
46
What specialized enzymes are required for movement for DNA-only transposons?
transposase
47
What specialized enzymes are required for movement for Retroviral-like retrotransposons?
- reverse transcriptase
| - integrase
48
What specialized enzymes are required for movement for Nonretroviral retrotransposons?
- reverse transcriptase
- endonuclease
requires enzymes but doesn't code for their enzymes
49
What is the mode of movement for DNA-only transposons?
moves as DNA either by:
- cut and paste
- replicative pathways
50
What is the mode of movement for Retroviral-like retrotransposons?
moves via an RNA intermediate produced by a promoter in the long terminal repeats (LTRs)
51
What is the mode of movement for Nonretroviral retrotransposons?
moves via an RNA intermediate that is often produced from a neighboring promoter
52
What are some examples of DNA-only transposons?
- P element (Drosophila)
- Ac-Ds (maize)
- Tn3 and Tn10 (E. coli)
- Tam3 (snapdragon)
53
What are some examples of Retroviral-like transposons?
- Copia (Drosophila)
- Ty1 (yeast)
- THE1 (human)
- Bs1 (maize)
54
What are some examples of Nonretroviral retrotransposons?
- F element (Drosophila)
- L1 (human)
- Cin4 (maize)
55
Which transposon type exists ONLY as DNA in their movement?
DNA-only transposons
56
What organism does DNA-only transposons predominate in?
bacteria
57
DNA-only transposons is largely responsible for the spread of _____________________
antibiotic resistance
58
What do DNA-only transposons contain?
- gene encoding transposase
| - sequences recognized by the enzyme necessary for movement (short inverted repeat sequences)
59
How does the cut and paste transposition work for DNA-only transposons?
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
What is a transposome?
A loop juxtaposing two ends of an element created when 2 transposases come together
61
What does the central intermediate of DNA-only transposons do?
catalyze direct attack on random site of target DNA
62
What does the attack on random site of target DNA create?
creates a staggered break between 2 phosphodiester bonds
creates new phosphodiester bonds as it joins the DNA together
63
What repairs the gaps created by staggered breaks in the target DNA?
DNA pol and DNA ligase
64
What is the result of the repair of the staggered breaks/gaps in DNA created by central intermediate?
results in duplication of the target's insertion site
65
What is the "hole" in the donor chromosome repaired by?
- 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
What will double-stranded break repair do to the DNA-only transposons?
will restore the transposon
67
What will homologous recombination repair do to the DNA-only transposons?
will NOT restore the transposon
68
What till non homologous end joining do at the break site of the DNA-only transposons?
will produce a mutation at the break site
69
Some viruses are considered ___________
mobile elements
70
How do retroviruses integrate into the host genome?
use transposition
71
How are retroviruses different from transposons?
they encode proteins that package their genetic information into virus particles that can infect other cells
(HIV)
72
What do retroviruses consist of?
SS RNA genome packed into protein caspid with a virus-encoded reverse transcriptase (RT) enzyme
73
What does reverse transcriptase do?
synthesizes a DNA copy from RNA
74
What happens during the infection process for retroviruses?
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
How are Retrovirus-like transposons different from Retroviruses?
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
Transposition steps
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
What enzyme is used to integrate the ds DNA into the site on the chromosome?
integrase
encoded by the Retrovirus-like transposons
78
What does integrase cut?
one strand at each end of the viral sequence
79
Each exposed 3' OH end attacks a _____________ of the target DNA
phosphodiester bond
80
What process allows viral DNA to insert into the target?
each exposed 3' OH end attacking a phosphodiester bond of the target DNA
81
What happens to the DNA once the viral DNA attacks the target?
it leaves gaps to be filled/ligated
82
What happens after the gaps are filled by DNA repair and the viral DNA has fully integrated into the target?
leaves short repeats on each side of the integrated DNA segment
83
What type of transposons comprise a large portion of our genome?
Nonretroviral transposons
84
What are mutated and truncated Nonretroviral transposons?
repeated sequences in the genome
85
Which transposons are mostly immobile but few retain the ability to move?
Nonretroviral transposons
L1 element (LINE)
86
What 2 enzymes are required for Nonretroviral transposons to move?
- endonuclease
| - reverse transcriptase
87
Do Nonretroviral transposons encode for their required enzymes?
NO
They use enzymes from other transposons
88
What % of the human genome is made of Nonretroviral transposons?
40%
89
Steps of transposition by Nonretroviral transposons
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
What is the function of conservative site specific recombination?
- mediates rearrangements of other types of mobile DNA elements
- breaks and joins 2 DNA double helixes on each molecule
91
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
- DNA integration
- DNA excision
- DNA inversion
92
How is conservative site specific recombination different from transcription?
- 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
Does transposition use a covalent protein/DNA intermediate?
NO
94
What is different about the outcome of conservative site specific recombination?
the relative orientation of the DNA sites
95
What moves in and out of the host genome by the mechanism of conservative site specific recombination?
many bacterial viruses
bacteriophage lambda
96
If the DNA sites are in the same orientation what happens to the DNA sequence? > >
DNA sequence can be INTEGRATED or EXCISED
97
If the DNA sites are inverted in orientation, what happens to the DNA sequence? > <
DNA sequence is INVERTED instead of excised
98
What is the first mobile element to be understood in biochemical detail?
bacteriophage lambda
99
Steps of the lifecycle of bacteriophage lambda
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
What happens in the Prophage pathway of the bactriophage lambda lifecycle?
After integration of lambda DNA into host chromosome:
1) Cell division
2) Integrated lambda DNA replicates along with host chromosome
101
What happens in the Lytic pathway of the bacteriophage lambda lifecycle?
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
Steps of lambda phage insertion into bacterial chromosome
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
What can site-specific recombination be used for?
to turn genes on and off
tool for knocking out gene function in specific tissues in mice
