Chapter 12 - Site-Specific Recombination and Transposition of DNA Flashcards
Name two genetic processes that rearrange DNA sequences and lead to more dynamic genome structure.
Conservative site-specific recombination (CSSR) and transpositional recombination.
Which biological process does CSSR and transposition promote?
Insertion of viral genome into the DNA of the host cell during infection, inversion of DNA segments to alter gene structure, and the movement of transposable elements - often celled “jumping” genes.
What are recombinases?
Proteins that recognise specific sequences where recombination will occur within a DNA molecule. The recombinases bring these sites together to form a protein-DNA complex bridging the DNA site, known as the synaptic complex.
What are recombination sites?
Specific short sequence elements where DNA exchange occurs (often quite short - 20 bp).
Name the types of DNA rearrangements that CSSR generates?
- Insertion of a segment of DNA into a specific site.
- Deletion of a DNA segment.
- Inversion of a DNA segment.
What are the sequence elements within the recombination sites?
Recombinase recognition sequences that flank a central short asymmetric sequence, known as the crossover region, where DNA cleavage and rejoining occurs.
Why is the crossover region asymmetric?
To give the recombination site a defined polarity. The orientation of two sites present on a single DNA molecule will be related to each other in either an inverted repeat or a direct repeat manner. Recombination between a pair fo inverted sites will invert the DNA segment between the two sites. Recombination between two sites organised as direct repeats deletes the DNA segment between the two sites.
Name the two families of site-specific recombinases: the serine recombinases and the tyrosine recombinases. What are their DNA intermediate respectively?
Serine recombinases and the tyrosine recombinases.
For serine: the side chain of a serine residue within the protein’s active site attacks a specific phosphodiester bond in the recombination site. This reaction introduces a single-strand break in the DNA and simultaneously generates a covalent linkage between the serine and phosphate at this DNA cleavage site.
For tyrosine: side chain of the the active site tyrosine attacks and becomes joined to the DNA.
How is the cleaved phosphodiester bond rejoined in site-specific recombination?
The covalent protein-DNA intermediate conserves the energy of the cleaved phosphodiester bond. The strands can be rejoined by several of the cleavage process. An OH group from the cleaved DNA attacks the covalent bond that links the protein to the DNA. This process covalently seals the DNA break and regenerates the free recombinases.
Name two proteins that introduce double-stranded breaks into DNA to initiate homologous recombination during meisosis.
DNA topoisomerases and Spo11.
How do serine recombinases facilitate strand exchange?
Cleave all four strands –> one molecule of the recombinase protein promotes each of these cleavage reactions; therefore, four subunits of the recombinase are required. The breaks regenerate four double-stranded DNA segments (R1, R2, R3 and R4). For recombination to occur, the R2 segment of the top DNA molecule must recombine wit the R3 segment of the bottom molecule. Likewise, the R1 segment of the top molecule must recombination with the R4 segment of the bottom DNA molecule. Once this DNA swap has occurred, the 3’-OH ends if each of the cleaved DNA strands can attack the recombinase-DNA bound in their new partner segment.
How do tyrosine recombinases facilitate strand exchange?
Tyrosine recombinases cleave and rejoin two DNA strand first and only then cleave and rejoin the other two strands. The subunits of recombinase bound to the left recombinase-binding sites (marked R1 and R3). Each cleave the top strand of the DNA molecule to which they are bound. This cleavage occurs at the first nucleotide of the crossover region. The right top strands from the top DNA and the right top strand from the bottom molecule “swap” partners. “First-strand” exchange reaction generates a holiday junction. The “second-strand” exchange reaction undos the Holiday junction.
Explain Cre-lox recombination.
Cre in as enzyme encoded by phage P1, which functions to circularise the linear phage genome during infection. The recombination site on the DNA, where Are acts, are called lox sites. The Cre-lox is a simple example of tyrosine recombination.
It requires four subunits of Cre, with each molecule bound to one binding site on the substrate DNA molecules. Cre exists in two distinct conformations with one pair of subunits in conformation 1 and the other paring conformation 2.
Name some uses of site-specific recombination systems experimentally.
Delete genes in eukaryotic organisms.
