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The term recombination was originally used by geneticists to describe the __
In this context it refers specifically to the generation of __
- outcome of crossing over between pairs of homologous chromsomes
- new allele combos during meiosis
In the 1960s, it was realized that crossing over involves __, and “recombination” gradually became the name given to any event that involves __
- breakage and subsequent rejoining of DNA molecules
- breakage and reunion of DNA molecules
Recombination is also involved in __. It is also involved in the __, as well as the integration of __ and __.
integration into bacterial genomes of DNA acquired by transformation, conjugation, or transduction
episomes and lysogenic bacteriophage genomes
Homologous recombination begins with __
__ is the name given to the type of recombination that occurs during crossing over between homologous chromosomes. It occurs between__
- formation of a DNA heteroduplex
- Homologous recombination
- two double-stranded DNA molecules that have regions where the nucleotide sequences are the same or at least very similar
Homologous recombination begins when __.
Then, what happens?
the two double-stranded molecules line up adjacent to one another
A double-strand cut is made in one of the molecules, breaking this one into two pieces.
One strand in each half of this molecule is then shortened by removal of a few nucleotides, giving each end a 3’ overhang
The partnership between the two chromosomes is set up when __
one of the 3’ overhangs invades the uncut DNA molecule, displacing one of its strands and forming a D loop
__ is stabilized by __ between the transferred segment of polynucleotide and the intact polynucleotide of the recipient molecule
- Strand invasion
- base pairing
This base pairing is possible because of the __
The __ is extended by new __, enlarging the __, and at the same time, the other __
sequence similarity between the two molecules
broken strand is also extended
After strand extension, what happens, giving a structure called a __, in which the __, which is dynamic and can move along the heteroduplex. This __ results in the exchange of longer segments of DNA
- the free polynucleotide ends are joined together
- two double-stranded molecules are linked together by a pair of Holliday structures
- branch migration
__ results in recombination
__ of the heteroduplex back into individual double-stranded molecules occurs by __. Each Holliday junction can be cleaved in either of __, with very different outcomes
The true structure of a Holliday structure is the __.
Cleavage of the Holliday structures
Separation, or resolution,
cleavage of the Holliday structures
What is the first cleavage?
First, the cut can be made left to right across the chi form, which is equivalent to cleavage directly across the Holliday structures Cleavage of a Holliday structure in this way, followed by joining of the cut strands, gives two molecules that have exchanged short segments of polynucleotide
As the exchanged strands have __, the effect on the genetic constitution of each molecule is __, and this outcome is thought not to be relatively common during meiosis
- similar sequences
- relatively minor
What is the second cleavage?
More frequently, a Holliday structure is resolved by cutting the chi structure in the second of the two possible orientations, up-down.
This is the type of cleavage that is not readily apparent from the two-dimensional representation of the Holliday structure. But, it is vitally important because __
up-down cleavage results in reciprocal strand exchange, double-stranded DNA being transferred between the two molecules so that the end of one molecule is exchanged for the end of the other molecule.
This is the DNA transfer that occurs during __
True or False:
The biochemical pathways for homologous recombination have been studied in E. coli There are no between the recombination processes in bacteria and eukaryotes
False: There are important differences
At a biochemical level, there are three distinct recombination systems in E. coli, each involving a different set of proteins. These are __, __, and __ pathways.
RecBCD, RecE, and RecF
Explain the RecBCD pathway
The central player in the RecBCD pathway is the RecBCD enzyme, which is made up of three different protiens. Two of these—RecB and RecD—are helicases, enzymes capable of breaking the base pairs that hold polynucleotides together
To initiate homologous recombination, one copy of the RecBCD enzyme does what?
Using its __activity, RecBCD then __ at a rate of approximately 1 kb per second until it reaches the first copy of the __ 5’-GCTGGTGG-3’. This is called the __.
attaches to the free ends of a chromosome at a double-strand break
progresses along the DNA molecule
eight-nucleotide consensus sequence
The outcome of what happens at a chi site is that the __
RecBCD enzyme produces a double-stranded molecule with a 3’ overhang, as is required for the initiation of recombination
The next step is __. This stage is mediated by the __, which forms a __ that is able to __ and __
- establishment of the heteroduplex
- RecA protein
- protein-coated DNA filament
- invade the intact double helix
- set up the D loop
An intermediate in formation of the D loop is __, a __
- triplex structure
- three-stranded DNA helix in which the invading polynucleotide lies within the major groove of the intact helix and forms hydrogen bonds with the base pairs it encounters
Once the heteroduplex has been established, the subsequent events are probably common to all three recombination systems. If __ occurs, then it is catalyzed by the __, both of which attach to a __
- branch migration
- RuvA and RuvB proteins
- Holliday structure
i. __ bind directly to the __, forming a core to which two __, each consisting of __ attach, one to either side. The resulting structure might act as a molecular motor, doing what?
- Four copies of RuvA
- branch point
- RuvB rings
- eight proteins
- rotating the helices in the required manner so that the branch point moves
Branch migration is __. Instead, it stops at the sequence 5’-(A/T)TT(G/C)-3/. In this sequence, (A/T) and (G/C) denote that either of the two nucleotides can be present at the positions indicated.
The sequence occurs frequently in the E. coli genome so presumably migration does not halt at the __. When __ has ended, the __ detaches and is replaced by two __proteins
- first instance of the motif that is reached
- branch migration
- RuvAB complex
RuvC is a __and so can carry out the __
These cuts are made between the __
- cleavage that resolves the Holliday structure
second T and the (G/C) components of the recognition sequence
The biochemical events underlying recombination appear to be similar in all organisms
Studies of yeast have ID’ed proteins with functions equivalent to those of the participants in the __pathway.
Two yeast proteins called __ and __ are the homologs of __of E. coli
Although __ and __ are thought to have their own individual roles, the two proteins are found together at the same locations within nuclei that are undergoing __, and they are thought to work together in many __
- RAD51 and DMC1
- homologous recombination events
One puzzling aspect of homologous recombination n eukaryotes is the mechanism by which __. __that cut Holliday structures in the test tube were discovered, but the cleaved ends that they leave cannot be __to one another easily. It therefore seems unlikely that they have any role in recombination in living cells
- Holliday structures are resolved
During this search, it was also realized that it is possible to resolve a Holliday structure how? The combo of __ can separate the heteroduplex back into independent chromosomes
without the use of a nuclease enzyme
helicases and topoisomerases
__was a protein discovered that cuts the Holliday structure in almost exactly the same way as __, and, importantly, the cut DNA ends resulting from __cleavage can be joined together easily.