Biochemical Characterization of the Methanothermobacter thermautotrophicus Minichromosome Maintenance (MCM) Helicase N-terminal Domains

Kasiviswanathan, Rajesh; Shin, Jae‐Ho; Melamud, Eugene; Kelman, Zvi

doi:10.1074/jbc.m403202200

Cited by 56 publications

(79 citation statements)

References 36 publications

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“…A very similar static threefold symmetric ring is observed for the Nterminal domains of the DnaB homolog G40P [50]. Notably, the N-terminal domain of an archaeal MCM protein forms a nearly sixfold symmetric ring that is crucial for hexamerization of this complex [47,51]. This ring is formed by an OB-fold, which is interesting as this is the proposed exit side of the helicase [52], and this region could conceivably play a role in interacting with extruded single-stranded DNA.…”

Section: Intersubunit Interactionsmentioning

confidence: 53%

“…In the case of T7gp4, oligomerization is mediated by an extended 'tail' that appends the primase domain and sits on the adjacent subunit [36 ]. Oligomerization of DnaB, the SF3 helicases [46], and MCM proteins [47] appears to derive from a second domain that is static and also forms tight and extensive interactions with adjacent subunits through apparently inflexible interfaces. These properties classify this region as a 'collar' similar to that described earlier in the case of clamp-loaders [48,49].…”

Section: Intersubunit Interactionsmentioning

confidence: 99%

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On helicases and other motor proteins

Enemark

Joshua‐Tor

2008

Current Opinion in Structural Biology

192

238

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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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Section: Intersubunit Interactionsmentioning

confidence: 53%

Section: Intersubunit Interactionsmentioning

confidence: 99%

On helicases and other motor proteins

Enemark

Joshua‐Tor

2008

Current Opinion in Structural Biology

192

238

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“…As is typical for DNA helicases, but in contrast to Mcm2-7 (22,58,218), ATP hydrolysis is stimulated by the addition of either ssDNA or dsDNA (42,125,174).…”

Section: Atp Hydrolysis and Allosteric Interactionsmentioning

confidence: 99%

The Mcm Complex: Unwinding the Mechanism of a Replicative Helicase

Bochman

Schwacha

2009

Microbiol Mol Biol Rev

294

278

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SUMMARY The Mcm2-7 complex serves as the eukaryotic replicative helicase, the molecular motor that both unwinds duplex DNA and powers fork progression during DNA replication. Consistent with its central role in this process, much prior work has illustrated that Mcm2-7 loading and activation are landmark events in the regulation of DNA replication. Unlike any other hexameric helicase, Mcm2-7 is composed of six unique and essential subunits. Although the unusual oligomeric nature of this complex has long hampered biochemical investigations, recent advances with both the eukaryotic as well as the simpler archaeal Mcm complexes provide mechanistic insight into their function. In contrast to better-studied homohexameric helicases, evidence suggests that the six Mcm2-7 complex ATPase active sites are functionally distinct and are likely specialized to accommodate the regulatory constraints of the eukaryotic process.

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“…Biochemical studies with the various Mcm protein complexes in yeast and mammals have shown that a dimeric complex of Mcm467 heterotrimer contains 3Ј 3 5Ј helicase, ssDNA binding, and DNA-dependent ATPase activities, whereas its interaction with either Mcm2 or Mcm3-5 inhibit the helicase activity (19,22,62). The Mcm complex is believed to function as a replicative helicase of Eukaryote and Archaea (18,19).…”

Section: Figmentioning

confidence: 99%

Modulation of DNA Synthesis in Saccharomyces cerevisiae Nuclear Extract by DNA Polymerases and the Origin Recognition Complex

Mitkova

Biswas-Fiss

Biswas

2005

Journal of Biological Chemistry

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We have analyzed the modulation of DNA synthesis on a supercoiled plasmid DNA template by DNA polymerases (pol), minichromosome maintenance protein complex (Mcm), topoisomerases, and the origin recognition complex (ORC) using an in vitro assay system. Antisera specific against the four-subunit pol ␣, the catalytic subunit of pol ␦, and the Mcm467 complex each inhibited DNA synthesis. However, DNA synthesis in this system appeared to be independent of pol⑀. Consequently, DNA synthesis in the in vitro system appeared to depend only on two polymerases, ␣ and ␦, as well as the Mcm467 DNA helicase. This system requires supercoiled plasmid DNA template and DNA synthesis absolutely required DNA topoisomerase I. In addition, we also report here a novel finding that purified recombinant six subunit ORC significantly stimulated the DNA synthesis on a supercoiled plasmid DNA template containing an autonomously replicating sequence, ARS1.Studies on the replication of genomes of bacteriophages, plasmids, viruses, and prokaryotic and eukaryotic cells have established general mechanisms of DNA replication (1). The eukaryotic origins of DNA replication were first discovered in the budding yeast, Saccharomyces cerevisiae, and were named autonomously replicating sequences (ARS) 1 (2-4). Many yeast ARS elements have been characterized in detail, which has allowed the discovery of many cis-acting replication proteins and regulators of DNA replication (5, 6). The discovery by Bell and Stillman (7,8) that the in vivo initiation of DNA replication in S. cerevisiae requires a six subunit origin recognition complex (ORC) is a major advancement in understanding the mechanism of eukaryotic DNA replication. In the G 1 phase of the cell cycle, Cdc6p, Cdt1p, minichromosome maintenance (Mcm), and Cdc45p proteins bind sequentially to DNA to form a prereplication complex (pre-RC) (9 -11). At the G 1 /S boundary, S phase-specific cyclin-dependent protein kinases (Cdks) and particularly the Cdc7/Dbf4 kinase transform the pre-RC into an active replication complex (10 -12). Finally, the initiation coincides with the association of the polymerase ␣/primase (pol ␣/primase) complex to the RPA-coated unwound origins (10). S and M phase-specific Cdks block the re-binding of Mcm to the chromatin and prevent a new round of initiations until mitosis (13).The Mcm protein family that is required for DNA replication in eukaryotes consists of six proteins (Mcm 2-7) with highly conserved amino acids between the six polypeptides. All of these proteins are essential for the cell viability (13, 14). The Mcm467 proteins form a relatively stable core complex and other Mcm proteins are loosely associated to it (14 -17). It has been demonstrated that a DNA helicase activity is associated with the Mcm467 complex, suggesting that this complex is involved in the initiation of DNA replication as a DNA unwinding enzyme (18,19). Furthermore, Mcm467 remains associated with the replication fork during the elongation step of DNA replication (20). Among a number of candidate D...

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Biochemical Characterization of the Methanothermobacter thermautotrophicus Minichromosome Maintenance (MCM) Helicase N-terminal Domains

Cited by 56 publications

References 36 publications

On helicases and other motor proteins

On helicases and other motor proteins

The Mcm Complex: Unwinding the Mechanism of a Replicative Helicase

Modulation of DNA Synthesis in Saccharomyces cerevisiae Nuclear Extract by DNA Polymerases and the Origin Recognition Complex

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