DNA replication, repair and recombination 3 (lecture 5) Flashcards
Can crossing over and gene conversion occur in the same chromosome
yes
Meiotic recombination occurs with paired ____ and _____ chromosomes
maternal and paternal homologous
Meiotic recombination begins with a _______ by enzymes ____ and ____
double strand break, Spo11 (yeast), which breaks the ends, and Mre11 which process the ends
DNA intermediate containing four DNA strands from two different helices
Holliday junction
Homologous recombination is a means to generate DNA molecules of ____ sequence
novel
Holliday junctions are present only
transiently
Resolution of Holliday junctions is when strands of the helices are cleaved by ______
endonuclease (RuvC)
Resolution of Holliday junctions results in 2 outcomes
- Crossing over
- Gene conversion
90% of Holliday junctions in humans resolve in
gene conversion
_____ are rare events with only 1-2 per chromosome
Crossing over
If both strands in each holliday junction are cut in the same way, they will separate with
minimal exchange of sequences
If both strands in each Holliday junction are cut in opposite directions,
portions of each chromosome upstream and downstream are swapped, yielding crossover
in branch migration, base pairs are continually ____ and ___ as the branch point moves
broken and reformed
Once strand invasion occurs, the point of exchange can move through _____
branch migration
in branch migration ____ region of one single strand displaces a ____ region on the other
unpaired, paired
Branch migration happens _____ in both directions or can be catalyzed by ____ to move in one direction
spontaneously, special helicase (use of helicase requires energy)
Meiotic recombination results in regions of _____ DNA
heteroduplex
what is a strand with the DNA sequence of the paternal homolog is base-paired with a strand from the maternal homolog is called
Heteroduplex region
In meiosis, each parent should make _____ contribution to the genetic material of the offspring. This may not be the case due to ____
an equal, gene conversion
Divergence form the expected distribution of alleles during meiosis
gene conversion
How do gene conversions occur
- DNA synthesis during homologous recombination
- Repair of mismatches in regions of heteroduplex DNA. As a consequence of repair, one allele is lost, the other duplicated, resulting in “conversion” of one to the other
What are the two types of homologous recombination that do not require substantial regions of DNA homology
- Transpositional recombination
- Conservative-site-specific recombination
Transpositional Recombination
- DNA-only transposons
- Retroviral-like retrotransposons
- Nonretroviral retrotransposons
Conservative site- specific recombination
- Bacteriophage lambda
Mobile genetic elements
Transposons
Transposable elements are also called
- Selfish DNA
- Jumping genes
specialized segments of DNA that move form one position in the genome to another
Transposons
Transposons range in size form ____ to ______ of nucleotide pairs
100s to tens of thousands
Each transposon has ____ set of genes and encode enzymes that catalyzes ______ of transposon
unique, movement
Transposons can provide benefits to the cell such as in ____ in bacteria
antibiotic resistance
Transposons produce ______ variation by occasionally rearranging neighboring ___ of host and can induce _________
genetic, DNA, Spontaneous mutations
Do Transposons require sequence homology
No!!! They can insert anywhere in the genome
Transposons move ______. one in every ____ cell divisions in bacteria
infrequently. 10^5
Enzyme encoded by the transposon itself
Transposase
Functions of Transposase
- Acts on specific DNA sequence on each end of the transposon
- Allows insertion into a target DNA site
Transposons are grouped into ___ large classes: which are?
- 3
- DNA-only transposons
- Retroviral-like retrotransposons
- nonretroviral retrotransposons
What is the specialized enzyme required for the movement of DNA-only transposons
Transposase
What is the specialized enzymes required for the movement of Retroviral-like retrotransposons
Reverse transcriptase and Integrase
What is the specialized enzymes required for the movement of nonretroviral retrotransposons
Reverse transcriptase and endonuclease
Mode of movement for DNA-only transposons
moves as DNA, either by cut-and-paste or replicative pathways
Mode of movement for retroviral-like retrotransposons
Moves via an RNA intermediate whose production is driven by a promotor in the LTR
nonretroviral retrotransposons mode of movement
moves via an RNA intermediate that is often synthesized from a neighboring promotor
DNA only transposons exist only as ____ in their movement
DNA
DNA only transposons are predominate in _____ and are largely responsible for
bacteria, and are largely responsible for the spread of antibiotic resistance
DNA only transposons use ________ or ______ forms of movement
- cut-and-paste transposition
- replicative transposition
DNA only transposons contain
- gene encoding transposase
- sequences recognized by the enzyme necessary for movement
explain cut-and-paste transposition
- begins when transposase brings the two inverted DNA sequences of the DNA-only transposon together forming a DNA loop
- insertion into the target chromosome, also catalyzed by the transposase, occurs at a random site through the creation of staggered breaks in the target chromosome
- following the transposition reaction, the single-stranded gaps created by the staggered breaks are repaired by DNA polymerase and ligase
- the insertion site is marked by a short direct repeat of the target DNA sequence
