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Eukaryotic Replicative Helicase Subunit Interaction with DNA and Its Role in DNA Replication

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Eukaryotic Replicative Helicase Subunit Interaction with DNA and Its Role in DNA Replication G C A T T A C G G C A T genes Review Eukaryotic Replicative Helicase Subunit Interaction with DNA and Its Role in DNA Replication Matthew P. Martinez, Amanda L. Wacker, Irina Bruck and Daniel L. Kaplan * Department of Biomedical Sciences, Florida State University College of Medicine, 1115 W. Call St., Tallahassee, FL 32306, USA; [email protected] (M.P.M.); [email protected] (A.L.W.); [email protected] (I.B.) * Correspondence: [email protected]; Tel.: +1-850-645-0237 Academic Editors: Linda Bloom and Jörg Bungert Received: 17 February 2017; Accepted: 31 March 2017; Published: 6 April 2017 Abstract: The replicative helicase unwinds parental double-stranded DNA at a replication fork to provide single-stranded DNA templates for the replicative polymerases. In eukaryotes, the replicative helicase is composed of the Cdc45 protein, the heterohexameric ring-shaped Mcm2-7 complex, and the tetrameric GINS complex (CMG). The CMG proteins bind directly to DNA, as demonstrated by experiments with purified proteins. The mechanism and function of these DNA-protein interactions are presently being investigated, and a number of important discoveries relating to how the helicase proteins interact with DNA have been reported recently. While some of the protein-DNA interactions directly relate to the unwinding function of the enzyme complex, other protein-DNA interactions may be important for minichromosome maintenance (MCM) loading, origin melting or replication stress. This review describes our current understanding of how the eukaryotic replicative helicase subunits interact with DNA structures in vitro, and proposed models for the in vivo functions of replicative helicase-DNA interactions are also described. Keywords: DNA replication; helicase; protein-DNA interaction; DNA unwinding; DNA melting; replication stress; MCM; Cdc45; GINS 1. Introduction to DNA Replication and Mcm2-7 During late M and G1 phases of the cell cycle, the Mcm2-7 complex is loaded as a double-hexamer to encircle double-stranded DNA at an origin (Figure1). The origin recognition complex (ORC), Cdc6, Cdt1, and ATP are required for this activity, which is also known as origin licensing. This reaction has been reconstituted with purified proteins, and recent biochemical and biophysical experiments reveal the detailed mechanism for Mcm2-7 loading [1]. During S phase, the S-phase cell cycle kinases, S-CDK (S-phase cyclin-dependent kinase) and DDK (Dbf4-dpendent kinase), function with the replication initiation factors, including Sld2, Sld3, Dpb11, Mcm10, Pol ", and Sld7, to assemble Mcm2-7 with Cdc45 and GINS. The Cdc45-Mcm2-7-GINS complex (CMG), fully assembled in S phase, is the replicative helicase in eukaryotes [2]. The CMG helicase unwinds DNA bidirectionally from a replication origin, the site of replication initiation. The motor region of the eukaryotic replicative helicase is comprised of the heterohexameric Mcm2-7 protein complex. Furthermore, Mcm2-7 alone has some weak in vitro helicase activity under certain conditions [3], but fully active helicase activity requires Mcm2-7 assembly with Cdc45 and GINS [4,5]. By analyzing the crystal structure of MCM (minichromosome maintenance complex) by itself and in complex with nucleotides, DNA, and as the CMG complex, mechanisms of origin melting and DNA unwinding by the Mcm2-7 complex can be further understood. With its role in origin melting and DNA unwinding becoming clearer, more advanced models for its mechanism of unwinding can be developed. Here, the structure of Mcm2-7 and its implications in origin melting are reviewed Genes 2017, 8, 117; doi:10.3390/genes8040117 www.mdpi.com/journal/genes Genes 2017, 8, 117 2 of 13 Genes 2017, 8, 117 2 of 13 basedare reviewed on models based from on archaeal, models Saccharomycesfrom archaeal, cerevisiae Saccharomyces, Drosophila cerevisiae melanogaster, Drosophila, and melanogaster human systems., and Crystallography,human systems. FRETCrystallography, (fluorescence FRET resonance (fluorescence energy resonance transfer), energy structure transfer), function, structure andfunction,in vivo experimentsand in vivo haveexperiments provided have much provided of what much is of known what is about known the about structure the structure and mechanism and mechanism of this heterohexamericof this heterohexameric (composed (composed of six different of six different subunits) subunits) protein protein complex. complex. FigureFigure 1. 