Divergent S Phase Checkpoint Activation Arising from Prereplicative Complex Deficiency Controls Cell Survival

Abstract

The DNA replication machinery plays additional roles in S phase checkpoint control, although the identities of the replication proteins involved in checkpoint activation remain elusive. Here, we report that depletion of the prereplicative complex (pre-RC) protein Cdc6 causes human nontransformed diploid cells to arrest nonlethally in G1-G1/S and S phase, whereas multiple cancer cell lines undergo G1-G1/S arrest and cell death. These divergent phenotypes are dependent on the activation, or lack thereof, of an ataxia telangiectasia and Rad3-related (ATR)-dependent S phase checkpoint that inhibits replication fork progression. Although pre-RC deficiency induces chromatin structural alterations in both nontransformed and cancer cells that normally lead to ATR checkpoint activation, the sensor mechanisms in cancer cells seem to be compromised such that higher levels of DNA replication stress/damage are required to trigger checkpoint response. Our results suggest that therapy-induced disruption of pre-RC function might exert selective cytotoxic effects on tumor cells in human patients. INTRODUCTIONIn all eukaryotic cells, DNA replication initiates from multiple replication origins throughout the genome, and each segment of DNA replicates only once per cell cycle. Primary regulation of DNA replication is exerted at the initiation of DNA synthesis, when replication origins are licensed by assembly of prereplication complexes (pre-RCs) in early G1 (Bell and Dutta, 2002;Blow and Dutta, 2005). This process begins with origin recognition complex (ORC) binding to a nascent replication origin, resulting in recruitment of the loading factors Cdc6, and Cdt1, which function together to load the putative DNA replicative helicase minichromosomal maintenance (MCM) complex. Subsequently, phosphorylation of pre-RC and other DNA replication factors by the S phase-promoting kinases, cyclin-dependent kinases and Dbf4-dependent kinase Cdc7, in G1/S and S promotes loading of Cdc45, MCM10, GINS, RPA, and DNA polymerases to the pre-RC/origin, triggering the initiation of DNA replication.DNA replication fidelity is ensured by S phase checkpoint mechanisms that monitor aberrant DNA replication, replication stress, DNA damage, and chromatin structure alterations in S phase. The S phase checkpoints are mainly governed by the phosphoinositide 3-kinase-related kinases (PIKKs) ataxia telangiectasia mutated-(ATM) or ataxia telangiectasia and Rad3-related (ATR)-dependent signaling pathways. On activation, the ATM/ATR checkpoint pathways immediately suppress late origin firing to prevent further DNA replication and stabilize stalled replication forks to ensure proper replication restart once the replication block/DNA damage has been repaired or removed. Although the ATM-dependent checkpoint responds to DNA double-strand breaks (DSBs), the ATR-dependent checkpoint responds to a broad spectrum of DNA lesions, such as single-strand breaks, fork stalling, or chromatin structural alterations (Abraham, 2001;Yang and Zou, 2006;Cimprich and Cortez, 2008). Th...

Suppression of Reserve MCM Complexes Chemosensitizes to Gemcitabine and 5-Fluorouracil

“…Similar results were obtained by others in U2OS cells, where Chk1 activation and H2AX-p changes are not observed even though p53 is altered upon MCM loss and HU exposure (5). It has been shown that some cancer cells are apparently defective for fork stress/damage sensor mechanisms that involve ATR activation, resulting in sensitivity of such cancer cells to functional MCM loss in the absence of drug exposure (31). This suggests that tumor cells may differentially respond to MCM reduction and concurrent drug exposure dependent on the presence or absence of sensor pathways that ultimately produce changes in replication fork stress indicators.…”

“…Although the potential utility in suppressing MCM function is clear, an important problem must be considered in any clinical approach targeting MCM suppression over sustained time periods. Partial suppression of MCM function can result in reduced genomic stability and increased DNA damage (3, 5, 28, 31), and mice with sustained, partially defective MCM function display increased cancer risk (30). As such, extended MCM suppression during chemotherapy may be contraindicated for clinical management of cancer, with perhaps shorter durations of MCM co-suppression being more advantageous.…”

Differential Chromatin Structure Encompassing Replication Origins in Transformed and Normal Cells

“…64 The elevated in vivo association of pre-RC proteins with the 20mer1, 20mer2, and c-myc origins in the transformed cells may be the result of deregulation of the pre-RC checkpoint, causing certain origins to fire more than once per cell cycle, due to their improper licensing, leading to re-replication and thereby causing genetic instability and cancer. 65,66 In contrast, the equal in vivo association of pre-RC proteins with the lamin B2 origin in both transformed and normal cells indicates that this origin may remain unaffected by the aberrant deregulation of the pre-RC checkpoint.…”

CDC6 interaction with ATR regulates activation of a replication checkpoint in higher eukaryotic cells

“…Journal of Cell Science 123 (2) contrast, it was reported that, when CDC6 is further depleted with more siRNAs (~100 nM), MCM loading is further reduced, leading to cell-cycle arrest around G1-S and/or cell death (Feng et al, 2003;Lau et al, 2009). Under such conditions, spontaneous Chk1 phosphorylation is observed.…”