Spotlight on the Replisome: Aetiology of DNA Replication-Associated Genetic Diseases
Authors: Roberto Bellelli, Simon J. Boulton
Journal: Trends in Genetics (2020)

DNA replication is performed by a multiprotein complex known as the 'replisome,' which is assembled and regulated in a cell cycle–dependent manner.Hypomorphic mutations of components of the replisome lead to defective development, reduced growth, and altered tissue homeostasis.Whole-genome sequencing studies significantly expanded the repertoire of mendelian diseases caused by mutation of the replication machinery.Phenotypic analysis and mechanistic studies support defective replication origin assembly and activation and perturbation of replication fork stability as the pathogenetic mechanisms of replication-linked human genetic diseases. Human development and tissue homeostasis depend on the regulated control of cellular proliferation and differentiation. DNA replication is essential to couple genome duplication and cell division with the establishment and maintenance of cellular differentiation programs. In eukaryotes, DNA replication is performed by a large machine known as the 'replisome,' which is strictly regulated in a cell cycle-dependent manner. Inherited mutations of replisome components have been identified in a range of genetic conditions characterised by developmental abnormalities and reduced organismal growth in addition to an involvement of the immune and endocrine systems and/or heightened tumour predisposition. Here, we review the current knowledge of the molecular genetics of replisome dysfunction disorders and discuss recent mechanistic insights into their pathogenesis, with a focus on the specific steps of DNA replication affected in these human diseases. Human development and tissue homeostasis depend on the regulated control of cellular proliferation and differentiation. DNA replication is essential to couple genome duplication and cell division with the establishment and maintenance of cellular differentiation programs. In eukaryotes, DNA replication is performed by a large machine known as the 'replisome,' which is strictly regulated in a cell cycle-dependent manner. Inherited mutations of replisome components have been identified in a range of genetic conditions characterised by developmental abnormalities and reduced organismal growth in addition to an involvement of the immune and endocrine systems and/or heightened tumour predisposition. Here, we review the current knowledge of the molecular genetics of replisome dysfunction disorders and discuss recent mechanistic insights into their pathogenesis, with a focus on the specific steps of DNA replication affected in these human diseases. Efficient and accurate DNA replication is essential for the maintenance of genome stability and the accomplishment of developmental and differentiation programs in mammals. In vivo and in vitro studies in budding yeast and other lower eukaryotes have provided a clear understanding of the key steps involved in the control and execution of DNA replication [1.Bell S.P. Labib K. Chromosome duplication in Saccharomyces cerevisiae.Genetics. 2016; 203: 1027-1067Crossref PubMed Google Scholar]. Although redundancy has emerged during the evolution of higher eukaryotes, the basic mechanisms and players that perform DNA replication appear to be remarkably conserved. Over the last two decades, many of the basic components of the mammalian DNA replication machinery have been identified and functionally characterised. Proteomic studies have also identified several mammalian-specific factors stably or transiently associated with the replisome (see Glossary), particularly in the presence of replication stress, with a prominent role in maintaining genome stability and human health [2.Dungrawala H. et al.The replication checkpoint prevents two types of fork collapse without regulating replisome stability.Mol. Cell. 2015; 59: 998-1010Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar,3.Técher H. et al.The impact of replication stress on replication dynamics and DNA damage in vertebrate cells.Nat. Rev. Genet. 