An intact Mcm10 coiled-coil interaction surface is important for origin melting, helicase assembly and the recruitment of Pol-α to Mcm2-7.

Mcm10 is an essential eukaryotic factor required for DNA replication. The replication fork helicase is composed of Cdc45, Mcm2-7 and GINS (CMG). DDK is an S-phase-specific kinase required for replication initiation, and the DNA primase-polymerase in eukaryotes is pol α.

Dpb11 may function with RPA and DNA to initiate DNA replication.

Dpb11 is required for the initiation of DNA replication in budding yeast. We found that Dpb11 binds tightly to single-stranded DNA (ssDNA) or branched DNA structures, while its human homolog, TopBP1, binds tightly to branched-DNA structures. We also found that Dpb11 binds stably to CDK-phosphorylated RPA, the eukaryotic ssDNA binding protein, in the presence of branched DNA.

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

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.

Origin DNA Melting-An Essential Process with Divergent Mechanisms.

Origin DNA melting is an essential process in the various domains of life. The replication fork helicase unwinds DNA ahead of the replication fork, providing single-stranded DNA templates for the replicative polymerases. The replication fork helicase is a ring shaped-assembly that unwinds DNA by a steric exclusion mechanism in most DNA replication systems.

A Positive Amplification Mechanism Involving a Kinase and Replication Initiation Factor Helps Assemble the Replication Fork Helicase.

The assembly of the replication fork helicase during S phase is key to the initiation of DNA replication in eukaryotic cells. One step in this assembly in budding yeast is the association of Cdc45 with the Mcm2-7 heterohexameric ATPase, and a second step is the assembly of the tetrameric GINS (G-chi-ii-an) complex with Mcm2-7. Dbf4-dependent kinase (DDK) and S-phase cyclin-dependent kinase (S-CDK) are two S phase-specific kinases that phosphorylate replication proteins during S phase, and Dpb11, Sld2, Sld3, Pol ϵ, and Mcm10 are factors that are also required for replication initiation.

An Mcm10 Mutant Defective in ssDNA Binding Shows Defects in DNA Replication Initiation.

Mcm10 is an essential protein that functions to initiate DNA replication after the formation of the replication fork helicase. In this manuscript, we identified a budding yeast Mcm10 mutant (Mcm10-m2,3,4) that is defective in DNA binding in vitro. Moreover, this Mcm10-m2,3,4 mutant does not stimulate the phosphorylation of Mcm2 by Dbf4-dependent kinase (DDK) in vitro.

Insights into the Initiation of Eukaryotic DNA Replication.

The initiation of DNA replication is a highly regulated event in eukaryotic cells to ensure that the entire genome is copied once and only once during S phase. The primary target of cellular regulation of eukaryotic DNA replication initiation is the assembly and activation of the replication fork helicase, the 11-subunit assembly that unwinds DNA at a replication fork. The replication fork helicase, called CMG for Cdc45-Mcm2-7, and GINS, assembles in S phase from the constituent Cdc45, Mcm2-7, and GINS proteins.

Mcm10 coordinates the timely assembly and activation of the replication fork helicase.

Mcm10 is an essential replication factor that is required for DNA replication in eukaryotes. Two key steps in the initiation of DNA replication are the assembly and activation of Cdc45-Mcm2-7-GINS (CMG) replicative helicase. However, it is not known what coordinates helicase assembly with helicase activation.

The Replication Initiation Protein Sld3/Treslin Orchestrates the Assembly of the Replication Fork Helicase during S Phase.

The initiation of DNA replication is a highly regulated process in eukaryotic cells, and central to the process of initiation is the assembly and activation of the replication fork helicase. The replication fork helicase is comprised of CMG (Cdc45, Mcm2-7, and GINS) in eukaryotic cells, and the mechanism underlying assembly of the CMG during S phase was studied in this article. We identified a point mutation of Sld3 that is specifically defective for Mcm3 and Mcm5 interaction (sld3-m10), and also identified a point mutation of Sld3 that is specifically defective for single-stranded DNA (ssDNA) interaction (sld3-m9).

Conserved mechanism for coordinating replication fork helicase assembly with phosphorylation of the helicase.

Dbf4-dependent kinase (DDK) phosphorylates minichromosome maintenance 2 (Mcm2) during S phase in yeast, and Sld3 recruits cell division cycle 45 (Cdc45) to minichromosome maintenance 2-7 (Mcm2-7). We show here DDK-phosphoryled Mcm2 preferentially interacts with Cdc45 in vivo, and that Sld3 stimulates DDK phosphorylation of Mcm2 by 11-fold. We identified a mutation of the replication initiation factor Sld3, Sld3-m16, that is specifically defective in stimulating DDK phosphorylation of Mcm2.

Dpb11 protein helps control assembly of the Cdc45·Mcm2-7·GINS replication fork helicase.

Dpb11 is required for the initiation of DNA replication in budding yeast. Dpb11 binds to S-phase cyclin-dependent kinase-phosphorylated Sld2 and Sld3 to form a ternary complex during S phase. The replication fork helicase in eukaryotes is composed of Cdc45, Mcm2-7, and GINS.

The Dbf4-Cdc7 kinase promotes Mcm2-7 ring opening to allow for single-stranded DNA extrusion and helicase assembly.

The replication fork helicase in eukaryotes is composed of Cdc45, Mcm2-7, and GINS (CMG). The Dbf4-Cdc7 kinase phosphorylates Mcm2 in vitro, but the in vivo role for Dbf4-Cdc7 phosphorylation of Mcm2 is unclear. We find that budding yeast Dbf4-Cdc7 phosphorylates Mcm2 in vivo under normal conditions during S phase.

