Taken together, the results presented here indicate that Cdc6 ubiquitination induced by both UV irradiation and by DNA alkylation can be carried out by the Huwe1 enzyme. This study complements previous work on the cell cycle– and ionizing radiation–induced ubiquitination of Cdc6 carried out by APCCdh1
(Petersen et al., 2000
; Duursma and Agami, 2005
; Mailand and Diffley, 2005
) The demonstration of two independent ubiquitin ligases for Cdc6 is another example of a common theme in cell cycle regulation, namely multiple overlapping regulatory mechanisms. Presumably these multiple pathways evolved to ensure tight control over essential processes under a variety of conditions.
DNA damage sufficient to induce Cdk2 inhibition can accelerate Cdc6 degradation by inhibiting the protective phosphorylation at Ser54, but this mechanism can only operate when APC is active (Duursma and Agami, 2005
). On the other hand, we find that Huwe1 ubiquitination of Cdc6 can occur in S phase, even when Cdc6 has been altered to mimic phosphorylation by Cdk2. Therefore, like Cdt1, which associates with two different ubiquitin ligase complexes, Skp2 and Cul4, (Li et al., 2003
; Kondo et al., 2004
; Liu et al., 2004
; Sugimoto et al., 2004
; Takeda et al., 2005
; Hu and Xiong, 2006
; Senga et al., 2006
), Cdc6 is regulated by both APC and Huwe1. Because the human genome contains many thousands of potential origins, these different control pathways may be required to prevent relicensing at even a small percentage of origins. Multiple mechanisms may also need to be in place to adequately respond to a variety of cellular insults.
DNA damage activates an intracellular signaling pathway that culminates in cell cycle checkpoint arrest through inhibition of Cdks (reviewed in Abraham, 2001
; Sancar et al., 2004
). If the damage occurs during S phase or G2 and is subsequently repaired (instead of inducing apoptosis), then cells could recover with new preRCs on the already duplicated DNA. Even though geminin is not degraded after DNA damage (Higa et al., 2003
and our unpublished observations), the persistence of geminin during a checkpoint response is not sufficient to block substantial preRC reassembly. This assertion is supported by previous findings that direct inhibition of Cdk activity during G2, either pharmacologically or genetically, permits reloading of MCM proteins onto chromatin even in the presence of geminin (Bates et al., 1998
; Ballabeni et al., 2004
; Zhu et al., 2005
). Thus, there is a risk of genome instability if preRCs are permitted to assemble while Cdk activity is inhibited. Presumably to guard against this threat, both Cdc6 and Cdt1 are actively degraded after DNA damage, and we suggest that the degradation during S phase and G2 is particularly important.
The Huwe1-dependent mechanism of Cdc6 degradation after DNA damage caused by UV radiation or MMS is distinct from the APCCdh1
-dependent mechanism of Cdc6 degradation caused by ionizing radiation (IR). Substantial IR-induced Cdc6 degradation is only observed in p53-proficient cells and requires induction of p21 expression (Duursma and Agami, 2005
). In contrast, Cdc6 degradation induced by UV radiation, MMS, or adozelesin (another DNA alkylating compound) occurs equally well in both p53-proficient and p53-deficient cells (C and Blanchard et al., 2002
). The cellular response to IR is primarily mediated by the ATM and Chk2 kinases that are stimulated by double-strand DNA breaks. On the other hand, most other forms of DNA damage, including UV and MMS-induced damage, primarily trigger activation of the ATR and Chk1 kinases. These two kinase cascades are related but distinct in both the signals that trigger their activation and in their primary substrates. It is possible that differences in the kinase pathways that are activated by DNA damage account for the different mechanisms of Cdc6 degradation. In support of a contribution of ATR/ATM checkpoint kinases to Cdc6 degradation, we observe partial stabilization of Cdc6 after DNA damage in the presence of caffeine (Supplementary Figure S5).