2017; 18: 535-550Crossref PubMed Scopus (87) Google Scholar]. Even though the vast majority of replisome factors are required for viability of mammalian cells, hypomorphic mutations of replisome components have been increasingly identified in human genetic disease. Indeed, the application of whole-genome sequencing has led to a significant expansion in the identification of mutations in components of the replication machinery and factors required to maintain replication fork stability. While leading to a deeper understanding of the molecular genetics of these conditions, this has promoted new opportunities to investigate the pathological consequences of dysfunctional DNA replication in human biology and disease. Duplication of genomic DNA is performed by a large multiprotein assembly, known as the 'replisome,' whose activation and activity are strictly regulated during the cell cycle by a multistep process (Figure 1 and Box 1). The first step of DNA replication takes place during the G1 phase of the cell cycle, when DNA replication origins are 'licensed' by the sequential assembly of origin recognition complex 1–6 (ORC1–6) together with cell division cycle 6 (CDC6) and Cdc10-dependent transcript 1 (CDT1), which promote loading of inactive mini-chromosome maintenance 2–7 (MCM2–7) double hexamers at DNA replication origins [4.Riera A. et al.From structure to mechanism-understanding initiation of DNA replication.Genes Dev. 2017; 31: 1073-1088Crossref PubMed Scopus (0) Google Scholar] (Figure 1). Historically, the factors required for licensing of replication origins are also known as components of the pre-replication complex (PRE-RC) [1.Bell S.P. Labib K. Chromosome duplication in Saccharomyces cerevisiae.Genetics. 2016; 203: 1027-1067Crossref PubMed Google Scholar].Box 1Mechanisms of Control of Origin Licensing and ActivationSophisticated mechanisms have evolved to control the major steps of DNA replication, from PRE-RC complex formation and replication origin activation to replisome activity and replication fork stability. Defects in any one of these mechanisms can significantly impact genome stability and human development and are associated with congenital human disease. Regulation of licensing of DNA replication origins is particularly crucial for the maintenance of genome integrity. Accordingly, loading of MCM2–7 on chromatin is limited to the G1 phase of the cell cycle by several mechanisms that control overall levels and/or localisation of the licensing factors ORC1–6, CDC6, and CDT1 [110.Arias E.E. Walter J.C. Strength in numbers: preventing rereplication via multiple mechanisms in eukaryotic cells.Genes Dev. 2007; 21: 497-518Crossref PubMed Scopus (309) Google Scholar].Early studies in budding yeast defined two separate phases of the cell cycle, characterised by low or high CDK activity associated with licensing or activation of DNA replication origins in a mutually exclusive manner. CDK activity, while promoting activation of DNA replication origins, restrains PRE-RC complex formation and origin re-licensing through inhibitory phosphorylation [111.Siddiqui K. et al.Regulating DNA replication in eukarya.Cold Spring Harb. Perspect. Biol. 2013; 5a012930Crossref PubMed Scopus (138) Google Scholar]. In mammalian cells, several CDK-independent mechanisms have also emerged, targeting the licensing factor CDT1 [110.Arias E.E. Walter J.C. Strength in numbers: preventing rereplication via multiple mechanisms in eukaryotic cells.Genes Dev. 2007; 21: 497-518Crossref PubMed Scopus (309) Google Scholar]. Thus, in addition to CDK-dependent phosphorylation and SKP2-mediated ubiquitylation/degradation, the CRL4-CDT2 ubiquitin ligase targets CDT1 for replication-coupled destruction in a PCNA-dependent manner [112.Jin J. et al.A family of diverse Cul4-Ddb1-interacting proteins includes Cdt2, which is required for S phase destruction of the replication factor Cdt1.Mol. Cell. 2006; 23: 709-721Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar]. Furthermore, CDT1 association with the S phase–specific inhibitor, GEMININ, inhibits PRE-RC assembly by sequestering CDT1 in an inactive complex that is unable to interact with or recruit MCM2–7 [69.McGarry T.J. Kirschner M.W. Geminin, an inhibitor of DNA replication, is degraded during mitosis.Cell. 1998; 93: 1043-1053Abstract Full Text Full Text PDF PubMed Scopus (684) Google Scholar,70.Wohlschlegel J.A. et al.Inhibition of eukaryotic DNA replication by geminin binding to Cdt1.Science. 