Lecture 5 Flashcards
strands of the helices are cleaved by endonuclease (RuvC)
resolution
Holliday junctions
only present transiently
What are the two outcomes of resolution?
crossing over-complete crossing over of genetic material (rare(
and gene converstion- small portion of genetic material change (90%)
When does meiotic recombination occur?
With paired maternal and paternal homologous chromsomes
repari usually occurs btw newly duplicated and identical helicases
-begins with a double strand breaks
yeast Spo11
Mre11 identifies the DNA damage
strand invations and doubl HOlliday junction follow
resolusion
What happens when crossing over and gene converstion occur in the same chromosome?
homologous recombination… multiple opportunities for genetic reassortment
What are regions of heteroduplex DNA?
A result of recombination….region where the strand from the mternal homolog is base paired with a strand from the paternal homolog
divergence from the expected distribution of alleles during meiosis
both strands are cut in the same way causing minimal exchange
gene conversion
- this occures bc DNA synthesis during homologous recombinnation
- repair of mismatch
transposons, mobile genetic elements
very specialized DNA segments that move from one postion in the genome to another
each w unique sets of genes
encodes enzyme that catalyzes movement of transposons
Why can transposons benefit the cell?
antibiotic resistence genes…
can produce genetic variation
no sequence homology requried
enzyme encoded by the transposone intself
acts on specific DNA sequence on each end of the transposon
tranposase
- removes element
- forms central intermediate
DNA only transposons
stay DNA the entire time.
use transposase
moves as DNA, either by cut and paste or replicative pathways
contain: gene encoding transposase
sequences recongized by the enzyme necessary for movement
lgly responseible for antibiotic resistance
P element
Retroviral like retrotransposons
directly repeated long tnermal repeats (LTRs) at each end
revers transcriptase and integrase
moves via an RNA intermediate prodcued by a promoter in the LTR
Nonrtroviral retrotransposons
Poly A at 3’ end of RNA transcript; 5’ end is often truncated
reverse transcriptase and endonuclease
moves via an RNA intermediate that is often produced from a enighboring promoter
Cut and past transpostion of DNA only Transposons
sequences on each end of the element bind transposase
two transposase molecules come together forming a loop juxtaposing two ends of element= transposome
cuts at base of loop; removes element and forms central intermediate
DNA only Transposons
Central intermediate catalyzes direct attach on random site of targe DNA
What does the staggered braek in DNA only transposons create?
breaks 2 phosphodiester bonds, creating new
Retroviruses
they inject their RNA into the genome
consist of a SS RNA genome packed into protein capsid with a virus-endcoded reverse transcriptase enzyme
synthesizes a DNA copy form RNA
reverse transcriptase enzyme
creates 3’ OH ends on the DNA that attacks the host dNA for retroviruses
intergrase
Retroviruse stages
RNA
Retrovirus-like transposones
lack a coat, cannot leave resident cell
also uses integrase
life cycle of bacterophage lambda
….
means to generate DNA molecules of novel sequences
homologous recombination
-happens during meiosis
forms Holliday junctions- DNA intermediate containgin four DNA strands
crossing over and gene conversion can occur
in the same chromosome
-multiple opportunities for genetic reassortment
Regulation of HR
prevent inappropriate cross over
- if recombination occurs btw repeated sequences could scramble the genome
- mismatch repair pathway can interrupt HR btw poorly matched sequences preventing recombination events. Could help reserve speciation by blocking recombination btw closely related species
enzyme encoded by the transposomn itself
acts on specific DNA sequence on each end of the transposon
allows insertion into a traget DNA site
transposase
With DNA only transposons staggered breaks produce gaps that are repaired by DNA polymerase and sealed by ligase.
results in duplication of the target DNA’s insertion site
double stranded break repair, if chromosome has just been replicated and there is an identical copy
transposon will be restored
homologous recombination using homologus chromosome
transposon will not be restored
nonhomologous end joining
will produce a mutation at the break site
How is retroviruses unlike transposons?
they encode proteins that package their genetic info into virus particles that can infect other cells
-HIV
consist of a SS RNA genome packed into protein capsid with a virus encoded reverse transcriptase (RT) enzyme
-RT synthesizes a DNA copy from RNA
retrovirus
How does retroviruses respond during infection process?
viral RNA enters cell and is converted to a double stranded DNA molecule by RT activity
In retroviruses, what creates 3’OH ends on the DNA that attacks the host DNA
integrase
WHat are new viral RNAs synthesized by?
host’s RNA polymerase
Transposition by retrovirus or retroviral-like transposon
- integrase cuts one strand at each end of viral sequence
- each exposed 3’ OH ends 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
nonretroviaral transposons
- comprise a lg portion of our genome
- repeated sequences are mutated and truncated nonretroviral transposons
- few retain abillity to move
- L1 element (LINE)
What does a nonretroviral transposon require to move?
endonuclease
reverse transcriptase
-do not encode the enzyme
-use enzymes from other transposons
How do you transposition by a nonretroviral retrotransposon?
- endonuclease and reverse transcriptase bind to L1 RNA
- Endonuclease nicks the target DNA at insertion point
- releases 3OH to serve as primer in revers transcription step
- single-stranded DNA copy of L1 directly linked to target DNA
- insertion of double-stranded DNA cope of L1 at target site
Conservitive site specific recombination
mediates rearrangments of other tpes of mobile DNA elements
- break and join two DNA doulbe helices on each molecule
- depending on positions and relative orientations of recombination sites can get : DNA integration, DNA excision, or inversion
How is conservative site specific recomination different from transposition?
- need speicail sites on each DNA that serve as recognition sites for recominase
- only transposon sequence is requrired for transposition
- form transient high energy covalent bonds and use this energy to complete DNA rearrangment
- no covalent protein/DNA intermediate in transposition
- gaps must be filled by DNA polymerase and ligase
Conservative site, if sites are in same orientation,
DNA sequence can be integrated or excised
conservative site, if sites are invverted in orientation,
DNA sequneces is inverted instead of excised
Site- specific recombination can be used to turn genes on and off
great tool for knocking out gene function in specific tissues in mice
