Nbs1 Converts the Human Mre11/Rad50 Nuclease Complex into an Endo/Exonuclease Machine Specific for Protein-DNA Adducts

Abstract

The human Mre11/Rad50/Nbs1 (hMRN) complex is critical for the sensing, processing, and signaling of DNA double-strand breaks. The nuclease activity of Mre11 is essential for mammalian development and cell viability, although the regulation and substrate specificity of Mre11 have been difficult to define. Here we show that hMRN catalyzes sequential endonucleolytic and exonucleolytic activities on both 5' and 3' strands of DNA ends containing protein adducts, and that Nbs1, ATP, and adducts are essential for this function. In contrast, Nbs1 inhibits Mre11/Rad50-catalyzed 3'-to-5' exonucleolytic degradation of clean DNA ends. The hMRN endonucleolytic cleavage events are further stimulated by the phosphorylated form of the human C-terminal binding protein-interacting protein (CtIP) DNA repair enzyme, establishing a role for CtIP in regulating hMRN activity. These results illuminate the important role of Nbs1 and CtIP in determining the substrates and consequences of human Mre11/Rad50 nuclease activities on protein-DNA lesions.

DNA-dependent protein kinase promotes DNA end processing by MRN and CtIP

“…1B, lanes 4 and 5); thus, we conclude that the processing is done by the catalytic activity of Mre11. We have previously observed that ATP binding by Rad50 promotes Nbs1-dependent endonucleolytic cutting by Mre11 (8,19). Here we find that ATP is essential and the nonhydrolyzable AMP-PNP analog does not substitute for ATP in promoting MRN nuclease activity in the presence of DNA-PK (Fig.…”

“…Our previous studies of MRN and Ku dynamics on DNA ends using the DNA curtains system and ensemble assays showed MRN cleavage of Ku alone, but this reaction required manganese ions (8,12). Other studies also recently demonstrated yeast Mre11/Rad50/Xrs2(MRX) cleavage of various protein blocks including yeast Ku, which was also manganese-dependent in vitro (10, 11).…”

“…We recently showed that MRN can remove DNA end blocks by sequential endo-exoendo activities, generating a double-strand break adjacent to the block (8,12), a model first suggested by processing of meiotic DNA breaks (26). In the presence of DNA-PK bound to DNA ends, the MRN complex executes similar end-processing of the radiolabeled substrate.…”

“…At single-ended breaks during replication, the nuclease activity of Mre11 was shown to be important for removal of Ku (6), suggesting that MRN catalytic processing of ends during S phase is important for recombination-mediated repair of single-ended breaks. While we and others have shown that MRN endonuclease activity is specifically stimulated by protein blocks on DNA ends (8)(9)(10)(11), we considered the possibility that DNA-PK itself constitutes a powerful block to resection, and that DNA-PK may create a critical barrier to end processing at single-ended breaks where there is no other DNA end to participate in end joining.…”

“…1A, lanes 2 and 3). Formation of the cleavage product requires CtIP, which has been shown to promote the endonuclease activity of Mre11 on heterologous substrates (8,9). Mre11 nuclease activity in vitro is generally reported to be manganese-dependent; however, Mre11/Rad50 complexes from archaea and from T4 bacteriophage also exhibit nuclease activity in magnesium when physiologically relevant protein cofactors are present (13, 14).…”