Daniel L. Kaplan

DnaB Drives DNA Branch Migration and Dislodges Proteins While Encircling Two DNA Strands

DnaB is a ring-shaped, hexameric helicase that unwinds the E. coli DNA replication fork while encircling one DNA strand. This report demonstrates that DnaB can also encircle both DNA strands and then actively translocate along the duplex. With two strands positioned inside its central channel, DnaB translocates with sufficient force to displace proteins tightly bound to DNA with no resultant DNA unwinding. Thus, DnaB may clear proteins from chromosomal DNA. Furthermore, while encircling two DNA strands, DnaB can drive branch migration of a synthetic Holliday junction with heterologous duplex arms, suggesting that DnaB may be directly involved in DNA recombination in vivo. DnaB binds to just one DNA strand during branch migration. T7 phage gp4 protein also drives DNA branch migration, suggesting this activity generalizes to other ring-shaped helicases.

Dbf4-Cdc7 Phosphorylation of Mcm2 Is Required for Cell Growth

The Dbf4-Cdc7 kinase (DDK) is required for the activation of the origins of replication, and DDK phosphorylates Mcm2 in vitro. We find that budding yeast Cdc7 alone exists in solution as a weakly active multimer. Dbf4 forms a likely heterodimer with Cdc7, and this species phosphorylates Mcm2 with substantially higher specific activity. Dbf4 alone binds tightly to Mcm2, whereas Cdc7 alone binds weakly to Mcm2, suggesting that Dbf4 recruits Cdc7 to phosphorylate Mcm2. DDK phosphorylates two serine residues of Mcm2 near the N terminus of the protein, Ser-164 and Ser-170. Expression of mcm2-S170A is lethal to yeast cells that lack endogenous MCM2 (mcm2Δ); however, this lethality is rescued in cells harboring the DDK bypass mutant mcm5-bob1. We conclude that DDK phosphorylation of Mcm2 is required for cell growth.

Cdc45 Protein-Single-stranded DNA Interaction Is Important for Stalling the Helicase during Replication Stress

Background: Polymerase stalling is coupled with helicase stalling at a eukaryotic replication fork by an unknown mechanism. Results: When a Cdc45-ssDNA binding mutant is expressed in budding yeast cells exposed to hydroxyurea, there is uncoupling of the helicase from the polymerase. Conclusion: Cdc45-ssDNA interaction is important during replication stress. Significance: A new model explains how polymerase stalling is coupled with helicase stalling. 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. We identified a mutant of Cdc45 that does not bind to ssDNA. When this mutant of cdc45 is expressed in budding yeast cells exposed to hydroxyurea, cell growth is severely inhibited, and excess RPA accumulates at or near an origin. Chromatin immunoprecipitation suggests that helicase movement is uncoupled from polymerase movement for mutant cells exposed to hydroxyurea. These data suggest that Cdc45-ssDNA interaction is important for stalling the helicase during replication stress.

DnaB Helicase Activity Is Modulated by DNA Geometry and Force

The replicative helicase for Escherichia coli is DnaB, a hexameric, ring-shaped motor protein that encircles and translocates along ssDNA, unwinding dsDNA in advance of its motion. The microscopic mechanisms of DnaB are unknown; further, prior work has found that DnaBs activity is modified by other replication proteins, indicating some mechanistic flexibility. To investigate these issues, we quantified translocation and unwinding by single DnaB molecules in three tethered DNA geometries held under tension. Our data support the following conclusions: 1), Unwinding by DnaB is enhanced by force-induced destabilization of dsDNA. 2), The magnitude of this stimulation varies with the geometry of the tension applied to the DNA substrate, possibly due to interactions between the helicase and the occluded ssDNA strand. 3), DnaB unwinding and (to a lesser extent) translocation are interrupted by pauses, which are also dependent on force and DNA geometry. 4), DnaB moves slower when a large tension is applied to the helicase-bound strand, indicating that it must perform mechanical work to compact the strand against the applied force. Our results have implications for the molecular mechanisms of translocation and unwinding by DnaB and for the means of modulating DnaB activity.

