MRC National Institute for Medical Research, London
Science for Health
An unexpected mechanism of FHA function in DNA-repair signalling
13 January 2012
A recent paper by NIMR scientists and collaborators in Switzerland, Denmark and the UK, published in Nucleic Acids Research, provides new insights into the mechanisms by which the detection and repair of double-stranded DNA breaks is signalled to the cell-cycle machinery.
Double-stranded DNA breaks (DSBs) are generally accepted to be the most toxic lesions suffered by cell genomes. One of the earliest events marking the existence of DSBs in eukaryotic genomes is the phosphorylation of the C-terminal tail of a variant histone H2A (called H2AX) by ATM kinase. So-called γH2AX-containing nucleosomes decorate chromatin flanking DSBs over distances of megabases in human cells. Staining of DNA-damaged cell nuclei with phospho-specific antibodies reveals concentrated ‘spots’ of γH2AX accumulation known as ionization radiation-induced nuclear foci (IRIF) that serve as primary markers of DSB events.
Far from containing just γH2AX, IRIFs are associated with localization of many other DNA-damage response proteins and complexes. Among these Mdc1 (mediator of the DNA-damage checkpoint protein 1), a large, modular phosphoprotein containing both FHA (Forkhead-associated domain) and BRCT-repeat phospho-serine/threonine binding domains, is of central importance. A previous study established that chromatin tethering is the primary function of the C-terminal Mdc1 BRCT domain. However, a clear function for the Mdc1 FHA domain has remained elusive.
Steve Smerdon (pictured) has now shown that a previously unnoticed DNA-damage and ATM–dependent phosphorylation site at the extreme Mdc1 N-terminus unexpectedly sponsors tight FHA domain dimerisation both in vitro and in human cells. Structures of free and peptide-bound forms showed a head-to-tail arrangement that is related to that of the ATM-phosphorylated form of Chk2 kinase, a major transducer of DNA-damage signals. These data highlight a mechanism for mediator dimerisation distinct from those previously proposed for yeast Rad9 and human 53BP1 and suggest a more general significance of FHA-FHA interactions than has previously been appreciated. Perhaps more importantly, a combination of structural and biochemical data allied to a large scale quantitative SILAC-based proteomic screen of Mdc1 FHA interactors in DNA-damaged human cells suggests that Mdc1 dimerisation mediates FHA interactions with phospho-dependent or, indeed, phospho-independent targets. These interactions seem to be regulated in a cyclical mechanism that is synchronised with DNA repair and the establishment of and recovery from DNA-damage checkpoints.
Although great progress has been made towards understanding the enzymology of DNA repair, many questions remain about how the cell-cycle checkpoint machinery is connected to these processes. It is well established that ‘mediator’ proteins such as Mdc1 are extremely important in this context but their large size and complexity combined with their elaborate patterns of post-translational modification have made detailed mechanistic studies difficult. FHA domains have been a long-standing interest of the lab and using an arsenal of approaches we have now been able to provide a glimpse of how the FHA of Mdc1 might be functioning during the series of events that mark damage detection, repair and checkpoint regulation. The fact that it works through regulated dimerisation is really quite unexpected but we suspect that this kind of mechanism is likely to have real significance for understanding FHA activities in many other signalling contexts beyond the DNA-damage response.
Steve Smerdon
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A. Structure of the dimeric Mdc1 FHA/pThr-4 peptide complex
B. Co-localisation of an Mdc1 FHA-YFP fusion with endogenous, γH2AX-bound Mdc1 is abolished in cells by mutations that disrupt dimerisation in vitro.
Original article
Jungmichel S, Clapperton JA, Lloyd J, Hari FJ, Spycher C, Pavic L, Li J, Haire LF, Bonalli M, Larsen DH, Lukas C, Lukas J, Macmillan D, Nielsen ML, Stucki M, Smerdon SJ. (2012)
The molecular basis of ATM-dependent dimerization of the Mdc1 DNA damage checkpoint mediator
Nucleic Acids Research, epub ahead of print. Full text.
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