Organization of DNA replication origin firing inXenopusegg extracts : the role of intra-S checkpoint

Abstract

During cell division, the duplication of the genome starts at multiple positions called 14 replication origins. Origin firing requires the interaction of rate-limiting factors with potential 15 origins during the S(ynthesis)-phase of the cell cycle. Origins fire as synchronous clusters is 16 proposed to be regulated by the intra-S checkpoint. By modelling either the unchallenged or the 17 checkpoint-inhibited replication pattern of single DNA molecules from Xenopus sperm chromatin 18 replicated in egg extracts, we demonstrate that the quantitative modelling of data require: 1) a 19 segmentation of the genome into regions of low and high probability of origin firing; 2) that regions 20 with high probability of origin firing escape intra-S checkpoint regulation; 3) that the intra-S 21 checkpoint controls the firing of replication origins in regions with low probability of firing. This 22 model implies that the intra-S checkpoint is not the main regulator of origin clustering. The minimal 23 nature of the proposed model foresees its use to analyse data from other eukaryotic organisms. 24 25 2013; Rhind and Gilbert, 2013). The core motor component of the replicative helicase, the MCM2-33 7 complex, is loaded on chromatin from late mitosis until the end of G1 phase as an inactive 34 head-to-head double hexamer (DH) to form a large excess of potential origins (DePamphilis et al., 35 2006; Ticau et al., 2015). During S phase, only a fraction of the MCM2-7 DHs are activated to 36 form a pair of active Cdc45-MCM2-7-GINS (CMG) helicases and establish bidirectional replisomes 37 (DePamphilis and Bell, 2010). MCM2-7 DHs that fail to fire are inactivated by forks emanating from 38 neighboring fired origins (Blow et al., 2011). Origin firing requires S-phase cyclin-dependent kinase 39 (CDK) and Dbf4-dependent kinase (DDK) activities as well as the CDK targets Sld2 and Sld3 and the 40 1 of 29 Manuscript submitted to eLife replisome-maturation scaffolds Dpb11 and Sld7 in S. cerevisiae. The six initiation factors Sld2, Sld3, 41 Dpb11, Dbf4, Sld7 and Cdc45 are expressed at concentrations significantly lower than the MCM 42 complex and core replisome components, suggesting that they may be rate-limiting for origin firing 43 (Mantiero et al., 2011; Tanaka et al., 2011). Among these six factors, Cdc45 is the only one to travel 44 with the replication fork. 45 DNA replication initiates without sequence specificity in Xenopus eggs (Harland and Laskey, 1980; 46 Méchali and Kearsey, 1984), egg extracts (Mahbubani et al., 1992; Hyrien and Méchali, 1992; Carli 47 et al., 2016, 2018) and early embryos (Hyrien and Méchali, 1993; Hyrien et al., 1995) (for review see 48 Hyrien et al. (2003)). To understand how a lack of preferred sequences for replication initiation 49 is compatible with a precise S-phase completion time, investigators have studied replication at 50 the single DNA molecule level using the DNA combing technique (Lucas et al., 2000; Herrick et al., 51 2000; Blow et al., 2001; Marheineke and Hyrien, 2001, 2004). In contr...