Cdc6 Stability Is Regulated by the Huwe1 Ubiquitin Ligase after DNA Damage

Abstract

The Cdc6 protein is an essential component of pre-replication complexes (preRCs), which assemble at origins of DNA replication during the G1 phase of the cell cycle. Previous studies have demonstrated that, in response to ionizing radiation, Cdc6 is ubiquitinated by the anaphase promoting complex (APC Cdh1 ) in a p53-dependent manner. We find, however, that DNA damage caused by UV irradiation or DNA alkylation by methyl methane sulfonate (MMS) induces Cdc6 degradation independently of p53. We further demonstrate that Cdc6 degradation after these forms of DNA damage is also independent of cell cycle phase, Cdc6 phosphorylation of the known Cdk target residues, or the Cul4/DDB1 and APC Cdh1 ubiquitin E3 ligases. Instead Cdc6 directly binds a HECT-family ubiquitin E3 ligase, Huwe1 (also known as Mule, UreB1, ARF-BP1, Lasu1, and HectH9), and Huwe1 polyubiquitinates Cdc6 in vitro. Degradation of Cdc6 in UV-irradiated cells or in cells treated with MMS requires Huwe1 and is associated with release of Cdc6 from chromatin. Furthermore, yeast cells lacking the Huwe1 ortholog, Tom1, have a similar defect in Cdc6 degradation. Together, these findings demonstrate an important and conserved role for Huwe1 in regulating Cdc6 abundance after DNA damage. INTRODUCTIONDuplication of large mammalian genomes requires that DNA replication initiate at thousands of chromosomal origins. In order for an origin to be competent for replication, it must first be bound by a multiprotein complex, the prereplication complex (preRC). PreRCs are constructed in a stepwise process through the chromatin binding of the origin recognition complex (ORC), which then recruits both the Cdc6 ATPase and Cdt1, two proteins that are required for the stable loading of the minichromosome maintenance complex (MCM). The Cdc6 and Cdt1-dependent loading of MCM complexes at origins licenses them for replication during the G1 phase of the cell cycle. Sufficient preRCs must be assembled during G1 to promote complete replication, but new preRCs must not assemble after S phase begins because relicensing of previously fired origins leads to rereplication and genome instability (Vaziri et al., 2003;Melixetian et al., 2004;Zhu et al., 2004;Archambault et al., 2005). For these reasons, preRC assembly is one of the most highly regulated events in the control of DNA replication. Cells restrict preRC assembly to the G1 period through a combination of overlapping mechanisms that regulate individual preRC components (reviewed in Bell and Dutta, 2002;Blow and Hodgson, 2002;Nishitani and Lygerou, 2002;Diffley, 2004;Machida et al., 2005;Fujita, 2006).Cdc6 is not only an essential factor for preRC construction, but it has also been implicated in the activation of the cell cycle checkpoint that prevents entry into mitosis while DNA replication is incomplete (Clay-Farrace et al., 2003;Oehlmann et al., 2004;Lau et al., 2006). These observations suggest that Cdc6 functions not only during G1, but also in later cell cycle stages. Moreover, Cdc6 plays a role in setting the threshold for...

Spatio-temporal regulation of the human licensing factor Cdc6 in replication and mitosis

“…Evidence exists that Cdc6 is involved in the surveillance of the replication process during S phase via the checkpoint kinase Chk1, 28,40 and it was also shown, that Cdc6 is degraded in response to induced DNA damage during all cell cycle phases. 41 If Cdc6 plays a role in these processes it must either remain available in the cell nucleus during S phase at low levels, or it relocates to the cell nucleus in response to signals induced by stalled replication forks or extrinsic DNA damage. Future work is required to examine these aspects of Cdc6 regulation and their functional consequences.…”

Mule determines the apoptotic response to HDAC inhibitors by targeted ubiquitination and destruction of HDAC2

“…Mule is a HECT domain-containing ubiquitin ligase that ubiquitinates and degrades multiple cellular substrates (Adhikary et al 2005;Chen et al 2005;Hall et (Fig. 4A).…”

“…Mule was first identified as a ubiquitin ligase for an anti-apoptotic Bcl-2 family protein, Mcl-1 (Zhong et al 2005). Mule is also known as ARF-BP1, Ureb1, LASU1, HUWE1, or HectH9, and promotes ubiquitin-proteasomal degradation of multiple substrates, including p53, c-Myc, Cdc6, histones, N-Myc, Miz1, TopBP1, and Polb (Adhikary et al 2005;Chen et al 2005;Zhong et al 2005;Hall et al 2007;Liu et al 2007;Herold et al 2008;Zhao et al 2008;Parsons et al 2009;Yang et al 2010). However, whether any of these existing or unidentified substrates mediate the major function of Mule in the DNA damage response remains elusive.…”

Dichotomous role of pancreatic HUWE1/MULE/ARF-BP1 in modulating beta cell apoptosis in mice under physiological and genotoxic conditions

“…Alternatively, this difference in HUWE1 function is a reflection of its context-and cell-type-dependent specificity in its biological role, which is highlighted by our findings in this report. Similarly, while HUWE1 can facilitate degradation of anti-apoptotic MCL1 [21,24,40], deletion of HUWE1 in the pancreas did not change MCL1 protein expression. This is consistent with the only slight differences in MCL1 that were seen at steady state in B lymphocytes with Huwe1 deletion, suggesting MCL1 regulation is context-specific [18,24].…”

“…Apart from p53, HUWE1 has mul tiple other polyubiquitination substrates [18][19][20][21][22]. Among them is MCL1, which belongs to the anti-apoptotic B cell CLL/lymphoma 2 (BCL-2) family of proteins [20].…”