Domain within the helicase subunit Mcm4 integrates multiple kinase signals to control DNA replication initiation and fork progression

Eukaryotic DNA synthesis initiates from multiple replication origins and progresses through bidirectional replication forks to ensure efficient duplication of the genome. Temporal control of initiation from origins and regulation of replication fork functions are important aspects for maintaining genome stability. Multiple kinase-signaling pathways are involved in these processes. The Dbf4-dependent Cdc7 kinase (DDK), cyclin-dependent kinase (CDK), and Mec1, the yeast Ataxia telangiectasia mutated/Ataxia telangiectasia mutated Rad3-related checkpoint regulator, all target the structurally disordered N-terminal serine/threonine-rich domain (NSD) of mini-chromosome maintenance subunit 4 (Mcm4), a subunit of the mini-chromosome maintenance (MCM) replicative helicase complex. Using whole-genome replication profile analysis and single-molecule DNA fiber analysis, we show that under replication stress the temporal pattern of origin activation and DNA replication fork progression are altered in cells with mutations within two separate segments of the Mcm4 NSD. The proximal segment of the NSD residing next to the DDK-docking domain mediates repression of late-origin firing by checkpoint signals because in its absence late origins become active despite an elevated DNA damage-checkpoint response. In contrast, the distal segment of the NSD at the N terminus plays no role in the temporal pattern of origin firing but has a strong influence on replication fork progression and on checkpoint signaling. Both fork progression and checkpoint response are regulated by the phosphorylation of the canonical CDK sites at the distal NSD. Together, our data suggest that the eukaryotic MCM helicase contains an intrinsic regulatory domain that integrates multiple signals to coordinate origin activation and replication fork progression under stress conditions.Eukaryotic DNA replication initiates from multiple replication origins within each chromosome to duplicate the large genome efficiently. To ensure DNA synthesis occurs once and only once across the genome, cells adopt a two-step process to activate replication origins during two separate stages of the cell-division cycle. The first step is licensing of replication origins, which occurs only when cyclin-dependent kinase (CDK) activity is low. In Saccharomyces cerevisiae, origins of DNA replication are licensed in G1 by the formation of a prereplicative complex (pre-RC). The process begins with the origin recognition complex binding to replication origins and recruiting the licensing factor Cdc6, which facilitates loading of the Cdt1-bound minichromosome maintenance (MCM) complex composed of Mcm2–Mcm7 (Mcm2–7). The hexameric Mcm2–7 is the core of the replicative helicase that unwinds DNA during replication. Within the pre-RC Mcm2–7 is loaded as an inactive double hexamer. The next step, activation of licensed origins (origin firing), occurs throughout the S phase and requires the continuous presence of two kinases, the S phase CDKs and the Dbf4-dependent Cdc7 kinase (DDK). CDK phosphorylates Sld2 and Sld3 to allow their binding to Dpb11 (1, 2), facilitating recruitment of Cdc45 and GINS (composed of protein subunits Sld5, Psf1, Psf2 and Psf3; Go, Ichi, Nii, and San stand for five, one, two, and three in Japanese, respectively) to Mcm2–7 to create an active helicase. DDK phosphorylates Mcm2–7 and blocks an intrinsic initiation inhibitory activity residing in the N terminus of the Mcm4 subunit (3). The concerted action of these S-phase kinases transforms the inactive Mcm2–7 double hexamer into the active helicase complex composed of Cdc45, Mcm2-7, and GINS (the CMG complex) (4–6). Upon initiation, DNA polymerases and other components of the replication machinery are recruited to form replisomes and establish replication forks, where DNA synthesis ensues.Kinase-signaling pathways target various components of the replication machinery. Both CDK and DDK target replication proteins in addition to their essential targets described above. Furthermore, Ataxia telangiectasia mutated/Ataxia telangiectasia mutated Rad3-related (ATM/ATR) signaling targets components of the CMG helicase complex under replication stress (7–10). In the yeast S. cerevisiae, DNA damage activates the checkpoint kinase Rad53, which phosphorylates both Sld3 and Dbf4 to inhibit late origin firing (11, 12). The yeast ATM/ATR homolog Mec1 also targets Mcm4 (13). The stress-activated protein kinase Hog1 targets an auxiliary replisome component Mrc1 to regulate both origin firing and fork progression (14). Although we now have a better understanding of the essential functions of protein kinases in controlling the initiation of replication, we do not completely understand how the separate kinase signaling pathways are coordinated to regulate both initiation and replication fork progression.The structurally disordered N-terminal serine/threonine-rich domain (NSD) of Mcm4 is a target of multiple kinases, including DDK, CDK, and Mec1 (3, 13, 15, 16). Within this region we have identified two functionally distinct domains that exert different functions and are regulated by different kinase systems even though they overlap extensively in primary amino acid sequences. The segment of the Mcm4 NSD proximal to the DDK-docking domain (DDD) (15), and hence termed “proximal NSD,” blocks initiation until it is phosphorylated by DDK. In contrast, the distal segment of the NSD at the N terminus, away from the DDD, is targeted by additional kinases and contributes positively to promote S-phase progression. In this study we present a comprehensive analysis of the pattern of origin activation, replication fork progression, and the checkpoint response in cells under replication stress caused by the inhibition of ribonucleotide reductase (RNR). We show that the distal and proximal NSD segments contribute differently to origin activation and DNA replication fork progression. Furthermore, they exert opposing effects on checkpoint signaling under replication stress. All these effects are regulated by phosphorylation. We suggest that the Mcm4 NSD, a regulatory domain intrinsic to the replicative helicase, mediates the control of multiple aspects of DNA replication. Our data reveal a sophisticated mechanism to fine-tune S-phase progression in response to changing environments.