Neurodegeneration is a prominent pathologic feature of A-T3
that is not well explained by the cell cycle checkpoint deficiencies and other DNA damage signaling defects associated with an ATM malfunction. We show here that ATM suppresses an error-prone pathway of DNA double-strand break repair referred to as microhomology-mediated end joining (MMEJ). This pathway results in deletion of sequences surrounding the break site and rejoining at short regions of homology. Incorrect modulation of MMEJ may be the basis for the pathobiological mechanisms leading to neuronal degeneration in A-T. Neurocytes are postmitotic and therefore depend on mechanisms other than homologous recombination between sister chromatids to repair DSBs. One such mechanism is MMEJ which can lead to loss of significant genetic information if not kept in check. The accumulation of genetic damage could ultimately lead to loss of function and/or death of neuronal cells. In addition, the misregulation of error-prone repair discussed herein likely contributes to the genetic instability and propensity for leukemias and lymphomas observed in A-T.
Repair events via MMEJ are associated with disease processes resulting in chromosomal translocations and tumor-formation.32–36
We show here that DNA degradation and consequent microhomology-directed break rejoining involved in MMEJ are highly dependent on Mre11 and in particular on its nuclease function. Both Mre11 knockdown and immunodepletion resulted in a decreased level of DNA substrate degradation. This was prominent in ATM-deficient nuclear extracts which have a marked elevation in nuclease activity and in MMEJ. Treating these extracts with Mirin, an inhibitor of Mre11 nuclease activity,30
also resulted in decreased substrate degradation, but not to the extent seen with Mre11 knock down or immunodepletion. This may indicate incomplete inhibition of Mre11 or the participation of one or more accessory nucleases that require the physical presence of Mre11 at the DNA end but whose kinetics are less dependent on Mre11 nuclease function.
Immunodepletetion of Mre11 from nuclear extracts resulted in decreased levels of linearized plasmid rejoining in these extracts via MMEJ. Moreover, using an in vivo reporter assay system we observed a decrease in MMEJ levels after Mre11 knockdown and after treating cells with Mirin. This direct demonstration of Mre11 involvement as a nuclease in MMEJ in vivo in a mammalian system is consistent with recent studies.15,16
Overexpression of Mre11 was demonstrated to increase the frequency of deletions at DNA ends whereas a nuclease deficient mutant Mre11 failed to cause a similar increase. Previous reports have indicated that purified Mre11 degrades DNA in vitro up to regions of microhomology, where it stalls.14
Consistent with biochemical analyses, recent Mre11-DNA complex crystal structures suggest that Mre11 DNA bridging may provide the requisite synaptic DNA architectures for DNA end pairing and degradation reactions regulating MMEJ ( and B
We modeled the structure of an Mre11 dimer directly engaging DNA ends and the model shows how the 5′ ends of a bridged DSB are placed into close juxtaposition by the dimeric protein scaffold. In this model, Mre11 complex DNA unwinding and 3′–5′ exonuclease activities may collaborate to facilitate simultaneous pairing of extended 5′ termini and exonucleolytic degradation of the 3′ ends in MMEJ ( and B
Figure 8 Mre11-mediated microhomology-mediated end-joining and its regulation by ATM. (A) Schematic for Mre11 dimer-mediated MMEJ. The Mre11 homodimer is a symmetric structure that binds two DNA ends. 5′ termini are paired at the interface of the dimer, (more ...)
Other mediators also have been associated with this error-prone repair pathway and these include the BLM helicase,38
PARP-1, XRCC1, DNA ligase III,39
the FEN1 endonuclease, DNA polymerase ε 40
and the Exo1 exonuclease.41
Whether these mediators are involved in the Mre11-depedent pathway merits investigation. Given the complexity of DSB repair in mammalian cells it is possible that more than one pathway leads to the formation of MMEJ products.
Based upon the key roles identified here for ATM and Mre11, the regulation of MMEJ and suppression of error-prone repair can be better explored. ATM deficiency leads to an increase in MMEJ in vitro.11
Here we extend this observation in vivo and reveal that Mre11 is the target of this regulation. This increase in MMEJ is not simply due to a reduction in the ability to rejoin DNA ends in the absence of appropriate ATM function, as nuclear extracts from both A-T and control cells are equally efficient in rejoining DNA ends.11
The ATM kinase activity was necessary for repression of DNA degradation by Mre11. Thus, inhibition of degradation and MMEJ by ATM may be through phosphorylation of the nuclease complex. ATM phosphorylates Nbs1 in response to DNA damage,20,21
and ATM-dependent phosphorylation of Mre11,27
was reported. Structurally, the Nbs1 connects directly to the Mre11 adjacent to its ATM interaction motif, so the mechanical linkage of Nbs1 to both ATM and Mre11 is established structurally.42
Furthermore, the connection of Rad50 to Mre11 suggests ATP driven conformational change may help open the DNA for Mre11 processing in ways dependent upon the state of Rad50.43
Notably, examination of MMEJ in yeast revealed roles for both Mre11 and Tel1, the yeast ATM homologue, in promoting MMEJ.12,13
The observation that Tel1 promotes MMEJ in yeast, but ATM suppresses MMEJ in mammalian cells may indicate the divergence of these two proteins and of their roles. It may also demonstrate pathway differences between yeast and mammalian systems. The yeast Dnl4 (DNA ligase 4), for example is required for MMEJ, whereas its mammalian homologue, LIG4, is dispensable for the pathway.44–47
Together our results and prior observations provide an integrated understanding of the critical roles of both ATM and Mre11 nuclease in MMEJ. We conclude that a DSB leads to activation of ATM via the MRN complex. ATM then regulates, through its kinase activities, the degradation and rejoining of DNA ends by Mre11. In particular, it suppresses error-prone MMEJ repair of the DSB by inhibiting thomology-directed DNA degradation mediated by the Mre11 nuclease ().