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CMMB 411 > UNIT 3 > Flashcards

UNIT 3 Flashcards

1
Q

what is homologous recombination

A

physical exchange of DNA sequences between chromosomes

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2
Q

What does the frequency of crossing over depend on

A

physical distance between genes (long distances give highest exchange)

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3
Q

what is crossing over

A

genetic exchange between chromosomes

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4
Q

two things homologous recombination requires

A
  • long stretch of homology

- several enzymes including (RecA, RecBCD, and others)

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5
Q

what are transposons

A

moveable SBA segments

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6
Q

What are the Physiological functions of homologous recombination

A
  • create variation
  • repair damaged DNA
  • restart stalled replication forks
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7
Q

What are practical applications of homologous recombination

A
  • create knock out variants

- introduce engineered genes into genome

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8
Q

What are the 4 methods to generate double stranded breaks (DSBs)

A
  1. ionization radiation and chemical agents
  2. Spo 11
  3. Bacterial conjugation
  4. Phage transduction
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9
Q

describe ionization radiation

A

two types:
direct
indirect (via DNA replication)

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10
Q

describe Spo 11

A
  • 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)
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11
Q

Describe bacterial conjugation

A

ssDNA in recipient cell

  • converted to dsDNA
  • is is linear DNA format
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12
Q

describe phage transduction

A

dsDNA introduced to host cells by transducing phages

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13
Q

what does homologous mean

A

DNA sequences are identical, for at least 100bp

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14
Q

Steps of homologous recombination

A
  1. alignment of two homologous DNA molecules
  2. introduction of breaks in DNA (generate ssDNA)
  3. strand invasion: short regions of bp are formed between recombining DNA molecules (regions of duplex DNA generated)
  4. 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))
  5. 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
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15
Q

how to solve recombination intermediate

A

2 holliday junctions

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16
Q

describe non cross over products for ABxab

A

AB + ab

17
Q

describe cross over products for ABxab

A

aB+Ab

18
Q

functions of RecBCD emzyme

A

process broken DNA molecules to generate ssDNA and load RecA strand-exchange protein on ssDNA

19
Q

what are Chi sites

A

specific DNA sequence elements that stimulate frequency of of homologous recombination

20
Q

functions of RecB, RecC, RecD

A

RecB-3’-5’ helicase and multifunctional nuclease domain that digest DNA as it moves along
RecD-5’-3’ helicase
RecC-recognizes Chi sites

21
Q

describe generation of ssDNA 3’ tail from dsDNA

A

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

22
Q

function of RecA

A

catalyze pairing of homologous DNA molecules

23
Q

describe features of chi site

A
  • recognized in ssDNA format
  • read from 3’ end
  • 5’GCTGGTGG3’
  • efficiency of chi site recognition 30-40%
  • 1008-1009chi sites in E Coli
24
Q

What happens to dsDNA without chi sites
and
how do chi sites protect E coli

A

degraded

protect from linear phage DNA

25
Q

describe properties of RecA

A
  • 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
26
Q

describe polarity of RecA assembly

A
  • 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
27
Q

what are the 2 binding sites for RecA

A

primary : binds ssDNA

secondary : binds dsDNA

28
Q

describe branch migration

A 
-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
29
Q

describe the concave and convex side of RuvA

A

concave-+charged

convex–charged

30
Q

function of RuvAB

A

displaces RecA and drives branch migration

31
Q

whats functionally equivalent to RuvAB

A

RecG

32
Q

describe RuvC

A
  • 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*
33
Q

what mediates DNA joining

A

ligase

34
Q

overview of meiotic recombination pathway

A

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

CMMB 411 (2 decks)

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