Abstract
The replication licensing factor CDC6 recruits the MCM2-7 replicative helicase to the replication origin, where MCM2-7 is activated to initiate DNA replication. MCM2-7 is activated by both the CDC7-Dbf4 kinase and cyclin-dependent kinase and via interactions with CDC45 and go-ichi-ni-san complex (GINS) to form the CDC45·MCM2-7·GINS (CMG) helicase complex. TIMELESS (TIM) is important for the subsequent coupling of CMG activity to DNA polymerases for efficient DNA synthesis. However, the mechanism by which TIM regulates CMG activity for proper replication fork progression remains unclear. Here we show that TIM interacts with MCM2-7 prior to the initiation of DNA replication. TIM depletion in various human cell lines results in the accumulation of aberrant CMG helicase complexes on chromatin. Importantly, the presence of these abnormal CMG helicase complexes is not restricted to cells undergoing DNA synthesis. Furthermore, even though these aberrant CMG complexes interact with the DNA polymerases on human chromatin, these complexes are not phosphorylated properly by cyclin-dependent kinase/CDC7-Dbf4 kinase and exhibit reduced DNA unwinding activity. This phenomenon coincides with a significant accumulation of the p27 and p21 replication inhibitors, reduced chromatin association of CDC6 and cyclin E, and a delay in S phase entry. Our results provide the first evidence that TIM is required for the correct chromatin association of the CMG complex to allow efficient DNA replication.
Highlights
Used as the template for DNA synthesis by DNA polymerase
CMG Helicase Complexes Accumulate on Human Chromatin in Non-S Phase, TIM-depleted Cells—Surprisingly, even though the levels of the chromatin-bound CDC6 and the origin-bound MCM2-7 in TIM knockdown cells were reduced, we found that components of the CMG complex, including the go-ichi-ni-san complex (GINS) (SLD5 and PSF2) and CDC45, were still found on the chromatin (Fig. 3, A, fifth through seventh panels, and B)
When we purified chromatin-bound FLAG-CDC45 from control knockdown HEK293T cells synchronized to either early G1 (4 h) or S phase (12 h) after nocodazole release (Fig. 5A), we found that, as expected, MCM2-7 and GINS were co-purified with CDC45 during S phase but not during early G1 phase (Fig. 5B)
Summary
TIM-TIPIN in cohesion establishment is consistent with the discovery of Csm and Tof mutations in genetic screens for chromosome segregation defects [14, 25]. Despite the inefficient recruitment of MCM2-7 to the active replication origin during G1 phase in TIM-deficient cells, the levels of chromatin-bound CMG complexes remain unchanged, and the presence of these CMG complexes on the chromatin is no longer restricted to S phase. These CMG complexes interact with DNA polymerases, the MCM4 subunit has an altered phosphorylation pattern at the DDK- and CDK-dependent PG sites, which are important for efficient DNA replication [26, 27]. We propose that the presence of these non-S phase CMG complexes with altered post-translational modifications acts as a false negative feedback signal to prevent CDC6 and cyclin E from binding to DNA, thereby hindering DNA replication in TIM-deficient cells
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