Recombination of Dna
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Transcript of Recombination of Dna
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DNA Recombination
Roles
Types Homologous recombination in E.coli
Transposable elements
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Biological Roles for Recombination
1. Generating new gene/allele combinations
(crossing over during meiosis)
2. Generating new genes (e.g., Immuno-
globulin rearrangement)
3. Integration of a specific DNA element
4. DNA repair
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Practical Uses of Recombination
1. Used to map genes on chromosomes
(recombination frequency proportional
to distance between genes)
2. Making transgenic cells and organisms
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Map of Chromosome I of
Chlamydomonas reinhardtii
Chlamydomonas Genetics Center
cM = centiMorgan; unit of recombination frequency
1 cM = 1% recombination frequency
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Types of Recombination
1. Homologous - occurs between sequences
that are nearly identical (e.g., during
meiosis)
2. Site-Specific - occurs between sequences
with a limited stretch of similarity; involves
specific sites
3. Transposition ± DNA element moves fromone site to another, usually little sequence
similarity involved
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Examples of Recombination
Fig. 22.1
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Holliday Model
R. Holliday (1964)
- Holliday Junctions
form during
recombination
- HJs can be resolved2 ways, only one
produces true
recombinant
molecules
Fig. 22.2 patch
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Fig. 22.5 a-e
The recBCD
Pathway of
Homologous
Recombination
Part I: Nicking and
Exchanging
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recBCD Pathway of Homologous
Recombination
Part I: Nicking and
Exchanging
1. A nick is created in one strand by recBCD at a Chi
sequence (GCTGGTGG), found every 5000 bp.
2. Unwinding of DNA containing Chi sequence byrecBCD allows binding of SSB and recA.
3. recA promotes strand invasion into homologous
DNA, displacing one strand.
4. The displaced strand base-pairs with the singlestrand left behind on the other chromosome.
5. The displaced and now paired strand is nicked
(by recBCD? ) to complete strand exchange.
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recBCD Pathway
of Homologous
Recombination
Part II: Branch
Migration and
Resolution
Fig. 22.5 f-h
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recBCD Pathway of Homologous Rec.
Part II: Branch Migration and Resolution
1. Nicks are sealed Holliday Junction2. Branch migration (ruvA + ruvB)
3. Resolution of Holliday Junction (ruv C )
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R ecBCD : A complex enzyme
R ecBCD has:
1. Endonuclease subunits (recBCD) that cut
one DNA strand close to Chi sequence.
2. DNA helicase activity (recBC subunit) and
3. DNA-dependent ATPase activity
± unwinds DNA to generate SS regions
Activity 2 and 3 linked
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R ecA
38 kDa protein that polymerizes onto SS DNA 5¶-3¶
Catalyzes strand exchange, also an ATPase
Also binds DS DNA, but not as strongly as SS
Fig. 22.6
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R ecA Function Dissected
3 steps of strand exchange:
1. Pre-synapsis: recA coats single strandedDNA (accelerated by SSB, get more relaxed
structure, Fig. 22.8)2. Synapsis: alignment of complementary
sequences in SS and DS DNA (paranemicor side-by-side structure)
3. Post-synapsis or strand-exchange: SS DNAreplaces the same strand in the duplex toform a new DS DNA (requires ATPhydrolysis)
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R uvA and R uvB
DNA helicase that catalyzes branch migration
R uvA tetramer binds to HJ (each DNA
helix between subunits)
R uvB is a hexamer ring, has helicase & ATPase
activity
2 copies of ruvB bind at the HJ (to ruvA and 2 of
the DNA helices) Branch migration is in the direction of recA
mediated strand-exchange
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http://www.sdsc.edu/journals/mbb/ruva.html
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R uv C : resolvase
Endonuclease that cuts 2 strands of HJ
Binds to HJ as a dimer Consensus sequence: (A/T)TT (G/C)
- occurs frequently in E. coli genome
- branch migration needed to reachconsensus sequence!
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R uv C
bound to
Holliday junction
Fig. 22.31a
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Transposable Elements
(Transposons)
DNA elements capable of moving ("transposing")
around the genome
Discovered by Barbara McClintock, largely from
cytogenetic studies in maize, but since foundin most organisms
She was studying "variegation" or sectoring in
leaves and seeds
She liked to call them "controlling elements³
because they effected gene expression in
myriad ways
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Mutant Kernel Phenotypes
Pigmentation mutants ± affect anthocyanin pathway
± elements jump in/out of transcriptionfactor genes (C or R)
± sectoring phenotype - somatic mutations ± whole kernel effected - germ line
mutation
Starch synthesis mutants- stain starch with iodine, see sectoring in
endosperm
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Start with mutant kernels defective in starch synthesis (endosperm
phenotypes) or anthocyanin synthesis (aleurone and pericarp
phenotypes).
Some maize phenotypes caused by transposable
elements excising in somatic tissues.
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Somatic Excision of Ds from C
Fig. 23.19
SectoringWild type Mutant
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Other Characteristics of McClintock's
Elements
Unstable mutations that revert frequently but often
partially, giving new phenotypes.
Some elements (e.g., Ds) correlated with
chromosome breaks.
Elements often move during meiosis and
mitosis. Element movement accelerated by genome
damage.
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Molecular analysis of transposons
Transposons isolated by first cloning a gene thatthey invaded. A number have been cloned this way,
via "Transposon trapping³.
Some common molecular features: ± Exist as multiple copies in the genome
± Insertion site of element does not have extensive
homology to the transposon
± Termini are an inverted repeat ± Encode ³transposases´ that promote movement
± A short, direct repeat of genomic DNA often
flanks the transposon : ³Footprint´
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Ac and Ds
Ds is derived from Ac by internal deletions
Ds is not autonomous, requires Ac to move
Element termini are an imperfect IR Ac encodes a protein that promotes
movement - Transposase
Transposase excises element at IR, and also
cuts the target
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Structure of Ac and Ds deletion
derivatives
Fig. 23.20D
s is not autonomous, requires Ac to move!
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How duplications
in the target site
probably occur.
Duplication
remains whenelement excises,
thus the
Footprint.
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utator (A Retrotransposon)
Discovered in maize; differs significantly from Ac by structure and transposing mechanism
Autonomous and non-autonomous versions;
many copies per cell contains a long terminal IR (~200 bp)
transposes via a replicative mechanism,instead of a gain/loss mechanism
A ³retrotransposon´ ± Similarities with retroviruses
± move via an RNA intermediate
± encode a reverse transcriptase activity
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Fig. 7.34 in Buchanan et al.
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Control of Transposons Autoregulation: Some transposases are
transcriptional repressors of their own
promoter(s) e.g., TpnA of the S pm element
Transcriptional silencing: mechanism not well
understood but important; correlates with
methylation of the promoter
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Biological Significance of
Transposons
They provide a means for genomic change
and variation, particularly in response to
stress (McClintock¶s "stress" hypothesis)(1983 Nobel lecture, Science 226:792)
or just "selfish DNA"?
No known examples of an element playing a
normal role in development.