Homologous Recombination Transposons and NHEJ Flashcards
What is NHEJ?
Cellular pathway that repairs double-strand breaks in DNA
What does NHEJ compete with to repair ds breaks?
homologous recombination
NHEJ does not require
complementary template DNA
what are the types of NHEJ?
classical NHEJ or alternative NHEJ
What is the classical NHEJ pathway?
- 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
What is the alternative MMEJ pathway?
- DNA ends require processing by MRE11 5’ to 3’ exonuclease complex
- DNA loss, insertions, deletions, chromosome translocations
Blunt DNA ends and compatible overhangs containing DNA ds BREAKS are repaired by
cNHEJ
all others aNHEJ
Transposable elements are source of genetic variability
because of their
mobility
transposons have Illegitimate Recombination because
there is no homology required
Mechanism by which some transposable elements
move is
site-specific recombination
- The lack of homology in the process makes this very dangerous: can insert into essential genes
What are the classes of transposons?
- Class I: retrotransposons (movement involves reverse transcriptase; moves through RNA intermediate)
- Class II: DNA transposons (2 types, moves through a DNA intermediate)
What are the 2 types of DNA transposons?
- replicative transposition
- non-replicative transposition
What is replicative transposition>
transposon makes a copy of itself resulting in an increase in the number of transposable elements
- enzymes: transposase, resolvase
What is non-replicative transposition>
transposone cuts and pastes itself from one position to a new target site
- enzyme: transposase
What is the simplest transposon?
insertion sequence:
inverted repeats flanks the IS element which is surrounded by duplicated, identical target sequences
Where does the duplicated target sequence arise?
- 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
What is a bacterial Tn transposon?
- carries other genes between inverted repeats that are unrelated to transposition
- genes are often antibiotic resistance
What are composite transposons?
- arose from two adjacent transposons
- larger gene-rich central region
- IS-lke module
What is Tn5?
PROCESS?
- composite transposon that encodes 3 antibiotic resistance genes
- inverted IS-like modules
- transposition by a cut-and-paste mechanism
- dimerization where IS-like module base pairs
- makes synaptic complex and that loop is cleaved by transposase (endonuclease)
- NHEJ to put the target DNA back together
why is 5’ end labelling use for a 3’ to 5’ exonuclease?
you can see a ladder
- if it was 3’ end labelled, you would only see the full DNA
why are all phosphates not labelled?
you would see a smear on the gel (too much signalling)
if a gel is run over time, what would darker bands represent?
- 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
What is recombination?
breaking and rejoining polynucleotides (for repair or other processes)
recombination is important because it is
a source of genetic variability
