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3382 > Homologous Recombination Transposons and NHEJ > Flashcards

Homologous Recombination Transposons and NHEJ Flashcards

1
Q

What is NHEJ?

A

Cellular pathway that repairs double-strand breaks in DNA

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2
Q

What does NHEJ compete with to repair ds breaks?

A

homologous recombination

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3
Q

NHEJ does not require

A

complementary template DNA

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4
Q

what are the types of NHEJ?

A

classical NHEJ or alternative NHEJ

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5
Q

What is the classical NHEJ pathway?

A
  • Ku protein recognize only blunt DNA ends or overhanging ends that are compatible
  • little or no end processing
  • DNA ends ligated together
  • minimal loss of genetic information
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6
Q

What is the alternative MMEJ pathway?

A
  • DNA ends require processing by MRE11 5’ to 3’ exonuclease complex
  • DNA loss, insertions, deletions, chromosome translocations
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7
Q

Blunt DNA ends and compatible overhangs containing DNA ds BREAKS are repaired by

A

cNHEJ

all others aNHEJ

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8
Q

Transposable elements are source of genetic variability
because of their

A

mobility

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9
Q

transposons have Illegitimate Recombination because

A

there is no homology required

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10
Q

Mechanism by which some transposable elements
move is

A

site-specific recombination
- The lack of homology in the process makes this very dangerous: can insert into essential genes

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11
Q

What are the classes of transposons?

A
  1. Class I: retrotransposons (movement involves reverse transcriptase; moves through RNA intermediate)
  2. Class II: DNA transposons (2 types, moves through a DNA intermediate)
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12
Q

What are the 2 types of DNA transposons?

A
  1. replicative transposition
  2. non-replicative transposition
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13
Q

What is replicative transposition>

A

transposon makes a copy of itself resulting in an increase in the number of transposable elements
- enzymes: transposase, resolvase

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14
Q

What is non-replicative transposition>

A

transposone cuts and pastes itself from one position to a new target site
- enzyme: transposase

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15
Q

What is the simplest transposon?

A

insertion sequence:
inverted repeats flanks the IS element which is surrounded by duplicated, identical target sequences

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16
Q

Where does the duplicated target sequence arise?

A
  • transposons move from one position to another in the same genome
  • makes a stagerred cut in the target
  • gap is filled by transpositio reaction which makes the identical sequence around it
17
Q

What is a bacterial Tn transposon?

A
  • carries other genes between inverted repeats that are unrelated to transposition
  • genes are often antibiotic resistance
18
Q

What are composite transposons?

A
  • arose from two adjacent transposons
  • larger gene-rich central region
  • IS-lke module
19
Q

What is Tn5?

PROCESS?

A
  • composite transposon that encodes 3 antibiotic resistance genes
  • inverted IS-like modules
  • transposition by a cut-and-paste mechanism
  1. dimerization where IS-like module base pairs
  2. makes synaptic complex and that loop is cleaved by transposase (endonuclease)
  3. NHEJ to put the target DNA back together
20
Q

why is 5’ end labelling use for a 3’ to 5’ exonuclease?

A

you can see a ladder
- if it was 3’ end labelled, you would only see the full DNA

21
Q

why are all phosphates not labelled?

A

you would see a smear on the gel (too much signalling)

22
Q

if a gel is run over time, what would darker bands represent?

A
  • could be DNA thats folded over
  • could be enzyme slowing down on that region bc of high GC content
  • could be that the enzyme falls off at that location/loses specificity
23
Q

What is recombination?

A

breaking and rejoining polynucleotides (for repair or other processes)

24
Q

recombination is important because it is

A

a source of genetic variability

25
Q

What is Deinococcus radiodurans?

if the gene recA is knocked out…

A

bacteria that survives high radiation environments
- sustains huge # of DNA breaks, repairs, and survives

cell still recovers but to a lesser degree

26
Q

What are the types of recombination?

A

homologous recombination, NHEJ, transposition

27
Q

What is homologous recombination?

A

recomb between DNA segments that share extensive sequence homology

28
Q

transposition requires

A

short regions of sequence similarity

29
Q

where does a ds break appear in replication?

A

dsDNA nicked region of newly replicated lagging strand looks like a ds break

30
Q

What are the steps of general homologous recombination in E. coli?

A
  1. RecBCD makes DNA with 3’ free end
  2. RecA aligns homologous DNA to “broken” DNA
  3. Resolve Holliday Junctions
31
Q

How does DNA get a free 3’ end for homologous recombination?

A
  1. RecB = 3’-5’ helicase and 3’-5’ exonuclease and 5’-3’ endonuclease
  2. RecD = 5’-3’ helicase
  3. RecC = regulatory scaffold protein
  4. RebB nicks the 5’ and 3’ ends until Chi site
  5. Then, exonuclease (3’-5’ activity) stops and endonuclease (5’-3’ activity) continues
  6. RecA recruited to protect ss region and RecBCD dissociates

exonuc activity = short fragments bc 3’-5’ | endonuc is larger bc 5’-3’

32
Q

How is homologous DNA aligned to “ds break” DNA?

A
  1. RecA aligns homologous dsDNA to ssDNA and polymerizes ssDNA (from homologus DNA)
  2. forms a transient 3-stranded helix by rotating and ATP to break and reform new dsDNA
  3. RecA continues until it can’t tolerate anymore mismatches
33
Q

is RecA sequence specific?

A

no because ds breaks can happen anywhere; must bind to backbone

34
Q

What does RecA filament look like

A

extended helical structure that spools in dsDNA (homologous) and spools out one ssDNA (from homologous) and dsDNA (homologous strand and broken DNA)

35
Q

How are Holliday junctions resolved in homologous recombination?

A
  1. DNAP extends 3’ ends using the spooled in template
  2. crossed over structure forms
  3. RuvABC complex moves strands away from the ds breaks
  4. RuvA binds to Holliday Junction
  5. DNA binds in 4 symmetry-related grooves
  6. central pin is -vely charged to facilitate separateion of ssDNA segments (drives DNA apart from ds because bb
  7. RuvB are 2 ATPase heximeric rings that dsDNA passes through
  8. RuvC = endonuclease that resolves junction

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