And here, the discussion of our latest papers! While in part 1 keep a little background for the genes / proteins, went off tonight into the plant genome and the question is whether these RTR complex in the model plant Arabidopsis thaliana .
make the most of our work with insertion mutants. These are plants in which using the soil bacterium Agrobacterium tumefaciens relatively large DNA fragments were introduced into the sequence of a gene. This leads ultimately to the fact that the protein product of this gene can not be established, the plant line is distinguished from wild-type plants only in the fact that a particular gene was turned off. The purpose behind the investigation of these knockouts is quickly evident: When comparing wild type and Knockoutlinie, it is very likely that any additional defect is in the Knockoutlinie on the failure of the gene. Now you have to rethink just yet - the failure of a gene leads to certain failure, then this gene is normally fulfilled this specific function that prevents the development of the defect.
First, it is natural to ask: Is it the genes of the RTR complex at all in Arabidopsis? Here, you can now save a lot of work, thanks to very good bioinformatics and the freely accessible genome databases of many organisms. What we found is a good fit to the previously described genes, particularly from the yeast Saccharomyces cerevisiae and man.
RMI1 for the gene (or BLAP75) there are two possible candidates Arabidopsisgenom. One of them (the gene At5g19950 ) seems to be not expressed, and examined by us insertion mutants were completely normal. It could therefore be a pseudogene . The second candidate ( At5g63540) was all the more interesting, as I will show today and in the following posts to our papers. This gene was therefore named AtRMI1 of us [1].
on the T from RTR - the topoisomerase. Here it was easier because of TOP3 or a homologue TOPO3α was only one candidate gene in question, or At5g63920 AtTOP3α.
A little more complicated it is for the RecQ helicase. In this family we already know that there are seven members in Arabidopsis. Which of these seven now but the functional homologue to the only RecQ helicase in yeast, SGS1? In humans, this is among five family members of the BLM gene, and armed with the development of these two genes already covered some of the RecQ helicases Arabdopsis out. Of the rest, then remains only RECQ4A ( At1g10930) left when we include already known properties of members of the RecQ family in Arabidopsis with a (Hartung et al., 2007).
Fortunately for these genes were then also available insertion mutants, we were able to work with.
Finally now some "real" data. The description of how and why certain genes have been found at all and used, and how the mutants are built is certainly an important part of a paper - it's a bit boring.
There is a relatively simple method, the involvement of a gene or its protein product from the DNA repair study: Intentionally damaging the DNA, then these damages are repaired. But a gene is turned off, which is involved in the repair of DNA, then the repair does not logically so well. This can be measured in different ways, we determine the relative fresh weight of seedlings treated compared with untreated same old seedlings. For problems of DNA repair result such as death of the affected cells, which can then no longer part or waxes. The result: Smaller, lighter seedlings.
The range of possible damage to DNA is incredibly high, accordingly, it is also a lot of repair pathways. By the organisms various substances with known harmful effects on the DNA is exposed if you can make with this experiment is also a first classification of the studied genes in these repair pathways. A Knockoutmutante Namely only more sensitive than the wild type response to a substance if it is active in the repair pathway is affected.
In this figure, the sensitivities of Knockoutmutanten Gene RECQ4A, TOP3α RMI1 and shown to various mutagens. Against methyl methanesulfonate (MMS, A in the figure), many genes in homologous recombination are sensitive, so our three genes. MMS generates Adducts in DNA by a methyl group attached to nitrogen atoms in the bases of DNA. This has to mean that the normal double helix is distorted, and basic processes such as transcription and replication can not take place. This is probably also related to the homologous recombination, as it assists directly to resolve problems at the replication fork. Apart from this damage by alkylation but also by the repair pathways of base excision repair and nucleotide excision be resolved [2]. The inaccuracy of these paths leads the way through the detour of mutations in a cancer-promoting effects of MMS.
cisplatin (cis -specific Diamindichlorplatin, B in the figure) is an interesting material. About the rather accidental discovery of this small molecule I have already written . Today it is one part of one of the most commonly used chemotherapeutic agents, on the other hand, however, the most studied substances in terms of the mechanisms of DNA damage. What happens when cisplatin enters the cell? The central platinum atom reacts happily with just about any molecule that gets it hands on. Therefore, one then finds Platinaddukte of sugars, fats, proteins, but also the DNA. Stupid thing is though, that cisplatin on his two chloride groups can also react twice, and thus two previously unconnected Molecules covalently linked. So then ever proteins bound to DNA, or even worse, two bases of DNA covalently linked. This is called cross-linking, and there are two possibilities: either two bases from different strands of DNA are linked (= interstrand cross-link), or two bases are linked within a line (= intra-strand cross-link). The latter is more frequent in the cisplatin product, and here the nucleotide excision repair tasks are in and the homologous recombination (particularly during replication). Last
still camptothecin (CPT, C in the figure). This is an alkaloid from the plant such as Camptotheca acuminata is made for pest resistance. And the pests that nibble on the plant do'm really sorry: In the previous part I have just described what makes a topoisomerase to the DNA. Hinder this process of camptothecin topoisomerase first Just in the state when TOP1 is covalently linked to the DNA and there is a single strand break in DNA, CPT binds to it and prevents the topoisomerase closes the gap and come off the DNA. As a result, the replication of a hand, a protein linked covalently to the DNA is located and disabled the replication proteins, and also that by the sudden unwinding of the double strand of this gap in the DNA a double-strand break is. Major problem and most likely the death of the hungry pests [3].
As you can see, all mutants more sensitive than the wild type (Col-0 in the figure) to MMS and cisplatin. Against CPT contrast, only the Knockoutmutante of TOP3α is sensitive, the other two do not. top3A-2 reacts strictly speaking also on MMS and cisplatin sensitive than recq4A-4 and rmi1-2 . While these sensitivities, one of the most common indications now that the three proteins perform their functions together (that may be in a RTR complex) to interpret the results also indicate a special role for TOP3α. will on the I received but in the next or the next post - with NEM Cliffhanger have to return it that easy?
[1] So really new and creative name gives the genes rather rare, but it does happen. See, for example, the gene and its antagonist SUPERMAN KRYPTONITE ...
[2] If I start now to describe in detail each repair pathway I will not even finished the post. I have here, however a nice idea: I could devote the mutagens own posts, and then enter it on the relevant repair pathways.
[3] The camptothecin-producing plants have way TOP1 genes with slightly different sequences, so that even nicht anfällig gegenüber CPT sind (Sirikantaramas et al, 2008).
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