Recombination Flashcards
DSBR
1) the 5’ ends of each strand are chewed back to make overhangs (form overhangs)
2) the 3’ overhangs from each (chewed-back) end invade the undamaged strand (form D-loop)
3) Elongation
The Two Resolutions of DSBR
1) SDSA (Synthesis Dependent Strand Annealing)- helicases free intertwined strands
2) Holliday Junction Resolution
Holiday Junction Resolution
- A Class of endonucleases called Holliday junction resolvases facilitate the resolution
- X/X & Y/Y resolutions (same kind of cleavage) result in the non-cross over product
- X/Y & Y/X (cleavage in different directions) result in the cross-over product
Branch Migration
- possible when (at least) one DNA strand is simultaneously paired with another DNA strand
- Branch Migration can happen in either direction but enzymes involved in DNA homologous recombination catalyze movement in one direction
- hydrogen bonds break at the same rate that new ones form (no change to number of hydrogen bonds! Only location)
Fork Collapse and Repair Procedure
- If there is a lesion that occurs on a replication fork, a portion of the fork is not covalently attached and only associated with non-covalent interactions
- No strand extension!
1) The 5’ end of the damaged strand is chewed back by a nuclease to give an 5’ overhang extension
2) The chewed back strand is then invaded by the analogous strand of the undamaged DNA
3) Holliday junction forms by branch migration
4) Holliday junction resolved
Fork Regression (2 Pathways)
The fork moving backwards causes the template to reform W-C base pair interactions and causes daughter DNA to form new W-C base pair interactions
1) If repair has occurred, nuclease digestion of the base paired daughter (the conservative DNA) allows the replication fork to proceed. (The daughter strand DNA that is folded back is removed)
2) If repair has not occurred, the daughter DNA that is folded back is extended. Branch migration (forwards) restarts the fork. The elongation of the DNA allows the daughter DNA to bypass the lesion when the branch moves forward. However, the original lesion is still present on the template strand.
Gap Repair
- Occurs when the fork does bypass the lesion. Lesion creates a gap in the daughter DNA that is then filled in by gap repair.
- Gaps can also be repaired with TLS, but at a loss of fidelity, so gap repair is preferred.
1) Strand invasion happens. The damaged daughter strand uses its complimentary undamaged strand (also daughter) as a template.
2) Branch migration positions the DNA for replication.
3) Elongation happens, thus filling in the gap
4) The holliday junction is resolved and ligases seal the gaps
Class I (Retrotransposons)
Function by transcribing DNA from one locus to RNA, reverse transcribing the RNA back to DNA and introducing DNA into a new genomic locus.
Insertion Sequences (IS elements) [BACTERIAL transposons]
simple transposons which carry both the sequences necessary for transposition and the genes for transposases
Composite Transposons [BACTERIAL transposons]
more complex transposons that include mandatory sequences, transposase gene and additional genes (i.e. antibiotic resistance) which improve cell survivability but that are not strictly required for cell growth
Complex Transposons [BACTERIAL transposons]
Larger transposons that include transposition sequences, auxiliary genes (that facilitate transposition), other enzymes and additional proteins (such as those that facilitate spread between different cells
Homologous Recombination During Prophase I [Steps]
1)Chromosome are broken intentionally by SpoI (dimer, each monomer takes care of one strand and cuts it, then is attached to strand by 5’-phosphomonoester bond
2) nuclease/helicase complex catalyzes removal of DNA at 5’ end [5’ end chewed back]
3) RecA (helical filament) promotes strand exchange
4) Holliday junction resolved either cross over or non cross over
Features of site-specific binding site [Site specific recombination]
1) There are two binding sites (the two are inverted repeats of the other) separated by non symmetric sequence
Binding sites in opposite direction: inversion
Binding sites in same direction: deletion
Ty cycle [Ty/GYPSY transposons]
1) Transcription of Ty element into an RNA (in nucleus)
2) RNA leaves nucleus and is translated into TY proteins (proteins of ty cycle)
3) Gag proteins construct shell around RNA
4) Reverse transcription (RNA to DNA) happens within the shell in cytosol
5) Shell re-enters the nucleus, where it is disassembled
6) DNA released and integrated into the genome
Rag mediated Recombination [Steps]
1) Rag 1/2 and HMG proteins bind to the signal sequences
2) DNA is bent (process called synapsis) and signal sequences are made adjacent to follow the 12/23 rule
3) The bent DNA is cut to reform dsDNA. Most (but not all) of the signal sequence is cleaved off and the remaining ends of DNA are sealed (signal joint forms which gets destroyed)
4) The open ends from the cutting contain the genes and some residual signal sequence. These ends need to be ligated together.
-NHEJ is used because ends are not that complimentary except for the signal sequence. -This also needs polymerase TdT. TdT adds diversity by adding random nucleotides
What adds to the diversity of the antibody?
- DNA sequence between V and J region (or any of the genes) is lost with the signal joint.
- TdT adds diversity by adding random nucleotides
- NHEJ does not high fidelity and edits the ends for ligation
Steps for how CRISPR/Cas protects bacteria from being re-infected by a phage
1) Each time a phage infects a bacteria, small portions of the phage genome are inserted into the prokaryotic bacterial genome as spacer segments between palindromic repeats. This is called the CRISPR array. (CRISPR array forms).
2) CRIPSR array is made into RNA (pro-crRNA), which is then broken into short pieces (crRNA) each containing one spacer (complimentary to phage DNA) and repeat sequence
3) tracrRNA positions crRNA on a CAS nuclease. crRNA has complimentarity to phage DNA!
4) CAS nuclease then uses crRNA as a guide to cut the infecting phage DNA.
How does the bacteria prevent degrading its own DNA [CRISPR/CAS]?
-The CAS nuclease only tries to unwind and cleave DNA that has pam sequences.
-Pam sequences are only found in the phage DNA, so CAS will not operate on the bacterial genome that does have these spacers (that are essentially the same as the phage)
tracrRNA + crRNA makes what kind of RNA?
Guide RNA (gRNA)
CRISPR can have practical applications. [Name two and what is necessary for each]
1) Rendering a gene non-functional. You need a guide RNA and cas to target a specific gene. This gene will then be broken across both strands.
2) Introducing a new DNA sequence into an organism. Yes there will be a double stranded break, but by providing a desired DNA molecule, homologous repair can happen.