- The break in the donor chromosome is repaired (note that this often alters the DNA sequence causing a mutation at the original site of the excised transposable element
DNA-only transposons can be recognized in chromosomes by the
inverted repeat DNA sequences present at their ends
Two transposase molecules come together forming a loop juxtaposing two ends of element this is referred to as the
Transpososome
After the transpososome is formed transposase introduces cuts at base of loop then_____ and ____
removes element and forms central intermediate
The central intermediate of DNA only transposons catalyzes ________ creating a ______
direct attack on random site of target DNA, create a staggered break (breaks 2 phosphodiester bonds, creates new ones as it joins DNA together
Staggered breaks produce gaps that are repaired by _____ and sealed by ____. Resulting in
DNA polymerase and sealed by ligase. Resulting in duplication of the target DNA’s insertion site
The hole in the donor chromosome after DNA only transposon is removed is repaired by
- double-stranded break repair, if chromosome has just been replicated and there is an identical copy thus Transposon will be restored
- homologous recombination using homologous chromosome (transposon will not be restored)
- Nonhomologous end joining (will produce a mutation at the break site)
Retrovirus-like transposons resemble a retrovirus but lack a
coat
Retrovirus-like transposons move in and out of chromosome the same way but
they are unable to leave the resident cell
what is the first step in retroviral-like retrotransposons transposition
is the transcription of the entire transposon, producing an RNA copy of the element that is typically serval thousand nucleotides long.
What is the second step in retroviral-like retrotransposons transposition
The translation of the RNA transcript as messenger RNA by the host cell, encodes a reverse transcriptase enzyme
What is the third step in retroviral-like retrotransposons transposition
Reverse transcriptase makes a double strand DNA copy of the RNA molecule via an RNA-DNA hybrid intermediate. This double strand DNA can then integrates onto a site of the chromosome using the enzyme integrase which is encoded by the element as well
The entire Retrovirus-like transposon is transcribed by the
host
Explain how integrase integrates retrovirus or retroviral-like transposon
- integrase cuts on strand at each end of viral sequence
- Each exposed 3’ OH end attacks a phosphodiester bond of target DNA
- This inserts viral DNA into target, leaving gaps to be filled/ligated
- leaves short repeats on each side of integrated DNA segment
Nonretroviral transposons make up ____% of human genome
40%
Nonretroviral transposons comprise a large portion of
our genome
a significant fraction of many vertebrate chromosome is made up of repeated DNA sequences. In human chromosomes, these repeats are mostly mutated and truncated versions of _______
nonretroviral retrotransposons
Relatively recent movements of _____ (a nonretroviral retrotransposons) have been identified, some of which result in human disease, for example a particular type of hemophilia results from an L1 insertion into the gene encoding the blood-clotting protein Factor VIII
L1 element
Most nonretroviral Transposons are ____ but few retain the ability to ____
immobile, move
Nonretroviral transposons require ____ and ____ to move
endonuclease and reverse transcriptase
Nonretroviral transposons _____ the endonuclease and reverse transcriptase they need to move instead they
do not encode thrown enzymes
instead they use enzymes form other transposons
Explain Transposition by nonretroviral retrotransposons
- endonuclease and reverse transcriptase bind to L1 RNA
- Endonuclease nicks the target DNA at insertion point
- this releases 3’OH to serve as primer in reverse transcription step
- Single-Stranded DNA copy of L1 directly linked to target DNA
- Insertion of double-stranded DNA copy of L1 at target site
Conservative site specific recombination mediates rearrangements of other types of ______
mobile DNA elements (not transposons)
Conservative site-specific recombination can _____ rearrange DNA
Reversibly
in conservative site-specific recombination breakage and joining occur at ____ special sites, ____ on each participating DNA molecule
2 , 1
Depending on position and relative orientations of recombination sites Conservative site-specific recombination can lead to
- DNA integration
- DNA excision
- DNA inversion
Conservative site-specific recombination differs from transposition by
- need special sites on each DNA that serve as recognition sites for recombinase, which will catalyze there rearrangement (only transposon sequence is required for transposition and they can use any sequence)
- Recombinases resemble topoisomerases in the sense that they form transient high energy covalent bonds and use this energy to complete DNA rearrangement (No covalent protein/DNA intermediate in transposition and Gaps must be filled by DNA polymerase and ligase)
______ recombination is often used by DNA viruses to move their genomes in and out of the genomes of their host cells ex. ____
conservative site-specific recombination. Ex. Bacteriophage lambda
Conservative site specific recombination can produces two types of DNA rearrangements
- if sites are in same orientation, DNA sequence can be integrated or excised
- If sites are inverted in orientation, DNA sequence is inverted instead of excised
salmonella uses conservative site-specific recombination to control the expression of ______ genes
flagellin
