6.5 and 6.6 Flashcards
Recombination prefers homologous because
no loss of nucleotides at repair site
Homologous Recombination used in
Meiosis and DNA Damage Response
Homologous Recombination specific for
Double Stranded Breaks (DSB).
Homologous Recombination exchanges
similar/identical genetic information across two separate strands of DNA
Meselson-Weigle Experiment
Bacteria (Lambda) was grown/maintained in
“Light” medium
Lambda was chosen bc
Half DNA by weight so density of the whole virus reflects the density of its DNA
Meselson-Weigle proved
crossing over also involves breakage and reunion of DNA molecule
Yeast as a Model Organism
*Cheap
*Abundant
*Fast to grow
*Eukaryotic
*Easy to Genetically Manipulate
*Can be studied as Haploid or Diploid
–Yeast can undergo Meiosis
Yeast
has help in the understanding of recombination
Spo11
protein that makes a double strand break on on of the chromatids by cleaving the phosphodiester bond
Exonuclease
enzyme that removes nucleotides from an end of a DNA molecule
Dmc1
takes 3’ tail and promotes invasion into homologous chromatid – forms heteroduplex
Heteroduplex
region of double stranded DNA in which two strands have nonidentical sequences
Formed during crossing-over
Held by hydrogen bonds
D-loop
Structure where the two strands of a double stranded DNA molecule are separated for a stretch and held apart by a third strand of DNA
D-loop stabilized by
binding of replication protein A (RPA)
Spo11 is highly
conserved
D-Loop connects with other 3’ tail to form
2nd heteroduplex
Holliday junctions
interlocked regions of two sister chromatids in recombination intermediates
Heteroduplex Regions
Expand outwards on both chromatids
Resolvase
enzyme that breaks and joins DNA strands at Holliday junctions to separate nonsister chromatids during crossing-over
Anticrossover helicase
helps disentangle the invading strand thus interrupting Holliday junction formation and preventing crossing-over
If a cell were to respond to a DNA damage
event during homologous recombination,
where would there most likelybe an error?
Heteroduplex
Branch migration
Holliday juncations move away from each other and enlarge the heteroduplx region between them
exonuclease produces
two 3’ single-stranded tails
Homologous Recombination steps
*Step 1: Spo11 causes DSB
*Step 2: Exonuclease degrades 5’end of breaks.
*Step 3: Strand invasion – 1st heteroduplex
*Step 4: Holliday Junctions – 2nd heteroduplex
*Step 5: Expansion of heteroduplex regions
*Step 6: Resolving Holliday Junctions
Spo11 prefers to cleave
particular genomic sites, resulting in hot spots for cross-over
Gene Conversion
Where one allele is changed to another allele during recombination as a result of heteroduplex formation and mismatch repair during recombination
Mitotic cells do not make
Spo11 (no homologous recombination
Mitotic recombinantion occurs bc
damage by environment agents that result in double strand breaks or single strand nicks in DNA
Tetrad analysis
alleles segregate equally into gametes
Gene conversion produces
tetrads will not segregate equally (breaks Mendel’s first low)
Some organisms find it useful to have
site-specific recombination
site-specific recombination is due to
recombinases
site-specific recombination occurs only DNA target sites that are
less 200 base pairs long
site-specific recombination is much
similar that homologous recombination
Integration
insertion of one DNA molecule into another
Cre-Recombinase
Recognizes LoxP sequences to perform recombination
system becoming more frequently used in mouse-based research
FLP-FRT
FLP-FRT system
using flippase (FLP) recombinase, derived from the yeast Saccharomyces cerevisiae
FLP recognizes a pair of FLP recombinase target (FRT) sequences that flank a genomic region of interest
Two types of site specific recombination
FLP-FRT
Uses of site specific recombo
turn on or off expression of a specific gene
Clustered Regularly
Interspaced Short
Palindromic Repeats
CRISPR
CRISPR
Prokaryotic Anti-viral
defense mechanism
CRISPR/Cas9
induce double strand breaks at almost any position in the genome
CRISPR/Cas9 allows for
edit genomes in vicinity of the breakage