While in the last post to the repair aspect of our mutants were, should be considered in the next their involvement in DNA recombination. But this will be without a little background knowledge for homologous recombination as easily, so I've decided not to hope for this complex slot. recombination means, first, that genetic information between two DNA molecules is replaced. Of the many recombination, homologous recombination is the most conservative. That was not a political statement, it just means that ideally it will be no change in the sequence. This is possible because used for homologous recombination identical (= homologous) DNA sequences. These sequences occur in organisms such as humans or from Arabidopsis homologous chromosome diploid. Or, even better, according to the doubling of the chromosomes during replication, the sister . The starting point of homologous recombination is always a double-strand break. This may be deliberate, as in meiosis (which in another part of the overview paper issues, probably # 5). Alternatively, a double strand break but also be induced by ionizing radiation or chemicals. This would be the combination of homologous recombination with DNA repair.
What really happened during homologous recombination, we do not know one hundred percent. You can follow this process does not live on a DNA molecule. But you can build up his attempts to use specific processes can be excluded because of the result, but other processes are likely. Many of these basic experiments were made with baker's yeast Saccharomyces cerevisiae , the clever constructs defined in the genome have been introduced that allow for a homologous recombination, shares to be determined by specific procedures. This sounds very bland, so I just want to show on a beautiful example of what I mean.
The "Double beach Break Repair model of homologous recombination
preconceived 1983 Szostak et al. in a review article the then current state of Rekombinationsforschung together. They have found that previously proposed models of homologous recombination events during meiosis auftrende often unpredictable. If one considers, for example, two markers [1], there are heterozygous and coupled (ie sitting together on a chromosome, but not on the second chromosome of a diploid organism), then one would expect from the descendants of the simple splitting after Mendel in 1:1 - Half of the offspring has received the chromosome with two markers, the other half the homologous chromosomes without the markers. Because of the special situation of the chromosome then studied fungal species, the notation is traditionally not 1:1 but 4:4, the changes in the relationship but nothing. Then, can one see that happen sometimes, other divisions, such as 6:2 (or 2:6), or 5:3. This information should have been transferred from one chromosome to another, or an exchange between two chromosomes is to be such that the two markers are no longer linked on one chromosome and can be inherited independently. These two operations are referred to as gene conversion and crossing over, and they are in Figure 1 of Szostak et al. represented (albeit in reverse order):
Figure 1 of Szostak et al. (1983). State of two coupled heterozygous markers A and B and the effects of crossing over (a) and gene conversion (b). Click for larger version.
what's going on now? Szostak et al. propose a mechanism based on the repair of double strand breaks. This was new because the previous models were the immediate causes such as short single-stranded regions on the DNA (ssDNA nicks) from. From repair research was already known that the double-strand break repair in yeast very efficient expired. The new model should therefore start with a double-strand breaks, and with the possibility for both crossing over, as also end gene conversion. The idea of Szostak and colleagues looked like this:
double-strand break repair model (DSBR) by Szostak et al. (1983). Click for larger version.
Starting from a double strand break, the free ends by proteins ( exonucleases ) so cut back that there are single-stranded overhangs free. For example, a ssDNA is of course available for base pairing. If after a "homology" a homologous DNA sequence is found (like for example, said on the homologous chromosome, or may be also the sister), then is the so-called single-strand invasion: the single strand binds to the complementary sequence and displaces one of the existing strands. The resulting structure is D-loop (displacement loop) called. From here on the free end can be extended using a DNA polymerase, which increases the D-loop. Eventually, such a large area of DNA in the D-loop is displaced, that it can mate with the second free end of the double strand break. This also means that the first free end finally linked to the other side of the double strand break can be. The DSB is repaired now though, but we now have a problematic structure of DNA are present - two DNA molecules are crossed at two positions. Thus, a cruciform DNA structure known as the way to their first description Holliday Junction (AHA!), at the two crossings here is called a double Holliday junction (DHJ). Why this structure is problematic? Because a cell can not divide before the DHJ was dissolved!
and now takes up the idea of Jack Szostak and colleagues. A endonuclease, a protein that is the inside of a DNA molecule can be cut, set at the Holliday junction cuts to the two strands from each other to separate. And depending on whether the two symmetrical sections (bottom left) or asymmetric place (bottom right), is either a crossing over (CO, right) or gene conversion (also called noncrossover, so NCO, left).
Since 1983, much time passed, but the DSBR model has persisted. Meanwhile, for example, was shown to intentionally in meiosis, a special protein is double-strand breaks to initiate the homologous recombination: SPO11 . Also, Holliday junctions have been demonstrated experimentally already as intermediates of recombination.
"synthesis-dependent annealing-beach" and the "revised model "
order it now make a little more complicated, I will bring the sake of completeness the image up to date. In 1994, the groups made by William Engels and Gregory Gloor (Nassif et al., 1994) recombination with the fruit fly Drosophila melanogaster , they found results that with the DSBR model of Szostak et al. could not fully explain. Ultimately, their results were to be understood only if we allowed two free ends of the DSB, independent of one another to initiate the recombination with different partners. A resolution to their synthesis-dependent beach- annealing model (SDSA), therefore required no Holliday junction as an intermediate. Already after the extension of the free end, this would cast from the D-loop and the base-pairing with the second end are available. Through such a mechanism does not crossover products would be possible.
2001, the two competing models of DSBR and SDSA then combined simultaneously by two groups. In the revised model today as process called for in the yeast results obtained from Hunter and Kleckner (2001) and Allers and Lichten (2001) begins the first homologous recombination, as I have already described for DSBR and SDSA. The balance between the two arms of CO and NCO is, however, before the DHJ intermediate, namely at the level of the D-loop. From here from the recombination can be either dissolved the NCO, by SDSA model. Or just over twice the Holliday Junction to the CO, which, according to this model, the only result of the DSBR pathway.
revised model of homologous recombination by Hunter and Kleckner (2001) and Allers and Lichten (2001). Click for larger version.
would like at this level I then left for today. What I have mentioned at all here, are the many proteins that populate the whole way. By some very well known its position in the scheme to and their role in many other known only approximately, in which half of the model fit. In the next
correct post this short series I will then introduce armed with the background for homologous recombination here, our studies mutants for recombination.
[1] Before the era of rapid sequencing, and even before the triumph of the PCR were such markers often spore color genes of the tested fungi, or antibiotic-resistance genes.
JW Szostak, TL Orr-Weaver, RJ Rothstein, FW Stahl (1983). The double-beach-break repair model for recombination Cell, 33 (1), 25-35 DOI: 10.1016/0092-8674 (83) 90331-8
N Nassif, J Penney, S Pal, WR Engels, GB Gloor (1994). Efficient copying of nonhomologous sequences from ectopic sites via P-element-induced gap repair. Mol Cell Biol., 14 (3), 1613-1625
Neil Hunter, Nancy Kleckner (2001). The Single-End InvasionAn Asymmetric Intermediate at the Double-Strand Break to Double-Holliday Junction Transition of Meiotic Recombination Cell, 106 (1), 59-70 DOI: 10.1016/S0092-8674(01)00430-5
T Allers, M Lichten (2001). Intermediates of Yeast Meiotic Recombination Contain Heteroduplex DNA Molecular Cell, 8 (1), 225-231 DOI: 10.1016/S1097-2765(01)00280-5
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