2000; 290: 2309-2312Crossref PubMed Scopus (548) Google Scholar] (Figure 1).Importantly, during the G1 phase an excess of MCM2–7 double hexamers are loaded onto genomic DNA. Known as the MCM paradox, this phenomenon has been associated with the presence of accessory or 'dormant' replication origins. Dormant origins can be activated under conditions of replicative stress [113.Ge X.Q. et al.Dormant origins licensed by excess Mcm2-7 are required for human cells to survive replicative stress.Genes Dev. 2007; 21: 3331-3341Crossref PubMed Scopus (360) Google Scholar,114.Ibarra A. et al.Excess MCM proteins protect human cells from replicative stress by licensing backup origins of replication.Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 8956-8961Crossref PubMed Scopus (281) Google Scholar] in order to rescue replication fork stalling and facilitate completion of genome-wide replication.Initiation of DNA replication, upon DDK and CDK activation, takes place at thousands of genomic sites in a strictly regulated manner [115.Fragkos M. et al.DNA replication origin activation in space and time.Nat. Rev. Mol. Cell Biol. 2015; 16: 360-374Crossref PubMed Google Scholar]. Although specific sequence and chromatin features recently emerged as regulators of replication origin activity, in metazoans, flexibility in origin usage couples DNA replication and genome structure and function (e.g., transcriptional programs) [115.Fragkos M. et al.DNA replication origin activation in space and time.Nat. Rev. Mol. Cell Biol. 2015; 16: 360-374Crossref PubMed Google Scholar]. In addition to this, a series of chromatin marks, including histone acetylation and methylation, have been associated with PRE-RC activity and/or origin activation [115.Fragkos M. et al.DNA replication origin activation in space and time.Nat. Rev. Mol. Cell Biol. 2015; 16: 360-374Crossref PubMed Google Scholar]. Finally, replication origins are activated in a temporally regulated manner to prevent exhaustion of replication factors and dNTPs and allow accurate and complete DNA replication before transition into G2/M phase [115.Fragkos M. et al.DNA replication origin activation in space and time.Nat. Rev. Mol. Cell Biol. 2015; 16: 360-374Crossref PubMed Google Scholar]. Sophisticated mechanisms have evolved to control the major steps of DNA replication, from PRE-RC complex formation and replication origin activation to replisome activity and replication fork stability. Defects in any one of these mechanisms can significantly impact genome stability and human development and are associated with congenital human disease. Regulation of licensing of DNA replication origins is particularly crucial for the maintenance of genome integrity. Accordingly, loading of MCM2–7 on chromatin is limited to the G1 phase of the cell cycle by several mechanisms that control overall levels and/or localisation of the licensing factors ORC1–6, CDC6, and CDT1 [110.Arias E.E. Walter J.C. Strength in numbers: preventing rereplication via multiple mechanisms in eukaryotic cells.Genes Dev. 2007; 21: 497-518Crossref PubMed Scopus (309) Google Scholar]. Early studies in budding yeast defined two separate phases of the cell cycle, characterised by low or high CDK activity associated with licensing or activation of DNA replication origins in a mutually exclusive manner. CDK activity, while promoting activation of DNA replication origins, restrains PRE-RC complex formation and origin re-licensing through inhibitory phosphorylation [111.Siddiqui K. et al.Regulating DNA replication in eukarya.Cold Spring Harb. Perspect. Biol. 2013; 5a012930Crossref PubMed Scopus (138) Google Scholar]. In mammalian cells, several CDK-independent mechanisms have also emerged, targeting the licensing factor CDT1 [110.Arias E.E. Walter J.C. Strength in numbers: preventing rereplication via multiple mechanisms in eukaryotic cells.Genes Dev. 2007; 21: 497-518Crossref PubMed Scopus (309) Google Scholar]. Thus, in addition to CDK-dependent phosphorylation and SKP2-mediated ubiquitylation/degradation, the CRL4-CDT2 ubiquitin ligase targets CDT1 for replication-coupled destruction in a PCNA-dependent manner [112.Jin J. et al.A family of diverse Cul4-Ddb1-interacting proteins