Impact of age-associated cyclopurine lesions on DNA repair helicases.

8,5' cyclopurine deoxynucleosides (cPu) are locally distorting DNA base lesions corrected by nucleotide excision repair (NER) and proposed to play a role in neurodegeneration prevalent in genetically defined Xeroderma pigmentosum (XP) patients. In the current study, purified recombinant helicases from different classifications based on sequence homology were examined for their ability to unwind partial duplex DNA substrates harboring a single site-specific cPu adduct. Superfamily (SF) 2 RecQ helicases (RECQ1, BLM, WRN, RecQ) were inhibited by cPu in the helicase translocating strand, whereas helicases from SF1 (UvrD) and SF4 (DnaB) tolerated cPu in either strand.

DNA sequences proximal to human mitochondrial DNA deletion breakpoints prevalent in human disease form G-quadruplexes, a class of DNA structures inefficiently unwound by the mitochondrial replicative Twinkle helicase.

Mitochondrial DNA deletions are prominent in human genetic disorders, cancer, and aging. It is thought that stalling of the mitochondrial replication machinery during DNA synthesis is a prominent source of mitochondrial genome instability; however, the precise molecular determinants of defective mitochondrial replication are not well understood. In this work, we performed a computational analysis of the human mitochondrial genome using the "Pattern Finder" G-quadruplex (G4) predictor algorithm to assess whether G4-forming sequences reside in close proximity (within 20 base pairs) to known mitochondrial DNA deletion breakpoints.

The replication initiation protein Sld2 regulates helicase assembly.

Assembly of the Cdc45-Mcm2-7-GINS (CMG) replicative helicase complex must be regulated to ensure that DNA unwinding is coupled with DNA synthesis. Sld2 is required for the initiation of DNA replication in budding yeast. We identified a mutant of Sld2, Sld2-m1,4, that is specifically defective in Mcm2-7 binding.

Cdc45 protein-single-stranded DNA interaction is important for stalling the helicase during replication stress.

Replicative polymerase stalling is coordinated with replicative helicase stalling in eukaryotes, but the mechanism underlying this coordination is not known. Cdc45 activates the Mcm2-7 helicase. We report here that Cdc45 from budding yeast binds tightly to long (≥ 40 nucleotides) genomic single-stranded DNA (ssDNA) and that 60mer ssDNA specifically disrupts the interaction between Cdc45 and Mcm2-7.

Single-molecule and bulk approaches to the DnaB replication fork helicase.

Motor proteins are enzymes that accomplish mechanical work in a wide variety of biological processes. In this review we focus on bulk and single molecule methods to study how motor proteins function. We discuss in detail the analysis of the motor protein DnaB, a hexameric helicase that unwinds DNA at a replication fork in Gram-negative bacteria.

DNA repair and replication fork helicases are differentially affected by alkyl phosphotriester lesion.

DNA helicases are directly responsible for catalytically unwinding duplex DNA in an ATP-dependent and directionally specific manner and play essential roles in cellular nucleic acid metabolism. It has been conventionally thought that DNA helicases are inhibited by bulky covalent DNA adducts in a strand-specific manner. However, the effects of highly stable alkyl phosphotriester (PTE) lesions that are induced by chemical mutagens and refractory to DNA repair have not been previously studied for their effects on helicases.

Enabling association of the GINS protein tetramer with the mini chromosome maintenance (Mcm)2-7 protein complex by phosphorylated Sld2 protein and single-stranded origin DNA.

The Cdc45-Mcm2-7-GINS (CMG) complex is the replication fork helicase in eukaryotes. Synthetic lethal with Dpb11-1 (Sld2) is required for the initiation of DNA replication, and the S phase cyclin-dependent kinase (S-CDK) phosphorylates Sld2 in vivo. We purified components of the replication initiation machinery and studied their interactions in vitro.

Origin single-stranded DNA releases Sld3 protein from the Mcm2-7 complex, allowing the GINS tetramer to bind the Mcm2-7 complex.

The replication fork helicase in eukaryotic cells is comprised of Cdc45, Mcm2-7, and GINS (CMG complex). In budding yeast, Sld3, Sld2, and Dpb11 are required for the initiation of DNA replication, but Sld3 and Dpb11 do not travel with the replication fork. Sld3 and Cdc45 bind to early replication origins during the G(1) phase of the cell cycle, whereas Sld2, GINS, polymerase ε, and Dpb11 form a transient preloading complex that associates with origins during S phase.

GINS and Sld3 compete with one another for Mcm2-7 and Cdc45 binding.

Sld3 is essential for the initiation of DNA replication, but Sld3 does not travel with a replication fork. GINS binds to Cdc45 and Mcm2-7 to form the replication fork helicase in eukaryotes. We purified Sld3, Cdc45, GINS, and Mcm2-7 and studied their interaction and assembly into complexes.

Sld2 binds to origin single-stranded DNA and stimulates DNA annealing.

Sld2 is essential for the initiation of DNA replication, but the mechanism underlying its role in replication is not fully understood. The S-phase cyclin dependent kinase (S-CDK) triggers the association of Sld2 with Dpb11, and a phosphomimetic mutation of Sld2, Sld2T84D, functionally mimics the S-CDK phosphorylated state of Sld2. We report that Sld2T84D binds directly to the single-stranded (ss) DNA of two different origins of replication, and S-CDK phosphorylation of Sld2 stimulates the binding of Sld2 to origin ssDNA.