Lecture 5 Flashcards
What is a DNA intermediate containing four DNA strands from two different helices?
Holliday Junction
What are the means to generate DNA molecules of novel sequence
Homologous recombination
Holliday junctions are structures that are present only _______?
Transiently
How are Holliday junctions separated?
The strands of the helices are cleaved by endonuclease (RuvC)
What is resolution?
When the holliday junctions are cleaved
What are the two outcomes of resolution?
Crossing over
-rare event that only 2 cross over events/chromosome occurs
Gene conversion
-90% of Holiday junctions in humans resolve this way
When does meiotic recombination occur?
Occurs with paired maternal and paternal homologous chromosomes
When recombination is needed to create diversity, how does meiotic recombination begin?
With a double strand break with Spo11(yeast).
What recognized DNA break in meiotic recombination process?
Mre11 identifies the DNA damage
What follows the break and identification of the damaged DNA in meiotic recombination?
Strand invasion and double Holliday junction formation
Resolution
If both strands in each HJ are cut in the same way, how much exchange will occur?
They will separate with minimal exchange of sequences
If strands in each HJ are cut in opposite direction, how much exchange occur?
Portions of each chromosome upstream and downstream are swapped, yielding crossover
True or False?
Crossing over and gene conversion can occur in the same chromosome
True
What does crossing over and gene conversion allow opportunities for?
Genetic reassortment
What is heteroduplex DNA regions on meiotic chromosomes?
Region where the strand from the maternal homolog is base-paired with a strand from the paternal homolog
In meiosis, each parent should contribute how much to the offspring?
Each parent should make an equal contribution to the genetic material of the offspring
In yeast, analysis of all four haploid gametes reveled what rare cases?
Rare cases where each parent did not make an equal contribution to the genetic material of the offspring. 3 came from maternal alleles, and 1 from paternal.
What is gene conversion?
Divergence from the expected distribution of alleles during meiosis.
How does gene conversion occur?
DNA synthesis during homologous recombination
Heteroduplex generated during meiosis and repair of mismatches in regions causes one allele to be lost and the other duplicated. This results in conversion of one to the other.
Why must HR in meiosis must be regulated?
To prevent inappropriate crossovers.
What is an example of an inappropriate cross over?
When recombination occurs between repeated sequences that could scramble the genome
What can interrupt HR between poorly match sequences preventing recombination?
The mismatch repair pathway, if it recognizes too many mismatches then it aborts recombination
What can the interruption of HR by the mismatch repair pathway possibly preserve?
Could also preserve speciation by blocking recombination between closely related species
What are transposons?
Mobile genetic elements also called transposable elements, “selfish DNA”, and “jumping genes”
What are the characteristics of transposons?
Specialized segments of DNA that move from one position in the genome to another
Range in size from 100s to 1000s of nucleotide pairs
-Each with unique sets of genes
-Encodes enzyme that catalyzes movement of transposon
What can transposons do to the cell?
Can provide benefits to the cell (ex. antibotic resistance in bacteria)
Can produce genetic variation
-Occasionally rearrange neighboring DNA of host
-Can induce spontaneous mutations
What is not required in transposons?
No sequence homology required
It can insert anywhere in the genome
How often to transposons move?
Infrequently, one in every 10^5 cell divisions in bacteria
What is transposase?
Enzyme encoded by the transposon itself
- Acts on specific DNA sequence on each end of the transposon
- Allows insertion into a target DNA site
What are DNA only transposons?
Transposons that exist only as DNA in their movement
Contain: Gene encoding transposase, sequences recognized by the enzyme necessary for movement
What type of transposon is largely predominate in bacteria?
DNA only transposons and are largely responsible for spread of antibiotic resistance
How do DNA only transposons move?
Cut and paste transposition or replicative transposition
How do DNA only transposons move by cut and past transposition?
Sequences on each end of element bind transposase
Two transposase molecules come together forming a loop juxaposing two end of element (called a TRANSPOSOME)
Transposase introduces cuts at base of loop
Removes element
Forms central intermediate
How does the Central intermediate become integrated in target DNA in DNA only transposon pathway?
Central intermediate catalyzes direct attack on random site of target DNA by creating a staggered break (breaks 2 phosphodiester bonds, creating new ones as it joins the DNA together)
Staggered breaks produce gaps that are repaired by DNA polymerase and sealed by ligase (results in duplication of the target DNA’s insertion site)
What happens to the donor DNA that has holes after the DNA only transposon has been removed?
Hole in donor chromosome repaired by
double stranded break repair.
If chromosome has just been replicated and there is an identical copy, the 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
What is a retrovirus?
Viruses that encode proteins that package their genetic info into virus particles that can infect other cells
-HIV
Consists of a ssRNA genome packed into protein capsid with a virus-encoded reverse transcriptase enzyme and RT synthesizes a DNA copy from RNA.
Integrase (viral encoded) creates 3’-OH ends on the Viral DNA and the DNA is synthesized by host’s RNA polymerase
How does retroviral-like transposons become integrated in target DNA?
Integrase cuts one strands at each end of viral sequence
Each exposed 3’-OH end attacks a phosphodiester bond of target DNA
Viral DNA inserts into target, leaving gaps to be filled/ligated
Leaves short repeats on each side of integrated DNA segment(b/c of cuts)
How much nonretroviral transponsons are in the human genome?
A large portion of our genome has repeated sequences that are mutated and truncated nonretroviral transposons.
Makes up 40% of human genome
How often do nonretroviral transposons move?
Most are immobile but few retain ability to move
-L1 element (line) does not move.
What do nonretroviral transposons require?
Endonuclease and reverse transcriptase to move but they do not encode the enzymes.
Enzymes used come from other transposons
How does transposition by nonretroviral retrotransposon occur?
- Endonuclease and reverse transcriptase bind to L1 RNA
- Endonuclease nicks the target DNA at insertion point and releases 3’-OH end 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 (multistep pathway produes second DNA strand)
What is conservative site specific recombination?
Mediates rearrangements f other types of mobile DNA elements
Breaks and joins two DNA double helices on each molecule
Depending on positions and relative orientations of recombination sites (Conservative site specific recombinations), what can we get?
DNA integration, DNA excision, or inversion
How does Conservative site specific recombination differ from transposition?
- Needs special sites on each DNA that serve as recognition sites for recombinase while only transposon sequence is required for transposition
- It forms transient high energy covalent bonds and use this energy to complete DNA rearrangement while there are no covalent protein/DNA intermediate in transposition and gaps must be filled by DNA polymerase and ligase
During conservative site specific recombination, if sites are in the same orientation, DNA sequence ___________?
Can be integrated or excised
During CSSR, if sites are inverted in orientation, DNA sequence is ______?
Inverted instead of excised
How do many bacterial viruses move in and out of host genome?s
By Conservative site specific recombination
-bacteriophage lambda
How is site-specific recombination useful in experimentation ?
It can be used to turn genes on and off. It is a tool for knocking out gene function in specific tissues in mice