includes Cdt2, which is required for S phase destruction of the replication factor Cdt1.Mol. Cell. 2006; 23: 709-721Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar]. Furthermore, CDT1 association with the S phase–specific inhibitor, GEMININ, inhibits PRE-RC assembly by sequestering CDT1 in an inactive complex that is unable to interact with or recruit MCM2–7 [69.McGarry T.J. Kirschner M.W. Geminin, an inhibitor of DNA replication, is degraded during mitosis.Cell. 1998; 93: 1043-1053Abstract Full Text Full Text PDF PubMed Scopus (684) Google Scholar,70.Wohlschlegel J.A. et al.Inhibition of eukaryotic DNA replication by geminin binding to Cdt1.Science. 2000; 290: 2309-2312Crossref PubMed Scopus (548) Google Scholar] (Figure 1). Importantly, during the G1 phase an excess of MCM2–7 double hexamers are loaded onto genomic DNA. Known as the MCM paradox, this phenomenon has been associated with the presence of accessory or 'dormant' replication origins. Dormant origins can be activated under conditions of replicative stress [113.Ge X.Q. et al.Dormant origins licensed by excess Mcm2-7 are required for human cells to survive replicative stress.Genes Dev. 2007; 21: 3331-3341Crossref PubMed Scopus (360) Google Scholar,114.Ibarra A. et al.Excess MCM proteins protect human cells from replicative stress by licensing backup origins of replication.Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 8956-8961Crossref PubMed Scopus (281) Google Scholar] in order to rescue replication fork stalling and facilitate completion of genome-wide replication. Initiation of DNA replication, upon DDK and CDK activation, takes place at thousands of genomic sites in a strictly regulated manner [115.Fragkos M. et al.DNA replication origin activation in space and time.Nat. Rev. Mol. Cell Biol. 2015; 16: 360-374Crossref PubMed Google Scholar]. Although specific sequence and chromatin features recently emerged as regulators of replication origin activity, in metazoans, flexibility in origin usage couples DNA replication and genome structure and function (e.g., transcriptional programs) [115.Fragkos M. et al.DNA replication origin activation in space and time.Nat. Rev. Mol. Cell Biol. 2015; 16: 360-374Crossref PubMed Google Scholar]. In addition to this, a series of chromatin marks, including histone acetylation and methylation, have been associated with PRE-RC activity and/or origin activation [115.Fragkos M. et al.DNA replication origin activation in space and time.Nat. Rev. Mol. Cell Biol. 2015; 16: 360-374Crossref PubMed Google Scholar]. Finally, replication origins are activated in a temporally regulated manner to prevent exhaustion of replication factors and dNTPs and allow accurate and complete DNA replication before transition into G2/M phase [115.Fragkos M. et al.DNA replication origin activation in space and time.Nat. Rev. Mol. Cell Biol. 2015; 16: 360-374Crossref PubMed Google Scholar]. At the G1–S transition, a series of Dbf4-dependent kinase (DDK) and cyclin-dependent kinase (CDK) phosphorylation events drive assembly of the eukaryotic processive helicase CMG, which comprises CDC45, MCM2–7, and the Go-Ichi-Ni-San (GINS) complex (composed of PSF1, PSF2, PSF3, and SLD5; also known as 'GINS1–4' in mammals [5.Moyer S.E. et al.Isolation of the Cdc45/Mcm2-7/GINS (CMG) complex, a candidate for the eukaryotic DNA replication fork helicase.Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 10236-10241Crossref PubMed Scopus (501) Google Scholar]). While MCMs represent the key targets of DDK activity, CDKs phosphorylate the essential pre-initiation factors TopBP1-interacting checkpoint and replication regulator (TICRR)/TRESLIN and RECQ-like helicase 4 (RECQL4; Sld3 and Sld2 in budding yeast), allowing BRCT-dependent binding to DNA topoisomerase II–binding protein 1 (TOPBP1; Dpb11 in Saccharomyces cerevisiae) and recruitment of CDC45 and GINS1–4 in concert with DNA polymerase epsilon (Pol ε) [6.Zegerman P. Diffley J.F.X. Phosphorylation of Sld2 and Sld3 by cyclin-dependent kinases promotes DNA replication in budding yeast.Nature. 2007; 445: 281-285Crossref PubMed Scopus (352) Google Scholar, 7.Tanaka S. et al.CDK-dependent phosphorylation of Sld2 and Sld3 initiates DNA replication in budding yeast.Nature. 