Lecture #5 Flashcards
Homologous Recombination
Alignment of homologous DNA molecules.
Introduction of breaks in DNA
- Ends are processed to make ssDNA.
Strand Invasion
- Single stranded DNA pairs with complimentary strand in homologous DNA molecule.
Formation of Holiday Junction
- DNA strands cross and branch migration.
Resolution of Holiday Junctions
- Regenerate DNA and finish genetic exchange through cleavage or dissolution.
Steps One & Two
Homologous DNA molecules align.
- Just before mitosis or meiosis.
Cells are at 4N copies of DNA.
Introduction of double strand (ds) breaks within the dsDNA.
- Part of the Double-Strand Break-Repair (DSB) Pathway
- Unknown mechanism in bacteria.
Due to damage?
- Specific proteins within eukaryotes.
Ends are degraded by a nuclease to generate a 3` ssDNA tails.
Step Three
Strand Invasion
- Occurs after end processing.
- Catalyzed by strand-exchange proteins.
Mechanism
- 3` ends invade the unbroken duplex.
- Broken strands pair with their complimentary strands.
- Stable enough to keep the two duplexes together.
Step Four
3` ends are extended by DNA polymerase
- Complementary strand acts as a template.
Holliday Junctions Form
- Invading strands rejoin their initial 5’ end.
Branch Migration
- Holliday Junctions move along the DNA sequence.
- Heteroduplex region extended.
Step Five
Resolution of the Holliday Junction (study figures)
Strands are cut near the site of the cross over.
- Can be cut in two different ways.
- Vertical vs. Horizontal
Two possible products. Spliced: recombination - "F" and "f" are switched. Patch: no recombination - "F" and "f" are not switched.
The proteins involved in HR (E. coli) Part 1
- E. coli* (need to know)
- RecBCD Pathway
- DSB may occur due to DNA damage
- RecBCD complex: helicase + nuclease activity; ATP dependent
- RecB: 3’–>5’ helicase (slow)
- RecD: 5’–>3’ helicase
DSB are processed
- DNA is unwound and strands degraded.
CHI sites recognition by RecC
- Recombination not spots 10X.
The proteins involved in HR (E. coli) Part 2
After CHI recognition
- Complex pauses – RecB catches up.
- Complex continues at a slower speed
- Nuclease activity for RecB shifts.
Other strands now cleaved.
- Generates a 3' overhang.
RecB recruits and interacts with RecA.
-SSB
RecB recruits and interacts with RecA
The proteins involved in HR (E. coli) Part 3
- RecA binds 3` end of overhang and initiates strand invasion* (need to know).
- Family of strand-exchange proteins.
- Cooperative assembly.
ssDNA aligns with homologous DNA duplex
- Each RecA protein binds a “triplet” of stacked bases
Sequence match of ~15bp is needed.
The proteins involved in HR (E. coli) Part 4
RuvA proteins recognize Holliday Junctions after strand invasion.
- Recruits RuvB
RuvB facilitates branch migration
- ATPase
RuvC resolves Holliday Junctions
Endonuclease.
- Endonuclease; cleaves DNA strands w/ the same polarity.
- Determines whether products are recombinant or non-recombinant.
The proteins involved in HR (Eukaryotes)
Similar to bacteria except for…
DSBs introduced by Spo11
MRX complex processes DNA ends
- MRE11, Rad50, Xrs2
- 5’ ends are processed to create 3’ overhangs.
Rad51 and Dmc1 – SSB proteins
- RecA homologues.
- Dmc1-meioisis only.
Rad51C and XRCC3
- Recognize holiday junctions.
Gene Conversion – A consequence of HR
An allele of a gene is replaced by another allele.
Occurs during meiosis.
Slight differences exist between the two DNA sequences.
Mismatch repair will randomly “fix” either top strand or the bottom strand.
Conservative Site-Specific Recombination (CSSR) Part 1
Rearrangement of a defined segment of DNA through the recombination between two defined sequence elements.
Key Characteristics: Recombination sites (RS) - Short sequences were exchange occurs.
Recombinase recognition sequences (RRS)
- Sequences within the RS flanking the crossover region (~20bp).
Crossover region
- Assymetrical region within RS where cleavage and rejoining occur.
Recombinases
- Enzymes that bind RRS and facilitate recombination.
Conservative Site-Specific Recombination (CSSR): Types
Three Types of CSSR:
Insertions
- RSS are on two different DNA molecules.
Deletions
- RSS are on one DNA molecule.
Direct repeats
Inversion
- RSS are on one DNA molecule.
No energy gain or loss.
- Every broken bond is resealed by recombinase.
- Inverted repeats.
Conservative Site-Specific Recombination (CSSR) Serine and Tyrosine Recombinases
Serine and Tyrosine Recombinases:
- Serine or tyrosine within the active site of the recombinase.
- Interacts with the DNA.
Serine Recombinases
- Cleaves all four strands of DNA before strand exchange occurs
Tyrosine Recombinases
- Top strands cleaved before 1st strand exchange
- Bottom strands cleaved before 2nd strand exchange
Transposition Part 1
Form of genetic recombination that moves specific genetic elements from one DNA site to another
- Transposon: genetic element (jumping gene).
Not always sequence specific at the insertion site.
- Can insert many places within the genome.
Most common source of mutations in some organisms.
Present in genomes of all life forms.
- Humans: 50% of human genome is composed of transposon related sequences; only 2% encodes for proteins.
