GENETIC RECOMBINATION/REARRANGEMENT
Recombination plays an important role in the 1)process of introducing genetic variation by allowing genes to be reassorted into different combinations. For example, genetic recombination results in the exchange of genes between paired homologous chromosomes during meiosis. (Fig. 3.4)
2) Also, an important mechanism for repairing damaged DNA (later lecture).
3) Recombination also is involved in rearrangements of specific DNA sequences that alter the expression and function of some genes during development and differentiation (for example immunoglobulins, the important antibodies of the immune system).
DNA molecules recombine by breaking and rejoining. In other words, phosphodiester bonds are broken and rejoined.
How can two double-stranded DNA molecules break exactly at the same sites so that no mutations occur? (otherwise there would be a gain of nucleotides for one DNA molecule and loss for the other).
Because of Homologous Recombination (recombination between DNA molecules with complementary sequences)
1964 Molecular model of recombination: Holliday Model
Named after Robin Holliday
This model has continued to provide the basis for current thinking about recombination mechanisims, although the model has been modified in recent years.
Original model:
1) Single-stranded nicks are introduced at the same position on both parental DNA molecules.
2) Each nicked strand then invades the other DNA molecule by complementary base pairing
3) Ligation produces a crossed-strand intermediate called a Holliday junction
4) Once a Holliday junction is formed, it can be resolved by nicking and rejoining of the crossed strands to yield recombinant or nonrecombinant heteroduplexes (Fig. 5.32).
There is direct EM evidence for formation of the Holliday junction (Fig. 5.31)
Recombinent heteroduplexes: Resulting DNA molecules are a combination of both parental DNA molecules. Nonrecombinent heteroduplexes: Except for heteroduplex region resulting DNA molecules contain only DNA from one parent molecule.
Different heteroduplexes are formed because the Holliday junction may rotate into different isomers before it is resolved, thus nicking either occurs in pure parental strands or in the complementary strands. The former leads to a separation of parental DNA and therefore recombinant heteroduplexes.
1975 revision of Holliday model
Recombination is initiated by one nick only (more likely!). The nicked strand is then displaced, and invades the other homologous DNA molecule by homologous base pairing. This process produces a displaced loop of single-stranded DNA, which can then be cleaved and joined to the other parental molecule (Fig. 5.33). The result is a crossed-strand Holliday junction as before, resolved as before into recombinant or nonrecombinant heteroduplex molecules.
Yet another variation of the model suggests that recombination is initiated by a double-strand break. Therefore, multiple alternatives may account for the initial stages of recombination between two DNA molecules. The details of the recombination mechanisms, particularly in eukaryotic cells, have not been fully elucidated. However, the crossed-strand Holliday junction through strand exchange leading to the formation of a heteroduplex region remains the central molecular intermediate in consideration of the recombination process.
Enzymes Involved in Homologous Recombination
Involved proteins were discovered through the analysis of recombination-defective mutants in E. coli
Necessary proteins: 1) general enzymes connected with DNA biochemistry (DNA polymerases, ligase etc.) 2) several specific proteins.
Central protein involved in homologous recombination is RecA
Rec A promotes the exchange of strands between homologous DNAs that causes heteroduplexes to form. The action of RecA occurs in three stages (Fig. 5.34):
1) First RecA binds to single-stranded DNA, coating the DNA to form a protein-DNA filament.
2) RecA has two DNA binding sites and is able to bind to a second, double
stranded DNA molecule, forming a complex between the two DNAs. This non-
specific interaction (between protein and DNA) is followed by specific base pairing between the single-stranded DNA and its complement.
3) RecA catalyzes strand exchange, with the single strand originally coated with RecA displacing its homologous strand to form the heteroduplex.
The RecA protein is therefore capable of catalyzing , by itself, the strand exchange reactions that are central to the formation of the Holliday junctions.
In yeast - protein designated RAD51 is structurally and functionally similar to RecA. Similar proteins have been found even in humans implying that this process and activity is highly conserved.
Most recombination events in E. coli also require the RecBCD enzyme (Fig. 5.35).
This enzyme is a complex of three proteins (RecB, C, and D). Seems to provide the single-stranded DNA to which RecA binds.
The enzyme first transiently unwinds the double-stranded DNA (binds to the end of a DNA molecule and then acts as a helicase) as it travels along the molecule. When it encounters the specific nucleotide sequence GCTGGTGG (the chi site), the enzyme acts as a nuclease to introduce a single-stranded nick. It then continues to unwind the double helix, forming a displaced single strand to which RecA can bind to initiate strand exchange.
Once the Holliday junction is formed, three other E. coli proteins (Ruv A, B, and C) become involved (Fig. 5.36). Ruv A and Ruv B together catalyze the movement of the crossed-strand site in Holliday junctions (branch migration).
RuvC resolves the Holliday junction by cleaving the crossed strands, which are then joined by ligase.
DNA Rearrangements
Site-Specific Recombination
Occurs between specific DNA sequences, which are usually homologous over only a short stretch of DNA. The principal interaction in this process is mediated by proteins that recognize the specific DNA target sequences rather than by complementary base pairing.
Examples:
Integration of bacteriophage DNA into host bacterial chromosome
Immunoglobulin and T Cell Receptor genes
DNA rearrangements via Transposition
involves the movement of sequences throughout the genome and has no requirement for sequence homology.
More Later!