2007; 445: 328-332Crossref PubMed Scopus (324) Google Scholar, 8.Kumagai A. et al.Treslin collaborates with TopBP1 in triggering the initiation of DNA replication.Cell. 2010; 140: 349-359Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 9.Sangrithi M.N. et al.Initiation of DNA replication requires the RECQL4 protein mutated in Rothmund-Thomson syndrome.Cell. 2005; 121: 887-898Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 10.Muramatsu S. et al.CDK-dependent complex formation between replication proteins Dpb11, Sld2, Pol ε, and GINS in budding yeast.Genes Dev. 2010; 24: 602-612Crossref PubMed Scopus (0) Google Scholar]. In addition to this, MDM two binding protein (MTBP; the ortholog of S. cerevisiae Sld7) interacts with TICRR/TRESLIN to promote CDC45 binding to MCMs and CMG assembly [11.Boos D. et al.Identification of a heteromeric complex that promotes DNA replication origin firing in human cells.Science. 2013; 340: 981-984Crossref PubMed Scopus (50) Google Scholar]. All together, these proteins form the so-called pre-initiation complex (PRE-IC) [1.Bell S.P. Labib K. Chromosome duplication in Saccharomyces cerevisiae.Genetics. 2016; 203: 1027-1067Crossref PubMed Google Scholar] (Figure 1). PRE-IC engagement with MCM10 triggers double-stranded DNA (dsDNA) melting and unwinding by the CMG. Replication protein A (RPA) is recruited to the resulting single-stranded DNA (ssDNA), and two separate replisomes are established, which translocate along ssDNA in a 3′–5′ direction [12.Douglas M.E. et al.The mechanism of eukaryotic CMG helicase activation.Nature. 2018; 555: 265-268Crossref PubMed Scopus (62) Google Scholar,13.Fu Y.V. et al.Selective bypass of a lagging strand roadblock by the eukaryotic replicative DNA helicase.Cell. 2011; 146: 931-941Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar] (Figure 1). After POLα-dependent synthesis of short RNA-DNA primers, leading and lagging strands are extended by the conserved polymerases POLε and POLδ [14.Burgers P.M.J. Kunkel T.A. Eukaryotic DNA replication fork.Annu. Rev. Biochem. 2017; 86: 417-438Crossref PubMed Scopus (148) Google Scholar] (Figure 2). In vitro reconstitution of DNA replication, as well as genetic and proteomic evidence in vivo have established that, in unchallenged conditions, POLε synthesises the majority of leading strand whereas POLδ is responsible for lagging strand extension [14.Burgers P.M.J. Kunkel T.A. Eukaryotic DNA replication fork.Annu. Rev. Biochem. 2017; 86: 417-438Crossref PubMed Scopus (148) Google Scholar]. Importantly, while POLδ is indirectly tethered to the replication fork, POLα appears to be physically coupled to the CMG by the AND-1/CTF4 trimer, a 'hub' that links multiple CTF4-interacting peptide (CIP) box–containing proteins to the CMG helicase [15.Villa F. et al.Ctf4 is a hub in the eukaryotic replisome that links multiple CIP-box proteins to the CMG helicase.Mol. Cell. 2016; 63: 385-396Abstract Full Text Full Text PDF PubMed Google Scholar] (Figure 2). Other essential components of the replication machinery are the replication factors C1–5 (RFC1–5) and chromosome transmission fidelity 18 (CTF18)–RFC2–5 complexes (or clamp loaders), which promote loading of the processive polymerase factor proliferating cell nuclear antigen (PCNA), a trimeric scaffold that encircles ssDNA–dsDNA junctions and promotes efficient synthesis on lagging and leading strands [14.Burgers P.M.J. Kunkel T.A. Eukaryotic DNA replication fork.Annu. Rev. Biochem. 2017; 86: 417-438Crossref PubMed Scopus (148) Google Scholar] (Figure 2). Upon extension of lagging-strand synthesis by POLδ, 5′ flap structures are generated that are processed by flap structure–specific endonuclease 1 (FEN1) or DNA replication helicase/nuclease 2 (DNA2; long flaps) before ligase 1–mediated ligation of end products [14.Burgers P.M.J. Kunkel T.A. Eukaryotic DNA replication fork.Annu. Rev. Biochem. 2017; 86: 417-438Crossref PubMed Scopus (148) Google Scholar] (Figure 2). In addition to this, CLASPIN (Mrc1 in S. cerevisiae) and the TIPIN/TIMELESS heterodimer (homologues of Csm3/Tof1) engage with the replisome to promote efficient DNA replication in unperturbed and perturbed conditions [1.Bell S.P. Labib K. Chromosome duplication in Saccharomyces cerevisiae.Genetics. 