1.Role Role of of Mcm2-7 Mcm2-7 in in DNA DNA replication.replication. In late late M M and and G1 G1 phases, phases, ORC, ORC, Cdc6, Cdc6, and and Cdt1 Cdt1 function function withwith ATP ATP to to load load Mcm2-7 Mcm2-7 as as a a double double hexamerichexameric ring surrounding double-stranded double-stranded DNA. DNA. Mcm2-7 Mcm2-7 doesdoes not not unwind unwind DNA DNA in in this this stage. stage. InIn SS phase,phase, cell cycle kinases kinases (S-CDK (S-CDK and and DDK) DDK) function function with with replicationreplication initiation initiation proteins proteins (Sld3, (Sld3, Sld2,Sld2, Dpb11,Dpb11, Sld7, Pol Pol ε," ,and and Mcm10) Mcm10) to to assemble assemble Mcm2-7 Mcm2-7 with with Cdc45Cdc45 and and GINS. GINS. The The CMG CMG complex complex isis the active replicative replicative helicase, helicase, unwinding unwinding DNA DNA bidirectionally bidirectionally fromfrom the the origin. origin. 2.2. DNA DNA Binding Binding Structures Structures of of Mcm2-7 Mcm2-7 Unlike previously studied homohexameric replicative helicases such as papilloma virus E1, Unlike previously studied homohexameric replicative helicases such as papilloma virus E1, SV40 SV40 Large T-antigen, archaeal MCM, and Escherichia coli DnaB [6], the eukaryotic Mcm2-7 helicase Large T-antigen, archaeal MCM, and Escherichia coli DnaB [6], the eukaryotic Mcm2-7 helicase forms forms a heterohexameric ring structure. The Mcm2-7 hexamer forms a double hexamer when loaded a heterohexameric ring structure. The Mcm2-7 hexamer forms a double hexamer when loaded onto onto origin DNA [7,8]. Mcm2-7 is a two-tiered ring comprised of the N-terminal domain (NTD), origin DNA [7,8]. Mcm2-7 is a two-tiered ring comprised of the N-terminal domain (NTD), required for required for double hexamer attachment, and a AAA+ C-terminal domain (CTD) that provides the doubleATPase hexamer activity attachment, of the helicase and a(Figure AAA+ 2) C-terminal (reviewed domain in [9]). (CTD)Both the that C-terminal provides the and ATPase N-terminal activity Genes 2017, 8, 117 3 of 13 Genes 2017, 8, 117 3 of 13 ofdomains the helicase of the (Figurering contain2) (reviewed DNA binding in [ 9]). region Both thes that C-terminal aid in DNA and unwinding N-terminal and domains are necessary of the ring cell containviability DNA [10–12] binding. regions that aid in DNA unwinding and are necessary cell viability [10–12]. While the N N-terminal-terminal domain does not provide any ATPase activity, it is composed of three subdomains that are necessary for DNA binding, double hexamer formation, and helicase activation (reviewed in [[2]2]):): a a largely largely helical helical subdomain subdomain A, A, Zn Zn-binding-binding motif subdomain B, B, and an OB (oligonucleotide/oligosaccharide-binding)-fold(oligonucleotide/oligosaccharide-binding)-fold subdomain C. Within thethe OB-fold,OB-fold, FroelichFroelich etet al.al. have identifiedidentified a region in archaeal MCM where two highly conserved a argininerginine residues, R124 R124 and and R186, R186, project from the β-barrel-barrel towards the interior of the MCM ring. They termed this region the MCM single-strandedsingle-stranded DNADNA bindingbinding motifmotif (MSSB) (MSSB) (Figure (Figure2). 2). They They have have demonstrated demonstrated that that these these residues residues of theof the MSSB MSSB are are necessary necessary for for helicase helicase loading loading and and activation activation [ 13[13]].. Additionally,Additionally, theythey havehave identifiedidentified these conserved residues in yeast (as either arginine or lysine) in Mcm4, Mcm6, and Mcm7, and have revealed how alanine mutations in these residues in at least two of the three MCM subunitssubunits show significantlysignificantly reducedreduced helicase helicase loading. loading. Further Further investigation investigation of the of N-terminal the N-terminal domain crystaldomain structure crystal hasstructure identified has identified a conserved a conserved loop in the loop OB-fold in the called OB- thefold Allosteric called the Communication Allosteric Communication Loop (ACL). ItLoop has been(ACL). suggested It has been that suggested the ACL that plays the aACL role plays in N- a androle C-terminalin N- and C domain-terminal communication, domain communication, with the ACLwith the positioned ACL positioned near a conserved near a conserved loop of theloop AAA+ of the domain, AAA+ domain, the pre-sensor-1- the pre-sensorβ-hairpin-1-β-hairpin (ps1β), of(ps1β), the adjacent of the adjacent subunit. subunit. The ACL The has ACL also has been also shown been shown to contact to contact the main-chain the main- amidechain amide atoms atoms of the helix-2-insetof the helix-2 (h2i)-inset within (h2i) within the same the subunit same subunit (Figure 2(Figure)[ 14]. This2) [14] interaction. This interaction was observed was observed in an inactive in an MCM,inactive revealing MCM, revealing a probable a probable function priorfunction to and prior during to and DNA during unwinding, DNA unwinding, discussed laterdiscussed on. In later fact, theon. In ACL fact, has the an ACL observed
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