DnaB from Thermus aquaticus Unwinds Forked Duplex DNA with an Asymmetric Tail Length Dependence

DnaB helicase is a ring-shaped hexamer of 300 kDa that is essential for replication of the bacterial chromosome. ThednaB gene from Thermus aquaticus was isolated and cloned, and its gene product was expressed and purified to homogeneity. A helicase assay was developed, and optimal conditions forT. aquaticus DnaB activity were determined using a forked duplex DNA substrate. The activity required a hydrolyzable nucleoside triphosphate and both 5′- and 3′-single-stranded DNA tail regions. Under conditions of single enzymatic turnover, the lengths of the 5′- and 3′-single-stranded regions were varied, and 6–10 nucleotides of the 5′-single-stranded tail and 21–30 nucleotides of the 3′-single-stranded tail markedly stimulated the unwinding rate. These data suggest that DnaB from T. aquaticus interacts with both DNA single-stranded tails during unwinding and that a greater portion of the 3′-tail is in contact with the protein. Two models are consistent with these data. In one model, the 5′-single stranded region passes through the central hole of the DnaB ring, and the 3′-tail makes extensive contact with the outside of the protein. In the other model, the 3′-single-stranded region passes through the DnaB ring, and the outside of the protein contacts the 5′-tail.

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 G1 phase of the cell cycle, whereas Sld2, GINS, polymerase ϵ, and Dpb11 form a transient preloading complex that associates with origins during S phase. We show here that Sld3 binds tightly to origin single-stranded DNA (ssDNA). CDK-phosphorylated Sld3 binds to origin ssDNA with similar high affinity. Origin ssDNA does not disrupt the interaction between Sld3 and Dpb11, and origin ssDNA does not disrupt the interaction between Sld3 and Cdc45. However, origin ssDNA substantially disrupts the interaction between Sld3 and Mcm2–7. GINS and Sld3 compete with one another for binding to Mcm2–7. However, in a mixture of Sld3, GINS, and Mcm2–7, origin ssDNA inhibits the interaction between Sld3 and Mcm2–7, whereas origin ssDNA promotes the association between GINS and Mcm2–7. We also show that origin single-stranded DNA promotes the formation of the CMG complex. We conclude that origin single-stranded DNA releases Sld3 from Mcm2–7, allowing GINS to bind Mcm2–7.

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.

Background: Mitochondrial DNA deletions are prominent in human genetic disorders and cancer. Results: Predicted mitochondrial G-quadruplex-forming sequences map in close proximity to known deletion breakpoints and form G-quadruplexes in vitro. Conclusion: The mitochondrial replicative helicase Twinkle inefficiently unwinds intra- and intermolecular G-quadruplexes. Significance: Mitochondrial G-quadruplexes are likely to cause genome instability by perturbing replication machinery. 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. We then used this information to map G4P sequences with deletions characteristic of representative mitochondrial genetic disorders and also those identified in various cancers and aging. Circular dichroism and UV spectral analysis demonstrated that mitochondrial G-rich sequences near deletion breakpoints prevalent in human disease form G-quadruplex DNA structures. A biochemical analysis of purified recombinant human Twinkle protein (gene product of c10orf2) showed that the mitochondrial replicative helicase inefficiently unwinds well characterized intermolecular and intramolecular G-quadruplex DNA substrates, as well as a unimolecular G4 substrate derived from a mitochondrial sequence that nests a deletion breakpoint described in human renal cell carcinoma. Although G4 has been implicated in the initiation of mitochondrial DNA replication, our current findings suggest that mitochondrial G-quadruplexes are also likely to be a source of instability for the mitochondrial genome by perturbing the normal progression of the mitochondrial replication machinery, including DNA unwinding by Twinkle helicase.

