UNIT 3 Flashcards
what is homologous recombination
physical exchange of DNA sequences between chromosomes
What does the frequency of crossing over depend on
physical distance between genes (long distances give highest exchange)
what is crossing over
genetic exchange between chromosomes
two things homologous recombination requires
- long stretch of homology
- several enzymes including (RecA, RecBCD, and others)
what are transposons
moveable SBA segments
What are the Physiological functions of homologous recombination
- create variation
- repair damaged DNA
- restart stalled replication forks
What are practical applications of homologous recombination
- create knock out variants
- introduce engineered genes into genome
What are the 4 methods to generate double stranded breaks (DSBs)
- ionization radiation and chemical agents
- Spo 11
- Bacterial conjugation
- Phage transduction
describe ionization radiation
two types:
direct
indirect (via DNA replication)
describe Spo 11
- in eukaryotic systems
- programmed generation of dsb’s to promote homologous recombination during meiosis
- Spo 11 cuts DNA during meiosis (i.e pairing of homologous chromosomes)
- Spo 11 cuts DNA with sequence specificity
- cuts located in less tightly packed nucleosomal regions (around promoters)
Describe bacterial conjugation
ssDNA in recipient cell
- converted to dsDNA
- is is linear DNA format
describe phage transduction
dsDNA introduced to host cells by transducing phages
what does homologous mean
DNA sequences are identical, for at least 100bp
Steps of homologous recombination
- alignment of two homologous DNA molecules
- introduction of breaks in DNA (generate ssDNA)
- strand invasion: short regions of bp are formed between recombining DNA molecules (regions of duplex DNA generated)
- Formation of Holliday: the two DNA molecules become connected by crossing DNA strands (this junction can move along DNA by repeated melting and formation of bp; each time the junction moves, bp in parent broken, while identical base pairs formed in recombination intermediate (called branch migration))
- resolution of Holliday junction: either by cleavage of Holliday junction or (in eukaryotes) process of “dissolution”
cleavage- cutting DNA strands within junction regenerates two separate duplexes
dissolution-convergence/collapse mechanism
how to solve recombination intermediate
2 holliday junctions
describe non cross over products for ABxab
AB + ab
describe cross over products for ABxab
aB+Ab
functions of RecBCD emzyme
process broken DNA molecules to generate ssDNA and load RecA strand-exchange protein on ssDNA
what are Chi sites
specific DNA sequence elements that stimulate frequency of of homologous recombination
functions of RecB, RecC, RecD
RecB-3’-5’ helicase and multifunctional nuclease domain that digest DNA as it moves along
RecD-5’-3’ helicase
RecC-recognizes Chi sites
describe generation of ssDNA 3’ tail from dsDNA
RecBCD enters DNA at site of DSBand moves along DNA unwinding strands
RecD runs faster than RecB
as RecB tries to keep up, loop of ssDNA from the 3’ end bulges out
upon entering the chi sequence, pauses (looped out DNA pulled back in/Rec B becomes primary motor, and conformational change due to uncoupling of RecD, and nuclease activity of RecBCD altered) for a sec, then continues at half speed
3’ ss extension terminating with chi sequence at 3’ end
RecBCD interacts with RecA and promotes its assembly
upon RecA binding, length of DNA molecule extended
FINAL PRODUCT:
short 3’-5’
long 5’-3’ with chi site at end
function of RecA
catalyze pairing of homologous DNA molecules
describe features of chi site
- recognized in ssDNA format
- read from 3’ end
- 5’GCTGGTGG3’
- efficiency of chi site recognition 30-40%
- 1008-1009chi sites in E Coli
What happens to dsDNA without chi sites
and
how do chi sites protect E coli
degraded
protect from linear phage DNA
describe properties of RecA
- 352aa (37kDa)
- structure:
- -n terminal tail
- -core (DNA and ATP binding)
- -c terminal tail (not visible)
- binds to both ssDNA and dsDNA
- -assembles to form helical RecA filament on DNA
- -6 RecA proteins/turn of RecA filament
- -binds 18 nucleotides of ssDNA/turn of RecA
- -functions as scaffold to bring dsDNA in elongated, right handed DNA structure
describe polarity of RecA assembly
- RecA-ATP has high affinity to ssDNA
- ATP hydrolysis ->dissociation of RecA from DNA
- assemble on single stranded 5’-3’
- after slow nucleation process, rate of assembly is 2-20 subunits/sec
what are the 2 binding sites for RecA
primary : binds ssDNA
secondary : binds dsDNA
describe branch migration
-catalyzed by RuvAB complex RuvA: --forms tetramer --recognizes Holliday junction structure in sequence independent manner --one subunit binds to one strand --prevents Holliday junction from excessive unwinding during branch migration --recruits RuvB RuvB: --hexameric ATP dependent helicase --encloses dsSNA but not ssDNA
describe the concave and convex side of RuvA
concave-+charged
convex–charged
function of RuvAB
displaces RecA and drives branch migration
whats functionally equivalent to RuvAB
RecG
describe RuvC
- forms dimer
- recognizes Holliday structure
- suggested to associate with RuvA to form RuvABC complex to scan for sequence
- cuts with low degree of sequence specificity
- mediated DNA cleavage*
what mediates DNA joining
ligase
overview of meiotic recombination pathway
formation of DSBS during meiosis requires presence of Spo 11 and the MRX complex
MRX protein responsible for resection of the 5’ ends at the break site
strand exchange proteins Dmc1 and Rad51 assemble onto ssDNA tail
