Irina Bruck

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.

The ring-type polymerase sliding clamp family

Ring-type polymerases consist of a DNA polymerase, a ring-shaped sliding clamp protein and a clamp-loading complex. Sliding clamp proteins are found in all organisms and are called proliferating cell nuclear antigen (PCNA) in eukaryotes and the β clamp in prokaryotes. Both PCNA and β form a ring around DNA, which is made up of two subunits of three domains each in β but three subunits of two domains each in PCNA. Despite this difference and a lack of detectable sequence homology, the structures of the two rings are very similar. The sliding clamp slides along DNA and tethers the polymerase to the DNA, enabling rapid and processive DNA replication.

The Essential C Family DnaE Polymerase Is Error-prone and Efficient at Lesion Bypass

DnaE-type DNA polymerases belong to the C family of DNA polymerases and are responsible for chromosomal replication in prokaryotes. Like most closely related Gram-positive cells, Streptococcus pyogenes has two DnaE homologs Pol C and DnaE; both are essential to cell viability. Pol C is an established replicative polymerase, and DnaE has been proposed to serve a replicative role. In this report, we characterize S. pyogenes DnaE polymerase and find that it is highly error-prone. DnaE can bypass coding and noncoding lesions with high efficiency. Error-prone extension is accomplished by either of two pathways, template-primer misalignment or direct primer extension. The bypass of abasic sites is accomplished mainly through “dNTP-stabilized” misalignment of template, thereby generating (–1) deletions in the newly synthesized strand. This mechanism may be similar to the dNTP-stabilized misalignment mechanism used by the Y family of DNA polymerases and is the first example of lesion bypass and error-prone synthesis catalyzed by a C family polymerase. Thus, DnaE may function in an error-prone capacity that may be essential in Gram-positive cells but not Gram-negative cells, suggesting a fundamental difference in DNA metabolism between these two classes of bacteria.

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.

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.

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.

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

Background: Sld2 is required for the initiation of DNA replication. Results: Sld2 inhibits assembly of replication fork helicase, but this inhibition is alleviated by origin single-stranded DNA and phosphorylation of Sld2. Conclusion: Phosphorylation of Sld2 and origin single-stranded DNA may be important for assembly of the replication fork helicase. Significance: We propose a new model for the assembly of the replication fork helicase. 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. We found that unphosphorylated or CDK-phosphorylated Sld2 binds to the mini chromosome maintenance (Mcm)2–7 complex with similar efficiency. Sld2 interaction with Mcm2–7 blocks the interaction between GINS and Mcm2–7. The interaction between CDK-phosphorylated Sld2 and Mcm2–7 is substantially inhibited by origin single-stranded DNA (ssDNA). Furthermore, origin ssDNA allows GINS to bind to Mcm2–7 in the presence of CDK-phosphorylated Sld2. However, unphosphorylated Sld2 blocks the interaction between GINS and Mcm2–7 even in the presence of origin ssDNA. We identified a mutant of Sld2 that does not bind to DNA. When this mutant is expressed in yeast cells, cell growth is severely inhibited with very slow progression into S phase. We propose a model wherein Sld2 blocks the interaction between GINS and Mcm2–7 in vivo. Once origin ssDNA is extruded from the Mcm2–7 ring and CDK phosphorylates Sld2, the origin ssDNA binds to CDK-phosphorylated Sld2. This event may allow the interaction between GINS and Mcm2–7 in vivo. Thus, CDK phosphorylation of Sld2 may be important to release Sld2 from Mcm2–7, thereby allowing GINS to bind Mcm2–7. Furthermore, origin ssDNA may stimulate the formation of the CMG complex by alleviating inhibitory interactions between Sld2 with Mcm2–7.

Crystal structure of a DNA polymerase sliding clamp from a Gram-positive bacterium

BackgroundSliding DNA clamps are processivity factors that are required for efficient DNA replication. DNA polymerases maintain proximity to nucleic acid templates by interacting with sliding clamps that encircle DNA and thereby link the polymerase enzyme to the DNA substrate. Although the structures of sliding clamps from Gram-negative bacteria (E. coli), eukaryotes, archaea, and T4-like bacteriophages are well-known, the structure of a sliding clamp from Gram-positive bacteria has not been reported previously.ResultsWe have determined the crystal structure of the dimeric β subunit of the DNA polymerase III holoenzyme of Streptococcus pyogenes. The sliding clamp from this Gram-positive organism forms a ring-shaped dimeric assembly that is similar in overall structure to that of the sliding clamps from Gram-negative bacteria, bacteriophage T4, eukaryotes and archaea. The dimer has overall dimensions of ~90 Å × ~70 Å × ~25 Å with a central chamber that is large enough to accommodate duplex DNA. In comparison to the circular shape of other assemblies, the S. pyogenes clamp adopts a more elliptical structure.ConclusionThe sequences of sliding clamps from S. pyogenes and E. coli are only 23% identical, making the generation of structural models for the S. pyogenes clamp difficult in the absence of direct experimental information. Our structure of the S. pyogenes β subunit completes the catalog of clamp structures from all the major sequence grouping of sliding clamps. The more elliptical rather than circular structure of the S. pyogenes clamp implies that the topological nature of encircling DNA, rather than a precise geometric shape, is the most conserved aspect for this family of proteins.