ATR signaling mediates an S-phase checkpoint after inhibition of poly(ADP-ribose) polymerase activity

Abstract

Human fibroblasts, capable of expressing a kinase-dead form of ATR (ATRkd), can be sensitized to the cytotoxic effects of methyl methanesulfonate (MMS) by the PARP inhibitor 4-amino-1,8-naphthalimide (4-AN). The combination of MMS+4-AN results in accumulation of cells in S-phase of the cell cycle and activation of Chk1. Inhibition of ATR activity by expression of ATRkd suppresses the S-phase accumulation and partially reverses the Chk1 phosphorylation. The results confirm involvement of an ATR-mediated damage response pathway in the MMS+4-AN-induced S-phase cell cycle checkpoint in human fibroblasts. Consistent with this hypothesis, the inhibitors caffeine and UCN-01 also abrogate the ATR- and Chk1-mediated delay in progression through S-phase. In the absence of ATR-mediated signaling, MMS+4-AN exposure results in a G(2)/M arrest, rather than an S-phase checkpoint. Thus, whereas ATR mediates the S-phase response, it is not critical for arrest of cells in G(2)/M.

DNA Replication Arrest in Response to Genotoxic Stress Provokes Early Activation of Stress-Activated Protein Kinases (SAPK/JNK)

“…[30][31][32] Therefore, ATR is a reasonable candidate for regulating signaling to stress kinases originating from DNA replication blockage. In line with this, pharmacological inhibition of ATR by caffeine 33 caused an ∼ 50% reduction of SAPK/JNK phosphorylation after UV exposure. Furthermore, irradiation of cells defective in BRCA2/FANCD1, which is a central player in HR and is regulated in an ATRdependent manner, 34,37,38 results in an enhanced level of SAPK/JNK phosphorylation 2 h after UV irradiation.…”

Methylated DNA Causes a Physical Block to Replication Forks Independently of Damage Signalling, O6-Methylguanine or DNA Single-Strand Breaks and Results in DNA Damage

“…ATR and PARP-1 are both activated at stalled replication forks 13,37,60 and both protect cells from cytotoxic effects caused by MMS. 40,61,62 The pronounced S-phase delay previously shown in BER-deficient cells requires damage signalling 14 and could be a result secondary replication-associated damage. We suggest that the DNA damage signalling is not involved in the formation of MMS-induced replication block, but rather has a role in the maintenance of the stability of the stalled fork, repair, replication restart and activation of cell death pathways.…”

“…14 However, the pronounced S-phase delay by MMS in combination with a PARP inhibitor can be reversed by addition of caffeine or expression of a dominant-negative ATR mutant. 40 Thus, there are conflicting data on the role of the DNA damage checkpoint on progression of the cell cycle.…”

Poly(ADP-ribose) binding to Chk1 at stalled replication forks is required for S-phase checkpoint activation

“…More specifically, PARP1 modifies many target ('acceptor') proteins with large amounts of PAR. PARP1 and poly(ADP-ribosyl)ation have been suggested to regulate ATM or ATR activation in response to double-strand breaks and SSBs [27][28][29] . PAR is a highly negatively charged moiety and it has been hypothesized to be a signal to other cellular targets for recruitment to the damage sites 21 .…”