H2ax

hRad17^>

Phosphoryiated by:

ATM Clfrer

SMC1 kinases

Pln2 c-Abi

CtIP I

MDM2 RadSt

I 8LM

I BF53

PLK3 TopBPI IMF-kB

BRCA1 SAPK3

FANCD2 c-jun

RPA E2F1

4E-BP1 Plk3 PHAS1

Cellular Responses

NHCJ Rlpslf

Gf/B Checkpoints

{JlRAOSiiR AD17)

S phaaa Checkpoints

(BRCAU1=ANa)2CHK2CDC2S) (NB81/8MC1)

G#M ChKfcpomta

(hRADl7)BfiCA1Ä>IK2)

SAPK3, c-jun, 4E-PB1

Figure 6 A DNA Damage Surveillance Network. As part of this signal transduction network, the ATM protein activates multiple cellular functions in response to the detection of spontaneous or induced DNA damage.

The primary abnormality in AT homozygotes presumably creates a defect in this network that prevents the activation of these cellular functions in response to DSBs, aberrant telomeres, and related DNA anomalies. This inability to respond to spontaneous and induced DNA damage can result in increased genomic instability. At the same time, this defect allows the triggering of apoptosis by otherwise nonlethal DNA damage in some cells but blocks damage-induced apopto-sis in others. These abnormalities contribute, in turn, to the multiple in vivo and in vitro abnormalities seen in AT homozygotes.

The ATM network appears to deal specifically with DSBs in genomic DNA, as AT cells are not particularly sensitive to other forms of DNA damage such as UV-induced photoproducts (161). Strand breaks containing modified 3' termini may be particularly good inducers of the ATM network, given the sensitivity of AT homozygotes to agents which induce strand breaks containing 3' phosphogly-colates (42-44). Short or aberrant telomeres also may activate the ATM network (103), suggesting that the ends of abnormally short telomeres may be perceived by the cell as DSBs. ATM-dependent functions also appear to respond to DSBs generated during meiotic synapsis and recombination, as meoisis ends in apopotic death during pachynema in Atm~y~ mice (96,97).

How far upstream ATM protein functions in the DSB surveillance network is not certain, but there is experimental support for the idea that ATM acts at an early stage. AT cells lack the majority of cellular responses to DSBs, thereby placing ATM at a point in the network prior to the branching of DNA repair, cell survival, and cell cycle checkpoint pathways. The identification of multiple protein targets for ATM-dependent phosphorylation also suggests an early role for ATM (see below).

Could ATM could also act as a DNA damage sensor? Indirect support for this possibility has been provided by studies showing that ATM has an affinity for binding to DSBs in vitro (136) and our own findings that ATM localizes to sites of DSBs in mouse spermatocytes and oocytes (135,162) and to regions of induced DSBs in human fibroblasts (see Fig. 5) (147). However, if ATM is a direct sensor of DSBs, then it does not appear to activate damage surveillance pathways alone, as a functional NBS1 is required for optimal radiation-induced ATM kinase activity in vivo (163-165).

The ATM surveillance network may use multiple sensors. Other candidate sensors include H2AX, the NBS1/Mre11/Rad50 complex, hRad17, and the hRad9/hHus1/hRad1 (9-1-1) complex. Molecules of the histone H2AX located at sites of DSBs undergo phosphorylation at serine 139 within 1 min of DSB induction (166), the most rapid known association of a protein with DSBs. The NBS1/Mre11/Rad50 complex rapidly associates with DSBs in vivo (167) and cells from patients defective in Mre11 (ATLD syndrome) and NBS1 (Nijmegen breakage syndrome) share similar cellular defects to AT (168,169), suggesting a role in early surveillance network events. The hRad17 protein and the hRad9/hHus1/hRad11 (9-1-1) complex, whose yeast homologues function as detectors of DNA damage, also are likely to serve as sensors of DSBs for the ATM-

dependent damage surveillance network, as hRad17 and hRad9 mutants are both cell cycle checkpoint defective and radiosensitive (170-172).

