Pax2 Transactivation domain interaction protein (PTIP) is a six-BRCT domain containing protein that has roles in transcription and the DNA damage response. The PTIP knockout mouse provided the first hint that PTIP might be involved in the DNA damage response [81
]. PTIP-/- embryos died at E9.5, and MEFs derived from PTIP-/- embryos failed to proliferate, recapitulating a phenotype similar to that seen for other DNA damage response proteins involved in DNA synthesis. Interestingly, PTIP-/- embryos exhibited increased TUNEL staining, however rather than the pyknotic morphology typically observed in apoptotic cells, TUNEL positive PTIP-/- cells had a diffuse morphology, possibly indicating the presence of fragmented chromosomes [81
]. The C-terminal tandem BRCT domains of PTIP were found to be a phosphopeptide binding module, with a preference for hydrophobic amino acids in the +3 position [15
]. PTIP was found to form IRIF, although the BRCT domains required for foci formation remains controversial. Two studies found that the phosphopeptide binding C-terminal BRCT domains are necessary for foci formation [15
], however, a recent study found the central BRCT domains are necessary for foci formation, and not the phosphopeptide binding BRCT domains [83
]. PTIP was also found to interact with 53BP1 in a phospho-dependent manner [15
], and this interaction likely requires the last four BRCT domains of PTIP [84
]. The requirement for four BRCT domains for phosphopeptide binding is a novel, however, it may be that the middle two BRCT domains stabilize the interaction with 53BP1, as it is a non-optimal phospholigand for the last two PTIP BRCT domains.
Exactly what PTIP does to control the DNA damage response and coordinate DNA repair remains elusive. Much of the work performed on PTIP has focused on the interaction of PTIP with MLL2, MLL3, and MLL4 complexes [85
] and the putative role of PTIP in transcription regulation. A recent study identified two PTIP containing complexes, a high molecular weight complex that co-purifies with MLL proteins, and a low molecular complex that purifies with PTIP-associated protein 1 (PA1). PA1 is recruited to IRIF by PTIP [83
]. In addition, Gong and colleagues demonstrated that PTIP did not form foci in the absence of H2AX, MDC1 or RNF8. Interestingly, despite the in vitro and in vivo interaction between PTIP and 53BP1, PTIP and 53BP1 appear to be recruited to foci independently [83
A number of important questions in the field remain unanswered, including the interplay between MCPH1 and MDC1, both of which seem to be interacting with γH2AX with similar affinities. It has been demonstrated that MCPH1 recruitment to foci does not depend on γ-H2AX; however, as MDC1 has been demonstrated to be a protein that coordinates and recruits several proteins to foci, the question remains, what is the role of MCPH1 in foci formation? Could MCPH1 be specifically involved in checkpoint exit? It has been demonstrated that MDC1 is degraded after DNA damage, suggesting that MCPH1 could bind to and facilitate the dephosphorylation of γH2AX after MDC1 destruction. It is also possible that MCPH1 regulates repair processes like HR, while MDC1 might functions primarily in the early stages of the DNA damage signaling response. Another facet of phosphopeptide binding in protein recruitment to foci after DNA damage is the question of uniqueness in ligand binding. It is not intuitive that recruitment of phosphopeptide binding proteins into nuclear foci should depend exclusively on a single ligand. Given the large number of ATM substrates, why should proteins like BRCA1 bind to such a limited number of ligands after DNA damage, especially when 14-3-3 proteins typically bind hundreds of distinct ligands. It seems likely that there are other factors involved in specifically localizing proteins to foci.