Ataxia-telangiectasia Mutated (ATM)-dependent Activation of ATR Occurs through Phosphorylation of TopBP1 by ATM

Abstract

ATM (ataxia-telangiectasia mutated) is necessary for activation of Chk1 by ATR (ATM and Rad3-related) in response to double-stranded DNA breaks (DSBs) but not to DNA replication stress. TopBP1 has been identified as a direct activator of ATR. We show that ATM regulates Xenopus TopBP1 by phosphorylating Ser-1131 and thereby strongly enhancing association of TopBP1 with ATR. Xenopus egg extracts containing a mutant of TopBP1 that cannot be phosphorylated on Ser-1131 are defective in the ATR-dependent phosphorylation of Chk1 in response to DSBs but not to DNA replication stress. Thus, TopBP1 is critical for the ATM-dependent activation of ATR following production of DSBs in the genome.In eukaryotic cells, a variety of checkpoint control mechanisms help to maintain genomic integrity (1-3). Elements of these regulatory systems scrutinize the genome for the existence of damaged or incompletely replicated DNA. ATM 2 (ataxia-telangiectasia mutated) and ATR (ATM and Rad3-related), which belong to the phosphoinositide kinase-related family of protein kinases, are key upstream players in checkpoint pathways (1). ATM responds principally to the occurrence of double-stranded DNA breaks (DSBs) in the genome. By contrast, ATR plays a distinct role in the detection of stalled DNA replication forks, but it also participates in DNA damage responses (1,4,5). ATR possesses a binding partner called ATRIP that is essential for checkpoint regulatory functions (6). It has been established that activation of Chk1 in response to DSBs, but not to DNA replication stress, also depends upon upstream regulation by ATM (7-9). This observation implied that certain DNA structures, but not others, rely on ATM to elicit the ATR-dependent phosphorylation of Chk1. In more recent studies, TopBP1 has been identified as a direct activator of the ATR-ATRIP complex (10). Here we demonstrate that TopBP1 has a direct and essential role in the pathway that connects ATM to ATR specifically in response to the occurrence of DSBs in the genome. EXPERIMENTAL PROCEDURESXenopus Egg Extracts-Xenopus egg extracts were prepared as described (11). Extracts were treated with 50 g/ml (dA) 70 ⅐(dT) 70 to induce checkpoint responses (12). To prepare extracts containing chromatin with double-stranded DNA breaks or DNA replication blocks, demembranated sperm nuclei (1000 -3000/ l) were incubated in extracts containing 0.05 unit/ l EcoRI or 100 g/ml aphidicolin, respectively. For preparation of nuclear fractions, egg extracts (50 l) containing 3000 sperm nuclei/ l were incubated under the indicated conditions. Nuclear fractions were prepared as described previously (13).Antibodies-For production of anti-P-Ser-1131 antibodies to phosphorylated XtopBP1, the peptide CLNTEPSQNEQI (containing residues 1126 -1136 of XtopBP1) was synthesized with a phosphate on Ser-1131 and an extra cysteine residue for conjugation to keyhole limpet hemocyanin. Anti-phosphopeptide antibodies were prepared as described (11). Other antibodies were described previously (10,14,15).Immunoprecipitation a...

Interplay between ATM and ATR in the regulation of common fragile site stability

“…However, recently the regulation of ATM and ATR was shown to be dependent one on the other in response to different DNA damaging conditions including IR, UV light and replication stalling by Hydroxyurea (HU) (Jazayeri et al, 2006;Myers and Cortez, 2006;Stiff et al, 2006;Cuadrado et al, 2006b;Yoo et al, 2007). Since 0.4 mM aphidicolin treatment activates both ATR and ATM pathways, we analysed whether under these conditions the activation of these proteins depends on each other.…”

Dpb11 activates the Mec1–Ddc2 complex

“…For Dpb11, our data indicate that this site serves as a positive-feedback amplification loop for Mec1 activation. For TopBP1, it is still unclear whether ATR autoamplifies by using this mechanism; however, ATM phosphorylation of this site potentiates the ability of TopBP1 to activate ATR (27).…”

DNA double-strand break repair and development

“…While there is cross talk between ATM and ATR (Jazayeri et al, 2006;Matsuoka et al, 2007;Yoo et al, 2007), they have separable biochemical roles and complete ATR loss of function, like any of the MRN components, is incompatible with viability (Brown and Baltimore, 2000). Interestingly, NBS shares more in common with ATR-Seckel syndrome than with A-T, despite clear evidence of ATM-Nbs1 interaction (Figure 1).…”