Mitotic UV irradiation induces a DNA replication-licensing defect that potentiates G1 arrest response.

Cdt1 begins to accumulate in M phase and has a key role in establishing replication licensing at the end of mitosis or in early G1 phase. Treatments that damage the DNA of cells, such as UV irradiation, induce Cdt1 degradation through PCNA-dependent CRL4-Cdt2 ubiquitin ligase. How Cdt1 degradation is linked to cell cycle progression, however, remains unclear. In G1 phase, when licensing is established, UV irradiation leads to Cdt1 degradation, but has little effect on the licensing state. In M phase, however, UV irradiation does not induce Cdt1 degradation. When mitotic UV-irradiated cells were released into G1 phase, Cdt1 was degraded before licensing was established. Thus, these cells exhibited both defective licensing and G1 cell cycle arrest. The frequency of G1 arrest increased in cells expressing extra copies of Cdt2, and thus in cells in which Cdt1 degradation was enhanced, whereas the frequency of G1 arrest was reduced in cell expressing an extra copy of Cdt1. The G1 arrest response of cells irradiated in mitosis was important for cell survival by preventing the induction of apoptosis. Based on these observations, we propose that mammalian cells have a DNA replication-licensing checkpoint response to DNA damage induced during mitosis.

Chromatin fractionation analysis of licensing factors in mammalian cells.

ORC, Cdc6, Cdt1, and MCM2-7 are replication-licensing factors, which play a central role in the once-per-cell cycle control of DNA replication. ORC, Cdc6, and Cdt1 collaborate to load MCM2-7 onto replication origins in order to license them for replication. MCM2-7 is a DNA helicase directly involved in DNA replication and dissociates from DNA as S phase progresses and each replicon is replicated. In the cell cycle, the loading of MCM2-7 is restricted during the end of mitosis and the G1 phase. Thus, the levels of chromatin-bound MCM2-7 and its loaders oscillate during the cell cycle. Chromatin association of these factors can be analyzed by separating a cell lysate into soluble and chromatin-enriched insoluble fractions in mammalian cells.

Imaging analysis to determine chromatin binding of the licensing factor MCM2-7 in mammalian cells.

S-CDK and DDK protein kinases initiate DNA replication at replication origins. Prior to the activation of these kinases, origins must become competent for replication by loading MCM2-7 DNA helicase on chromatin. This process is known as replication licensing or pre-replicative complex (pre-RC) formation. After the onset of S phase, however, licensing is inhibited to prevent re-replication of DNA. In this chapter, we describe a method to analyze origin licensing by imaging the chromatin-bound licensing factor MCM2-7. In a normal cell cycle, MCM2-7 is loaded at the end of mitosis or early G1 phase. As S phase progresses, MCM2-7 is dissociated from the replicated regions. When DNA replication is completed, cells in G2 phase have no chromatin-bound MCM2-7. The analysis of chromatin-bound MCM2-7 in each cell provides an insight into cell cycle stage and condition for cell cycle.

Checkpoint kinase ATR phosphorylates Cdt2, a substrate receptor of CRL4 ubiquitin ligase, and promotes the degradation of Cdt1 following UV irradiation.

The DNA replication-licensing factor Cdt1 is present during the G1 phase of the cell cycle. When cells initiate S phase or are UV-irradiated, Cdt1 is recruited to chromatin-bound PCNA and ubiquitinated by CRL4(Cdt2) for degradation. In both situations, the substrate-recognizing subunit Cdt2 is detected as a highly phosphorylated form. Here, we show that both caffeine-sensitive kinase and MAP kinases are responsible for Cdt2 phosphorylation following UV irradiation. We found that Cdt1 degradation was attenuated in the presence of caffeine. This attenuation was also observed in cells depleted of ATR, but not ATM. Following UV irradiation, Cdt2 was phosphorylated at the S/TQ sites. ATR phosphorylated Cdt2 in vitro, mostly in the C-terminal region. Cdt1 degradation was also induced by DNA damaging chemicals such as methyl methanesulfonate (MMS) or zeocin, depending on PCNA and CRL4-Cdt2, though it was less caffeine-sensitive. These findings suggest that ATR, activated after DNA damage, phosphorylates Cdt2 and promotes the rapid degradation of Cdt1 after UV irradiation in the G1 phase of the cell cycle.