Reconstitution of the Mcm2-7p Heterohexamer, Subunit Arrangement, and ATP Site Architecture

Helicases are molecular machines that utilize energy derived from ATP hydrolysis to move along nucleic acids and to separate base-paired nucleotides. The movement of the helicase can also be described as a stationary helicase that pumps nucleic acid. Recent structural data for the hexameric E1 helicase of papillomavirus in complex with single-stranded DNA and MgADP has provided a detailed atomic and mechanistic picture of its ATP-driven DNA translocation. The structural and mechanistic features of this helicase are compared with the hexameric helicase prototypes T7gp4 and SV40 T-antigen. The ATP-binding site architectures of these proteins are structurally similar to the sites of other prototypical ATP-driven motors such as F1-ATPase, suggesting related roles for the individual site residues in the ATPase activity. IntroductionHelicases are essential enzymes that unwind duplex DNA, RNA, or DNA-RNA hybrids. This unwinding is driven by consumption of input energy that is harnessed to separate base-paired oligonucleotides and also to maintain a unidirectional advancement of the helicase upon the nucleic acid substrate. This translocation can alternatively be described as an immobile helicase pumping nucleic acid. The energy for these transformations is derived from the hydrolysis of nucleotide triphosphate (NTP). Helicases can be depicted as an internal combustion engine with each individual NTPase site serving as one cylinder. Each individual cylinder follows a defined series of events: injection (ATP binding), compression (optimally positioning the site for hydrolysis), combustion (ATP hydrolysis/work generation), and exhaust (ADP and phosphate release). In a helicase, the individual combustion cylinders coordinate these actions to carry out the repetitive mechanical operation of prying open base pairs and/or actively translocating with respect to the nucleic acid substrate. Many other molecular motors utilize similar engines to carry out multiple diverse functions such as translocation of peptides in the case of ClpX, movement along cellular structures in the case of dyenin, and rotation about an axle as in F 1 -ATPase.Based upon conserved sequence motifs, helicases have been classified into six superfamilies [1,2 ]. An extensive review of these superfamilies has been provided recently [3 ]. Superfamily 1 (SF1) and superfamily 2 (SF2) helicases are very prevalent, generally monomeric, and participate in several diverse DNA and RNA manipulations. The other helicase superfamilies form hexameric rings (reviewed in [4]), as demonstrated by biochemistry [5][6][7][8] and electron microscopy studies [9][10][11][12][13][14][15][16][17], and often participate at the replication fork. All of these helicases bind and hydrolyze NTP at the interface between two recA-like domains. The binding site consists of a Walker A (P-loop) and a Walker B motif from the first domain and other elements such as an arginine finger from the other domain. The SF1 and SF2 helicases contain two recAlike domains coupled by a short linker, and t...

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“…and MCM3 appear to adopt positions on either side of MCM5 [58], all absent from the helicase-active MCM4/6/7 [59][60][61][62][63].…”

Section: Figurementioning

confidence: 98%

“…This position is occupied by the 'arginine finger' [18 ] in AAA+ proteins (SRC motif of clamp loaders [48] and SRF motif of MCM proteins [58]) and by b-R189 of F 1 -ATPase [44 ]. This position will be referred to as the 'piston' because of its ability to move in and out of the site.…”

Section: Atp Hydrolysis Permits Piston Departure From the Active Sitementioning

confidence: 99%

On helicases and other motor proteins

Enemark

Joshua‐Tor

2008

Current Opinion in Structural Biology

189

238

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“…The protein Mcm4 PK was purified, reconstituted into a complex with Mcm6 and Mcm7, and then radiolabeled to high specific activity using [γ 32 P]ATP and PKA. When Mcm4 PK /6/7 was applied to the gel-filtration column in the absence of ssDNA, the majority of the protein eluted in fractions [19][20][21][22][23][24][25][26][27][28][29] (Fig. 5a, triangles).…”

Section: Dna Binding By Mcm4/6/7mentioning

confidence: 99%

“…23,24 ATP hydrolysis by Mcm2 is dependent on Mcm6. 23,25 The ATP site of Mcm2/6 is located at the interface of the two proteins, with a conserved arginine residue from Mcm6 contributing to the hydrolysis of ATP bound to Mcm2 25 (Fig. 1a).…”

Section: Mutation Of the Mcm2/6 Interface Disrupts Atp Hydrolysismentioning

confidence: 99%

“…25,28 A study on Sulfolobus MCM has indicated that additional trans-acting residues, conserved in eukaryotic Mcm proteins, are important for the function of the helicase. 29 Furthermore, structural studies on viral SF3 helicases, E1 from papillomavirus and large T antigen (Tag) from simian virus 40 [also AAA + (ATPase associated with diverse cellular activities) proteins], illustrate that these trans-acting residues undergo conformational changes that are dependent on the nucleotide bound to the neighboring subunit.…”

Section: Atp Sites In MCM Proteinsmentioning

confidence: 99%

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ATP Binding and Hydrolysis by Mcm2 Regulate DNA Binding by Mcm Complexes

Stead

Sorbara

Brandl

et al. 2009

Journal of Molecular Biology

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The Eukaryotic CMG Helicase at the Replication Fork: Emerging Architecture Reveals an Unexpected Mechanism

O'Donnell

2018

BioEssays

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The eukaryotic helicase is an 11-subunit machine containing an Mcm2-7 motor ring that encircles DNA, Cdc45 and the GINS tetramer, referred to as CMG (Cdc45, Mcm2-7, GINS). CMG is "built" on DNA at origins in two steps. First, two Mcm2-7 rings are assembled around duplex DNA at origins in G1 phase, forming the Mcm2-7 "double hexamer." In a second step, in S phase Cdc45 and GINS are assembled onto each Mcm2-7 ring, hence producing two CMGs that ultimately form two replication forks that travel in opposite directions. Here, we review recent findings about CMG structure and function. The CMG unwinds the parental duplex and is also the organizing center of the replisome: it binds DNA polymerases and other factors. EM studies reveal a 20-subunit core replisome with the leading Pol ϵ and lagging Pol α-primase on opposite faces of CMG, forming a fundamentally asymmetric architecture. Structural studies of CMG at a replication fork reveal unexpected details of how CMG engages the DNA fork. The structures of CMG and the Mcm2-7 double hexamer on DNA suggest a completely unanticipated process for formation of bidirectional replication forks at origins.

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Reconstitution of the Mcm2-7p Heterohexamer, Subunit Arrangement, and ATP Site Architecture

Cited by 162 publications

References 46 publications

On helicases and other motor proteins

On helicases and other motor proteins

ATP Binding and Hydrolysis by Mcm2 Regulate DNA Binding by Mcm Complexes

The Eukaryotic CMG Helicase at the Replication Fork: Emerging Architecture Reveals an Unexpected Mechanism

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