Novel DNA repair mechanism described

07 oct

Damaged DNADNA repair is a very interesting field to me even if I didn’t go into for my PhD. I’m still following the news from it though. Here is a summary from a study published in Nature several days ago which describes a novel mechanism for DNA repair.

DNA is a highly active molecule and it reacts with other active molecules. Sometimes, it is not a good idea though. In those cases a damage occurs. In good days, in a human cell, something like 1 million of lesions can be counted per day. There are two types of damages: endogenous and exogenous. The former includes for instance, a reactive oxygen species is produced from normal metabolic byproducts that can attack the DNA or whatever replication errors may happen. The latter includes all external agents attacking DNA: UV radiation, high temperature, various mutagenic chemicals, cancer chemotherapy and radiotherapy.

Given the importance of DNA for many cells, it is vital to repair this damage. Therefore, various mechanisms exist to insure DNA integrity depending on the type of the damage inflicted. I will not do a catalogue of those, this is not of a real interest. I’ll just say several words about one given type of damage: the one the paper I discuss focuses on.

This damage is called alkylation. In brief, it is the addition of a group (usually, methyl) to one of the bases resulting for example in the production of N7-methylguanine (7mG) or N3-methyladenine (3mA). A class of drugs used in chemotherapy causes alkylation of cancer DNA. But alkylation can also be caused by endogenous factors. 3mA for example is highly toxic: it causes DNA polymerases inhibition during replication (this is the main reason of producing alkylating anti-cancer drugs). 7mG are are the most prevalent alkylation lesions and display a wide range of mutagenic properties. Given the positive charges at physiological pH, 3mA and 7mG are "especially susceptible to spontaneous depurination, which generates abasic sites in DNA that can ultimately lead to single- and double-strand breaks", explain the authors.

In general, what happens is that an enzyme, the DNA glycosylase, comes in the game: it is a part of the BER (Base-Excision Repair) machinery and will remove the N3- or N7-methylpurines it finds around. What DNA glycosylases do more precisely is to catalyze the first step of the BER process: they hydrolyse the N-glycosidic bond and thus, create an AP site (there is no more base, the sugar-phosphate backbone being intact). This reaction is insured by the DNA glycosylase flipping the base and keeping it in a special , complementarity shaped active site pocket while removing it.

AlkC and AlkD proteins were first identified in Bacillus cereus and afterwards, it became clear that all three "kingdoms of life" have them. Therefore, these 2 proteins are considered as "a unique DNA glycosylase superfamily specific for N3- and N7-alkylpurines", write the authors. AlkD is reported to increase the rate of 7mG depurination 100-fold over the spontaneous rate. But the mechanism behind the property to excise alkylated bases was not known up to now. So the authors were successful in obtaining the crystal structure of B. cereus AlkD in complex with 3mA-containing DNA. Indeed, the authors noticed that AlkD acts in a different fashion compared to the DNA glycosylase I told above: AlkD makes the alkylated base and the base paired with the latter to flip to the outside of the double helix.

To make long story short, the authors found several interesting features of the AlkD-DNA complexes :

The DNA is positioned along AlkD’s concave surface, which is lined with positively charged residues from the carboxy-terminal α-helix of each HEAT repeat. The C-shaped protein wraps halfway around the DNA helix with a footprint of ~10 bp. The contact surface is dominated by electrostatic interactions between side chains at the protein mid-region and the phosphoribose backbone of the DNA strand opposite the lesion. In contrast, contacts to the lesioned strand are limited to base pairs further removed from the lesion and the protein termini.

The most striking characteristic of these complexes is that the lesion resides on the face of the DNA duplex not in contact with the protein, "whereas the base opposite the lesion is nestled into a cleft on the protein’s concave surface". furthermore, the authors found several important differences in the lesion pocket that allow them to argue that AlkD is not as the traditional DNA glycosylases, thus becoming a second type of alkylation repair machinery.

This means that most if not all the organisms have 2 types of alkylation repair mechanisms: the DNA glycosylases and AlkD. This raises the question: why having a redundant alkylation repair machinery? The authors mention that its structure is quite similar to that of the DNA-dependent kinases. From one hand, it may be that AlkD is a general DNA-binding protein that "coincidentally accelerates hydrolysis of unstable N-glycosidic bonds" or may have a supporting role in general lesion detection. The authors speculate that keeping the lesion away from the protein has the advantage of make the damage accessible to the rest of the repair machinery.

From the other hand, one may ask whether these DNA-dependent kinases would have a yet unknown repair ability. Indeed, those HEAT motifs mentioned above are used by AlkD to grab hold the DNA and are found in these kinases. The HEAT motifs are of unknown function and this is the first time it is reported they have an enzymatic activity.

As mentioned by the press release, these findings are crucial for our fundamental knowledge of DNA repair mechanisms and can be extremely important for drug design.
Emily H. Rubinson, A. S. Prakasha Gowda, Thomas E. Spratt, Barry Gold & Brandt F. Eichman (2010). An unprecedented nucleic acid capture mechanism for excision of DNA damage Nature : doi:10.1038/nature09428


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Publié par le 7 octobre 2010 dans Science


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