Cdc6 is substantially overproduced in a wide variety of cancer cell types. This overproduction results in a longer time needed to eliminate Cdc6 protein from cancer cells experiencing DNA damage than from normal cells, leaving a longer window of opportunity for these cells to assemble preRCs. The abundant Cdc6 that arises from near ubiquitous deregulation of the E2F-RB transcriptional program in cancers could contribute to the observed genomic instability associated with transformation. We note that most cancer cells also have a disrupted p53 pathway that could blunt the APC-mediated mechanism of Cdc6 degradation. Thus Huwe1, which is also highly expressed in multiple tumor cell lines (Chen et al., 2005
; Yoon et al., 2005
), may represent the primary means of degrading Cdc6 in p53-null cells and may be particularly important in the response to chemotherapies that damage DNA.
Huwe1-depletion in the absence of DNA damage did not result in overt rereplication (data not shown). If the purpose of Cdc6 degradation after DNA damage is to prevent rereplication, then it is likely that in order to observe significant rereplication in these short-term assays, not only would Cdc6 degradation need to be blocked, and perhaps Cdt1 degradation, but Cdk activity would need to be maintained in order to permit origin firing. We suggest however that over the course of many cell cycles the degradation of both Cdc6 and Cdt1 when cells encounter DNA damage contributes to maintaining strict regulation of preRC assembly or to promoting appropriate checkpoint and apoptotic responses. Consistent with this model, modest overproduction of Cdt1 has minimal effects on the growth properties or genome stability in cultured mouse cells, but those cells have a higher propensity for tumorigenesis in vivo (Arentson et al., 2002
). We predict that stabilization of Cdc6 would have a similar phenotype, particularly in sensitized backgrounds or in cells subjected to repeated rounds of sublethal DNA damage.
Although most of the other known targets of Huwe1 (p53, Mcl-1, and c-myc) are specific to metazoan species, Huwe1-dependent regulation of Cdc6 may be ubiquitous in eukaryotes, because both human and yeast cells rely on this conserved enzyme for Cdc6 degradation. Cdc6 is certainly not the only target of Huwe1; Huwe1 is a very large protein with multiple protein–protein interaction domains for binding other proteins (Adhikary et al., 2005
; Chen et al., 2005
; Warr et al., 2005
; Zhong et al., 2005
). We propose the following model to accommodate what is currently understood concerning the regulation of human Cdc6 after DNA damage in the context of other recently identified Huwe1 substrates (illustrated in B): During normal cell growth, Huwe1 ubiquitinates p53 to induce p53 degradation (Chen et al., 2005
) and ubiquitinates and activates c-myc (Adhikary et al., 2005
). Huwe1 ubiquitinates the anti-apoptotic Mcl-1 protein but its access to Mcl-1 is not obvious until cells are exposed to DNA damage (Zhong et al., 2005
, and Zhong, unpublished observations). Hence, under normal growth conditions cells have active c-myc transcription, low p53, high Mcl-1, and cell cycle–regulated Cdc6 levels that are controlled by a combination of E2F-dependent transcription and APC-mediated degradation, and this combination supports robust proliferation (B, left). In response to DNA damage Huwe1 ubiquitinates Cdc6 and Mcl-1 to induce their degradation, but no longer ubiquitinates p53, resulting in low Cdc6 and Mcl-1 with stabilized p53, thus promoting cell cycle arrest, apoptosis, and inhibition of new preRC assembly (B, right).
Given the role of Huwe1 in the regulation of Cdc6, p53, c-myc, and Mcl-1, one might speculate that Huwe1 activity is regulated by DNA damage signals. Nevertheless we do not detect changes in Huwe1 protein levels after DNA damage (G and , A, B, D, and E) or during cell cycle progression (Supplementary Figure S2). Instead, we find that Cdc6 is lost from the chromatin fraction after DNA damage and may have greater access to the soluble Huwe1 under these conditions. The mechanism by which Cdc6 is released from chromatin is not yet known, but clearly does not involve degradation of Orc2 (A). It is tempting to speculate that one or more of the DNA damage signaling kinases regulates the chromatin association of Cdc6. This study prompts significant questions regarding the intracellular context of the Huwe1-Cdc6 interaction and the upstream regulators of Huwe1 and/or Cdc6 during a DNA damage response. The regulation of Cdc6 by Huwe1 is likely to be complex, and its elucidation is an important future goal.