Rif1 restrains the rate of replication origin firing in Xenopus laevis

Abstract

Metazoan genomes are duplicated by the coordinated activation of clusters of replication origins at different times during S phase, but the underlying mechanisms of this temporal program remain unclear during early development. Rif1, a key replication timing factor, inhibits origin firing by recruiting protein phosphatase 1 (PP1) to chromatin counteracting S phase kinases. We have previously described that Rif1 depletion accelerates early Xenopus laevis embryonic cell cycles. Here, we find that in the absence of Rif1, patterns of replication foci change along with the acceleration of replication cluster activation. However, initiations increase only moderately inside active clusters. Our numerical simulations suggest that the absence of Rif1 compresses the temporal program towards more homogeneity and increases the availability of limiting initiation factors. We experimentally demonstrate that Rif1 depletion increases the chromatin-binding of the S phase kinase Cdc7/Drf1, the firing factors Treslin, MTBP, Cdc45, RecQL4, and the phosphorylation of both Treslin and MTBP. We show that Rif1 globally, but not locally, restrains the replication program in early embryos, possibly by inhibiting or excluding replication factors from chromatin.

Dual DNA Replication Modes: Varying Fork Speeds and Initiation Rates within the spatial replication program inXenopus

“…In contrast, Rif1 depletion only moderately modified the pattern distribution, while Chk1 inhibition nor overexpression had any effect on this distribution. This is consistent with the fact that Plk1 depletion induced significant changes in initiation rates, IODs, and eye lengths when molecules of the same replicated fraction were compared [47], whereas Rif1 depletion [38] or Chk1 inhibition [31] did not. Therefore, these findings suggest that Plk1 promotes origin activation inside replication clusters.…”

“…Inhibition of the checkpoint effector kinase Chk1 by UCN-01 or Chk1 over-expression did not alter the partition of replicating DNA molecules into two separate classes (Figure 5B, C, Supplementary Figure S5 A-B). Another important negative regulator of the replication program in Xenopus is Rif1 [38]. However, using RepliCorr, we found that after Rif1 depletion, the separation of molecules into the two classes was maintained (Figure 5D, Supplementary Figure S5C).…”

“…DNA combing data were chosen from experiments during a control S phase and after Plk1 depletion [47], Rif1 depletion [38] or after Chk1 inhibition by UCN-01 and Chk1 overexpression [31]. Detailed experimental conditions and primary analysis are described in the respective publications.…”

“…On the other hand, the replication timing regulator Rap1-interacting factor (Rif1) has been shown to inhibit late replication in yeast [34, 35], mice [36], and human cell culture lines [37] at the level of large chromatin domains. In Xenopus , the depletion of Rif1 accelerates the replication program by accelerating origin cluster activation and increasing replication foci number [38]. Rif1 opposes replicative helicase activation by counteracting DDK-mediated MCM2-7 activation [39, 40, 41].…”

“…The first process shows a fast replication fork speed coupled with a low initiation rate, whereas the second process shows a slow replication fork speed associated with a high initiation rate. We used RepliCorr to analyze experiments in which three regulatory pathways were disrupted [31, 47, 38]. Chk1 inhibition or over-expression and Rif1 depletion did not affect the organization of these two processes.…”