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Holliday junctions

only present transiently

1

strands of the helices are cleaved by endonuclease (RuvC)

resolution

2

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%)

3

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

4

What happens when crossing over and gene converstion occur in the same chromosome?

homologous recombination... multiple opportunities for genetic reassortment

5

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

6

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

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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

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Why can transposons benefit the cell?

antibiotic resistence genes...
can produce genetic variation
no sequence homology requried

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enzyme encoded by the transposone intself
acts on specific DNA sequence on each end of the transposon

tranposase

-removes element
-forms central intermediate

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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

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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

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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

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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

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DNA only Transposons

Central intermediate catalyzes direct attach on random site of targe DNA

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What does the staggered braek in DNA only transposons create?

breaks 2 phosphodiester bonds, creating new

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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

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synthesizes a DNA copy form RNA

reverse transcriptase enzyme

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creates 3' OH ends on the DNA that attacks the host dNA for retroviruses

intergrase

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Retroviruse stages

RNA

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Retrovirus-like transposones

lack a coat, cannot leave resident cell
also uses integrase

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life cycle of bacterophage lambda

....

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means to generate DNA molecules of novel sequences

homologous recombination
-happens during meiosis
forms Holliday junctions- DNA intermediate containgin four DNA strands

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crossing over and gene conversion can occur

in the same chromosome
-multiple opportunities for genetic reassortment

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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

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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

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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

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double stranded break repair, if chromosome has just been replicated and there is an identical copy

transposon will be restored

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homologous recombination using homologus chromosome

transposon will not be restored

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nonhomologous end joining

will produce a mutation at the break site

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How is retroviruses unlike transposons?

they encode proteins that package their genetic info into virus particles that can infect other cells
-HIV

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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

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How does retroviruses respond during infection process?

viral RNA enters cell and is converted to a double stranded DNA molecule by RT activity

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In retroviruses, what creates 3'OH ends on the DNA that attacks the host DNA

integrase

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WHat are new viral RNAs synthesized by?

host's RNA polymerase

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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

36

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)

37

What does a nonretroviral transposon require to move?

endonuclease
reverse transcriptase
-do not encode the enzyme
-use enzymes from other transposons

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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

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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

40

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

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Conservative site, if sites are in same orientation,

DNA sequence can be integrated or excised

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conservative site, if sites are invverted in orientation,

DNA sequneces is inverted instead of excised

43

Site- specific recombination can be used to turn genes on and off

great tool for knocking out gene function in specific tissues in mice