2016; 203: 1027-1067Crossref PubMed Google Scholar] (Figure 2). Finally, termination of DNA replication occurs stochastically in the genome upon replication fork convergence, which triggers ubiquitylation-dependent CMG disassembly and ligation of end products (Box 2).Box 2Termination of DNA ReplicationAssembly of the CMG helicase and initiation of DNA replication have been studied extensively. However, less is known about the mechanisms that regulate convergence of replication forks and CMG disassembly at termination and the consequence of their deregulation [1.Bell S.P. Labib K. Chromosome duplication in Saccharomyces cerevisiae.Genetics. 2016; 203: 1027-1067Crossref PubMed Google Scholar]. The CMG helicase cannot be reloaded during S phase and appears to be remarkably stable even in conditions of replication stress [1.Bell S.P. Labib K. Chromosome duplication in Saccharomyces cerevisiae.Genetics. 2016; 203: 1027-1067Crossref PubMed Google Scholar]. Thus, its disassembly upon fork convergence must be regulated in a specific manner. Initial insights into this process came from the discovery of a dedicated ubiquitylation-dependent mechanism requiring the SCFDia2 ubiquitin ligase in budding yeast and Cdc48/p97 AAA+ ATPase in S. cerevisiae and Xenopus laevis, driving the disassembly of CMG helicase through ubiquitylation of its MCM7 subunit [116.Maric M. et al.Cdc48 and a ubiquitin ligase drive disassembly of the CMG helicase at the end of DNA replication.Science. 2014; 3461253596Crossref PubMed Scopus (117) Google Scholar,117.Moreno S.P. et al.Polyubiquitylation drives replisome disassembly at the termination of DNA replication.Science. 2014; 346: 477-481Crossref PubMed Scopus (102) Google Scholar]. Subsequent studies identified CUL-2LRR-1 as the higher eukaryotic mediator of CMG ubiquitylation in conjunction with the CDC-48 cofactors UFD-1 and NPL-4, which are required for its disassembly [118.Maric M. et al.Ufd1-Npl4 Recruit Cdc48 for Disassembly of Ubiquitylated CMG Helicase at the End of Chromosome Replication.Cell Rep. 2017; 18: 3033-3042Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 119.Sonneville R. et al.CUL-2LRR-1 and UBXN-3 drive replisome disassembly during DNA replication termination and mitosis.Nat. Cell Biol. 2017; 19: 468-479Crossref PubMed Scopus (33) Google Scholar, 120.Dewar J.M. et al.CRL2Lrr1 promotes unloading of the vertebrate replisome from chromatin during replication termination.Genes Dev. 2017; 31: 275-290Crossref PubMed Scopus (41) Google Scholar]. Importantly, CMG unloading at termination sites occurs after the formation of fully ligated DNA products during in vitro replication in X. laevis egg extracts, which suggested that a specific conformational change must occur to promote MCM7 ubiquitylation and p97-dependent unfolding and disassembly of the CMG [121.Dewar J.M. et al.The mechanism of DNA replication termination in vertebrates.Nature. 2015; 525: 345-350Crossref PubMed Google Scholar]. Consistent with this, in vitro reconstitution with purified budding yeast proteins recently showed that MCM7 ubiquitylation is normally repressed throughout fork elongation by the Y-shaped DNA structure of the replication fork itself, which is removed upon fork convergence and end-product ligation [122.Deegan T.D. et al.CMG helicase disassembly is controlled by replication fork DNA, replisome components and a ubiquitin threshold.Elife. 2020; 9e60371Crossref PubMed Google Scholar]. Efficient fork convergence and termination also necessitate activity of topoisomerase II and the Pif1 and Rrm3 DNA helicases [123.Deegan T.D. et al.Pif1-Family Helicases Support Fork Convergence during DNA Replication Termination in Eukaryotes.Mol. Cell. 2019; 74: 231-244.e9Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar]. In addition to CUL2LLR1, a mitotic backup pathway has recently been discovered that involves the ubiquitin ligase TRAIP [124.Deng L. et al.Mitotic CDK Promotes Replisome Disassembly, Fork Breakage, and Complex DNA Rearrangements.Mol. Cell. 2019; 73: 915-929.e6Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar,125.Priego Moreno S. et al.Mitotic replisome disassembly depends on TRAIP ubiquitin ligase activity.Life Sci. Alliance. 