The Dbf4-Cdc7 Kinase Promotes Mcm2-7 Ring Opening to Allow for Single-stranded DNA Extrusion and Helicase Assembly

Background: The Dbf4-Cdc7 kinase activates DNA replication, and the helicase is composed of Cdc45, Mcm2-7, and GINS. Results: Dbf4-Cdc7 phosphorylation of Mcm2 is required in vivo for DNA replication, single-stranded DNA accumulation, and GINS-Mcm2-7 interaction. Conclusion: The Dbf4-Cdc7 kinase promotes Mcm2-7 ring opening to allow for origin melting and helicase assembly. Significance: A mechanism for Dbf4-Cdc7 action is described. 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. Inhibiting Dbf4-Cdc7 phosphorylation of Mcm2 confers a dominant-negative phenotype with a severe growth defect. Inhibiting Dbf4-Cdc7 phosphorylation of Mcm2 under wild-type expression conditions also results in impaired DNA replication, substantially decreased single-stranded formation at an origin, and markedly disrupted interaction between GINS and Mcm2-7 during S phase. In vitro, Dbf4-Cdc7 kinase (DDK) phosphorylation of Mcm2 substantially weakens the interaction between Mcm2 and Mcm5, and Dbf4-Cdc7 phosphorylation of Mcm2 promotes Mcm2-7 ring opening. The extrusion of ssDNA from the central channel of Mcm2-7 triggers GINS attachment to Mcm2-7. Thus, Dbf4-Cdc7 phosphorylation of Mcm2 may open the Mcm2-7 ring at the Mcm2-Mcm5 interface, allowing for single-stranded DNA extrusion and subsequent GINS assembly with Mcm2-7.

Mcm subunits can assemble into two different active unwinding complexes.

The replication fork helicase in eukaryotes is a large complex that is composed of Mcm2-7, Cdc45, and GINS. The Mcm2-7 proteins form a heterohexameric ring that hydrolyzes ATP and provide the motor function for this unwinding complex. A comprehensive study of how individual Mcm subunit biochemical activities relate to unwinding function has not been accomplished. We studied the mechanism of the Mcm4-Mcm6-Mcm7 complex, a useful model system because this complex has helicase activity in vitro. We separately purified each of three Mcm subunits until they were each nuclease-free, and we then examined the biochemical properties of different combinations of Mcm subunits. We found that Mcm4 and Mcm7 form an active unwinding assembly. The addition of Mcm6 to Mcm4/Mcm7 results in the formation of an active Mcm4/Mcm6/Mcm7 helicase assembly. The Mcm4-Mcm7 complex forms a ringed-shaped hexamer that unwinds DNA with 3′ to 5′ polarity by a steric exclusion mechanism, similar to Mcm4/Mcm6/Mcm7. The Mcm4-Mcm7 complex has a high level of ATPase activity that is further stimulated by DNA. The ability of different Mcm mixtures to form rings or exhibit DNA stimulation of ATPase activity correlates with the ability of these complexes to unwind DNA. The Mcm4/Mcm7 and Mcm4/Mcm6/Mcm7 assemblies can open to load onto circular DNA to initiate unwinding. We conclude that the Mcm subunits are surprisingly flexible and dynamic in their ability to interact with one another to form active unwinding 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. Sld2T84D binds to a thymine-rich ssDNA region of the origin ARS1, and substitution of ARS1 thymines with adenines completely disrupts binding of Sld2T84D. Sld2T84D enhances the ability of origin ssDNA to pulldown Dpb11, and Sld2 binding to origin ssDNA may be important to allow Sld2 and Dpb11 to associate with origin DNA. We also report that Sld2T84D anneals ssDNA of an origin sequence. Dpb11 anneals ssDNA to low levels, and the addition of Sld2T84D with Dpb11 results in higher annealing activity than that of either protein alone. Sld2-stimulated annealing may be important for maintaining genome stability during the initiation of DNA replication.