Multiple kinases inhibit origin licensing and helicase

16 Meiotic cells undergo a single round of DNA replication followed by two rounds of 17 chromosome segregation (the meiotic divisions) to produce haploid gametes. Both DNA 18 replication and chromosome segregation are similarly regulated by CDK oscillations in mitotic 19 cells. Yet how these two events are uncoupled between the meiotic divisions is unclear. Using 20 Saccharomyces cerevisiae, we show that meiotic cells inhibit both helicase loading and helicase 21 activation to prevent DNA replication between the meiotic divisions. CDK and the 22 meiosis‐ specific kinase Ime2 cooperatively inhibit helicase loading, and their simultaneous 23 inhibition allows inappropriate helicase reloading. Further analysis uncovered two previously 24 unknown mechanisms by which Ime2 inhibits helicase loading. Finally, we show that CDK and 25 the polo‐ like kinase Cdc5 trigger degradation of Sld2, an essential helicase‐ activation protein. 26 Together, our data demonstrate that multiple kinases inhibit both helicase loading and activation 27 between the meiotic divisions, thereby ensuring reductive cell division. 28 29 Introduction 30 The production of haploid gametes is required for sexual reproduction. These gametes 31 are produced by meiosis, a specialized cell division program during which a single round of 32 DNA replication is followed by two rounds of chromosome segregation (the meiotic divisions), 33 Meiosis I (MI) and Meiosis II (MII). In contrast, mitotically‐ dividing cells maintain their ploidy by 34 strictly alternating rounds of DNA replication and chromosome segregation. The lack of DNA 35 replication between MI and MII is essential for the reduction in ploidy inherent to meiosis, but it 36 is unclear how the meiotic program differs from mitosis to allow for two sequential chromosome 37 segregation events without an intervening S phase. 38 In mitotic cells, both DNA replication and chromosome segregation require cyclin39 dependent kinase (CDK) activity to oscillate during the cell cycle. A low‐ CDK state during G1 40 3 phase allows both events to initiate, and a high‐ CDK state is required for their completion. 41 During meiosis, the CDK‐ oscillation dependence of both events presents a unique problem 42 between MI and MII, a period known as the MI‐ MII transition (Figure 1A). After MI has been 43 completed, CDK activity decreases, and then increases again upon entry into MII (Carlile and 44 Amon, 2008). This oscillation is required for multiple essential chromosome‐ segregation events, 45 including duplication of the spindle pole body (SPB, the yeast centrosome) (Buonomo et al., 46 2003; Fox et al., 2017; Marston et al., 2003). However, the DNA replication program must 47 remain inhibited between MI and MII to achieve the hallmark of meiosis, reductive cell division. 48 Given that an oscillation of CDK activity is sufficient for re‐ replication of the entire genome in 49 mitotic cells (Dahmann et al., 1995), it is not fully understood how meiotic cells reset the 50 chromosome segregation program while retaining inhibition of the DNA replication program. 51 Mitotic cells use oscillations of CDK activity to ensure that the genome is replicated 52 exactly once per cell division. During G1 phase, low CDK activity allows for the Mcm2‐ 7 53 complex, the core enzyme of the replicative helicase, to be loaded onto origins of replication in 54 an inactive state. This event, known as origin licensing or helicase loading, cannot occur in the 55 presence of high CDK activity and requires the cooperative action of three proteins: Cdc6, Cdt1, 56 and the Origin Recognition Complex (ORC) (Evrin et al., 2009; Remus et al., 2009). Upon 57 S‐ phase entry, S‐ CDK (CDK bound to S-phase cyclins Clb5/6) is activated and impacts DNA 58 replication in two ways. First, S‐ CDK phosphorylates two essential proteins, Sld2 and Sld3, that 59 subsequently promote helicase activation, replisome assembly, and chromosome duplication 60 (Masumoto et al., 2002; Tanaka et al., 2007; Zegerman and Diffley, 2007). Second, both 61 S‐ CDK and M-CDK (CDK bound to mitotic cyclins Clb1-4) inhibit new helicase loading during S, 62 G2, and M phases. These kinases directly phosphorylate Cdc6, Mcm3, and ORC to trigger the 63 proteolytic degradation of Cdc6, the nuclear export of Mcm2‐ 7‐ Cdt1, and inhibition of ORC 64 4 helicase‐ loading activity, respectively (Calzada et al., 2000; Chen and Bell, 2011; Drury et al., 65 2000; Labib et al., 1999; Nguyen et al., 2000). 66 CDK oscillations also ensure that chromosome segregation occurs once per mitotic cell 67 cycle (Winey and Bloom, 2012). At the end of G1 phase, G1‐ CDK (CDK bound to G1 cyclins 68 Cln1-3) is required for duplication of the SPB (Jaspersen et al., 2004). Later in the cell cycle, 69 S‐ CDK and M‐ CDK prevent re‐ duplication of SPBs, and M-CDK is essential for the assembly 70 of metaphase spindles (Avena et al., 2014; Elserafy et al., 2014; Haase et al., 2001). Finally, 71 downregulation of CDK activity is required for anaphase spindle disassembly upon completion 72 of chromosome segregation (Shirayama et al., 1999; Thornton and Toczyski, 2003; Wäsch and 73 Cross, 2002). From this point forward, we will refer only to total CDK activity without specifying 74 G1‐ , S‐ , or M‐ CDK, as the events we will be discussing are similarly regulated by all three 75 kinases. 76 Two models have been proposed to explain how meiotic cells uncouple DNA replication 77 and chromosome segregation during the MI‐ MII transition. The CDK‐ balance model suggests 78 that partially inactivating CDK is sufficient to reset the chromosome segregation program while 79 still inhibiting Mcm2‐ 7 loading and replication initiation (Iwabuchi et al., 2000). In contrast, the 80 alternative‐ kinase model suggests that a second kinase inhibits Mcm2‐ 7 loading during the 81 MI‐ MII transition, allowing the oscillation of CDK activity to reset the chromosome segregation 82 program without resetting the DNA replication program. Ime2, a yeast meiosis‐ specific kinase 83 that is evolutionarily related to CDK (Krylov et al., 2003), has been proposed to have this role 84 (Holt et al., 2007). 85 We set out to systematically address how DNA replication is inhibited between the 86 meiotic divisions using a combination of in vivo and in vitro approaches. We found that Mcm2‐ 7 87 loading is the earliest inhibited step of replisome assembly during the MI‐ MII transition, and that 88 this inhibition can be bypassed by simultaneous inhibition of CDK and Ime2. Furthermore, we 89