Regardless of whether ATM functions as a direct sensor of DNA damage, it is clear that a major function of the ATM protein is signal transduction. Our growing understanding of ATM biochemistry supports this hypothesis. As summarized in Table 1, 25 proteins are known to undergo ATM-dependent phosphorylation following induction of DSBs, and ATM has been shown to directly phosphorylate 15 of these proteins in vitro. These results support the idea that ATM is an upstream component of a kinase cascade that operates via phosphorylation and de-phosphylation of signaling molecules that links detection of DNA damage to modulation of the enzymatic machinery of cell cycle progression, genetic recombination, and apoptosis (see Fig. 6).

For ATM-dependent cell cycle checkpoints we now know the control pathways in some detail (see Fig. 6). ATM controls the G1/S cell cycle checkpoint through direct and indirect phosphorylation of p53 and mdm2, thereby modulating the activity of p21/waf1, which in turn controls the G1/S checkpoint by inhibiting the formation of cdc2/cyclinE complexes (see Fig. 6). Following induction of DSBs, ATM triggers the S phase checkpoint through parallel pathways dependent on NBS1 and Chk2 (173) and activates the G2/M checkpoint through p53 and BRCA1-dependent pathways (174,175). Exactly how ATM exerts control over DNA repair, homologous recombination, apoptosis, and stress responses are not as clear, although some individual steps have been identified (see Table 1 and Fig. 6).

Although ATM controls most cellular responses to DSBs, there are both early and late responses that are ATM independent. DSB-dependent induction of BLM protein does not require ATM (176), and the DSB-induced late G2/M checkpoint still functions in AT cells (67). Following induction of DSBs, many proteins involved in the ATM-dependent damage surveillance network form nuclear foci that are thought to be involved in DNA repair and other damage responses. The involvement of ATM in focus formation is variable. Optimal formation of foci of H2AX, Mre11, Rad50, and Chk2 appear to require functional ATM, whereas formation of Rad51, TopBP1, and FANCD2 appear to be independent of ATM function (147,177-181).

Eukaryotes from yeast to humans practice a certain economy in their damage-response networks. As a result, networks that respond to different types of DNA damage share some common components and end functions. For example, although the DSB and UV surveillance pathways use separate PI-3 kinases, ATM and ATR, to respond to different forms of DNA damage, they both use BRCA1 to control cell cycle checkpoints (182). Network components also can serve multiple biological roles. For example, BRCA1 is involved in the S phase and G2/M checkpoints as well as homologous recombination (67,183), whereas p53 activates cell cycle checkpoints, modulates cell survival, and plays a role in DNA repair (156,174,184,185). Differential phosphorylation may allow a single surveil lance network protein to respond differentially to different upstream transducers and to activate multiple downstream functions. For example, both ionizing and UV-irradiation induce phosphorylation of BRCA1 at multiple serine residues sites. However, phosphorylation of BRCA1 at serine 1387 is x-irradiation specific, whereas serine 1457 is predominately phosphorylated following UV-irradiation (182). Serine 1387 phosphorylation is a required step in BRCA1-dependent triggering of the S phase checkpoint by ionizing radiation, whereas phosphoryla-tion at serine1423 is necessary for BRCA1 to activate the G2/M checkpoint following irradiation (186).

A. Alternative Functions for ATM

Models such as the one presented in Figure 6 can account for much of the clinical and molecular phenotype of AT (84). However, ATM may have other functions as well. For example, it has been suggested that ATM is involved in oxidative stress responses in addition to reacting to DNA damage (187). ATM protein interacts with beta-adaptin in cytoplasmic vesicles (188), and ATM protein is primarily cytoplasmic in Purkinje cells (131), suggesting that ATM may have different, perhaps cytoplasmic, functions in neurons. Finally, ATM may have structural as well as signaling functions. In Atm~'~ pachytene spermatocytes, fragmentation of synaptonemal complexes occurs at sites of putative DSBs and is coincident with the first appearance of ATM foci along the axes of newly paired bivalents (189), suggesting that ATM might be required to maintain the physical integrity of DSBs so as to prevent their conversion into chromosome breaks and subsequent activation of apoptosis in meiotic cells.

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