2019; 2e201900390Crossref PubMed Scopus (14) Google Scholar], which is also required for replisome disassembly at converged replication forks during interstrand crosslink repair in X. laevis egg extracts [95.Wu R.A. et al.TRAIP is a master regulator of DNA interstrand crosslink repair.Nature. 2019; 567: 267-272Crossref PubMed Scopus (45) Google Scholar] and to allow mitotic DNA synthesis (MiDAS) and rescue of under-replicated DNA in mammalian cells [98.Sonneville R. et al.TRAIP drives replisome disassembly and mitotic DNA repair synthesis at sites of incomplete DNA replication.Elife. 2019; 8e48686Crossref PubMed Scopus (16) Google Scholar]. All together, these studies point to an essential role for regulated CMG disassembly in genome stability and human health; yet, the consequences of its dysfunction are just starting to be unveiled. Assembly of the CMG helicase and initiation of DNA replication have been studied extensively. However, less is known about the mechanisms that regulate convergence of replication forks and CMG disassembly at termination and the consequence of their deregulation [1.Bell S.P. Labib K. Chromosome duplication in Saccharomyces cerevisiae.Genetics. 2016; 203: 1027-1067Crossref PubMed Google Scholar]. The CMG helicase cannot be reloaded during S phase and appears to be remarkably stable even in conditions of replication stress [1.Bell S.P. Labib K. Chromosome duplication in Saccharomyces cerevisiae.Genetics. 2016; 203: 1027-1067Crossref PubMed Google Scholar]. Thus, its disassembly upon fork convergence must be regulated in a specific manner. Initial insights into this process came from the discovery of a dedicated ubiquitylation-dependent mechanism requiring the SCFDia2 ubiquitin ligase in budding yeast and Cdc48/p97 AAA+ ATPase in S. cerevisiae and Xenopus laevis, driving the disassembly of CMG helicase through ubiquitylation of its MCM7 subunit [116.Maric M. et al.Cdc48 and a ubiquitin ligase drive disassembly of the CMG helicase at the end of DNA replication.Science. 2014; 3461253596Crossref PubMed Scopus (117) Google Scholar,117.Moreno S.P. et al.Polyubiquitylation drives replisome disassembly at the termination of DNA replication.Science. 2014; 346: 477-481Crossref PubMed Scopus (102) Google Scholar]. Subsequent studies identified CUL-2LRR-1 as the higher eukaryotic mediator of CMG ubiquitylation in conjunction with the CDC-48 cofactors UFD-1 and NPL-4, which are required for its disassembly [118.Maric M. et al.Ufd1-Npl4 Recruit Cdc48 for Disassembly of Ubiquitylated CMG Helicase at the End of Chromosome Replication.Cell Rep. 2017; 18: 3033-3042Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 119.Sonneville R. et al.CUL-2LRR-1 and UBXN-3 drive replisome disassembly during DNA replication termination and mitosis.Nat. Cell Biol. 2017; 19: 468-479Crossref PubMed Scopus (33) Google Scholar, 120.Dewar J.M. et al.CRL2Lrr1 promotes unloading of the vertebrate replisome from chromatin during replication termination.Genes Dev. 2017; 31: 275-290Crossref PubMed Scopus (41) Google Scholar]. Importantly, CMG unloading at termination sites occurs after the formation of fully ligated DNA products during in vitro replication in X. laevis egg extracts, which suggested that a specific conformational change must occur to promote MCM7 ubiquitylation and p97-dependent unfolding and disassembly of the CMG [121.Dewar J.M. et al.The mechanism of DNA replication termination in vertebrates.Nature. 2015; 525: 345-350Crossref PubMed Google Scholar]. Consistent with this, in vitro reconstitution with purified budding yeast proteins recently showed that MCM7 ubiquitylation is normally repressed throughout fork elongation by the Y-shaped DNA structure of the replication fork itself, which is removed upon fork convergence and end-product ligation [122.Deegan T.D. et al.CMG helicase disassembly is controlled by replication fork DNA, replisome components and a ubiquitin threshold.Elife. 2020; 9e60371Crossref PubMed Google Scholar]. Efficient fork convergence and termination also necessitate activity of topoisomerase II and the Pif1 and Rrm3 DNA helicases [123.Deegan T.D. et al.Pif1-Family Helicases Support Fork Convergence during DNA Replication Termination in Eukaryotes.Mol. Cell. 2019; 74: 231-244.e9Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar]. In addition to CUL2LLR1, a mitotic backup pathway