Single Molecule Analysis of a Red Fluorescent RecA Protein Reveals a Defect in Nucleoprotein Filament Nucleation That Relates to Its Reduced Biological Functions

Fluorescent fusion proteins are exceedingly useful for monitoring protein localization in situ or visualizing protein behavior at the single molecule level. Unfortunately, some proteins are rendered inactive by the fusion. To circumvent this problem, we fused a hyperactive RecA protein (RecA803 protein) to monomeric red fluorescent protein (mRFP1) to produce a functional protein (RecA-RFP) that is suitable for in vivo and in vitro analysis. In vivo, the RecA-RFP partially restores UV resistance, conjugational recombination, and SOS induction to recA⁻ cells. In vitro, the purified RecA-RFP protein forms a nucleoprotein filament whose k_cat for single-stranded DNA-dependent ATPase activity is reduced ∼3-fold relative to wild-type protein, and which is largely inhibited by single-stranded DNA-binding protein. However, RecA protein is also a dATPase; dATP supports RecA-RFP nucleoprotein filament formation in the presence of single-stranded DNA-binding protein. Furthermore, as for the wild-type protein, the activities of RecA-RFP are further enhanced by shifting the pH to 6.2. As a consequence, RecA-RFP is proficient for DNA strand exchange with dATP or at lower pH. Finally, using single molecule visualization, RecA-RFP was seen to assemble into a continuous filament on duplex DNA, and to extend the DNA ∼1.7-fold. Consistent with its attenuated activities, RecA-RFP nucleates onto double-stranded DNA ∼3-fold more slowly than the wild-type protein, but still requires ∼3 monomers to form the rate-limited nucleus needed for filament assembly. Thus, RecA-RFP reveals that its attenuated biological functions correlate with a reduced frequency of nucleoprotein filament nucleation at the single molecule level.The fusion of native proteins to various fluorescent proteins has found widespread use in biology. If the fusion protein retains proper function, then the behavior and localization of the protein can be followed in living cells (1). Complementing the single-cell analysis, it is now possible to image the behavior of a fluorescent protein at the single molecule level (2–8). However, despite the growing popularity of fusion protein studies, a detailed biochemical analysis of the fusion protein is much less common, even though such examination is crucial for molecular interpretations. Thus, an in vivo and in vitro analysis of the function of a fusion protein relative to the wild-type protein is an essential prerequisite.Homologous recombination is an important process not only for generating genetic variation, but also for maintaining genomic integrity through the repair of DNA breaks. In Escherichia coli, recombinational repair of double-stranded DNA (dsDNA)³ breaks is mediated by the RecBCD pathway, whereas the repair of ssDNA gaps is mediated by the RecF pathway (9). Both of these recombination pathways require the functions of RecA protein.RecA protein is essential to recombinational DNA repair (9–11). RecA-like proteins are ubiquitous and highly conserved (12, 13). The ATP-bound form of the protein binds to ssDNA and polymerizes along the DNA to form an extended nucleoprotein filament (14–16). This is the functional form of the protein that interacts with dsDNA to search for a homologous sequence. Upon finding homology, RecA protein promotes the exchange of identical DNA strands to produce the heteroduplex joint molecules. The joint molecules can be converted into Holliday junctions and resolved by the RuvABC proteins to produce recombinant DNA products (17).The binding of RecA protein to ssDNA is competitive with the ssDNA binding (SSB) protein (18, 19). The assembly of RecA protein onto ssDNA that is complexed with SSB protein is a kinetically slow process, which is catalyzed by so-called mediator or loading proteins (20). RecBCD enzyme is one such RecA-loading protein (21, 22), but an additional set of loading proteins are the RecF, RecO, and RecR proteins that can form various subassemblies to facilitate the RecA-mediated displacement of SSB from ssDNA (23–26). In addition, a class of mutations that map to recA itself were isolated as suppressors of RecF function (srf) that produced mutant RecA proteins with an enhanced intrinsic ability to displace SSB from ssDNA (27). One such mutant is the RecA803 protein, in which valine 37 is mutated to methionine (28, 29). This mutant RecA protein displays a higher intrinsic rate of nucleoprotein filament assembly on ssDNA, which is responsible for its enhanced capacity to displace DNA-bound SSB protein.RecA protein was successfully fused to green fluorescent protein (GFP) and was visualized in living bacteria (30). The RecA-GFP protein foci were seen to appear after UV irradiation and to be dependent on the recB and recF gene products. Although this protein is clearly functional in vivo, it was unfortunately, largely insoluble in vitro, thereby limiting large scale purification.⁴ Therefore, to facilitate biochemical use, an alternative fusion protein was constructed. In the present study, the monomeric red fluorescent protein (mRFP1 (31)) was fused to the carboxyl terminus of the RecA803 protein (referred to as RecA-RFP). The hyperactive RecA803 was used because it assembles on ssDNA more rapidly and competes better with SSB than the wild-type proteins and, as will be shown below, fusion to mRFP1 resulted in attenuated activity; thus, fusion to a hyperactive RecA protein permitted retention of at least partial function. The purified fluorescent protein binds to DNA but shows attenuated ATP and dATP hydrolysis activities. Although nucleoprotein filament assembly is inhibited by SSB protein under typical reaction conditions, we found that nucleoprotein filament formation and enzymatic activities are restored when dATP is substituted for ATP, or when the pH is lowered to 6.2. These characteristics are similar to those of the partially defective RecA142 mutant protein (32, 33), thereby showing that the RFP fusion converted a hypermorphic protein to a hypomorphic RecA fusion protein. Fortunately, because the behavior of this RecA-RFP protein closely fits the biochemical profile of a previously characterized mutant RecA protein, we could understand its behavior. By observing assembly on single molecules of dsDNA, we could see that nucleation of a RecA-RFP filament was ∼3-fold slower than for the wild-type protein. Importantly, these findings lend direct single molecule support to conclusions from ensemble studies where it was shown that biological function of the RecA protein correlates with its ability to displace SSB protein that, in turn, is related to the rate of RecA protein nucleation onto DNA (34).     

Integrator3, a Partner of Single-stranded DNA-binding Protein 1, Participates in the DNA Damage Response

Single-stranded DNA-binding protein 1 (SSB1) plays an important role in the DNA damage response and maintenance of genomic stability. Here, by using protein affinity purification, we have identified Integrator3 (INT3) as a novel partner of SSB1. INT3 forms a complex with SSB1 by constitutively interacting with SSB1 regardless of DNA damage. However, following DNA damage, along with SSB1, INT3 relocates to the DNA damage sites and regulates the accumulation of TopBP1 and BRCA1 there. Moreover, INT3 controls DNA damage-induced Chk1 activation and G₂/M checkpoint activation. In addition, INT3 is involved in homologous recombination repair by regulating Rad51 foci formation following DNA damage. Taken together, these results demonstrate that INT3 plays a key role in the DNA damage response.The DNA damage response, including DNA damage checkpoint activation and DNA damage repair, ensures genomic stability under genotoxic stress. Among various types of DNA damage, DNA double-strand breaks (DSBs)³ are the most deleterious, easily causing chromosomal loss, fusion, and translocation. However, cells can sense and repair DNA DSBs by activating evolutionarily conserved pathways (1–3). Following DNA DSBs, ATM, ATR, and DNAPK, a family homologous to phosphoinositide 3-kinases (4, 5), are activated and phosphorylate histone H2AX at the DNA damage sites (6). Subsequently, phospho-H2AX (γH2AX) provides the platform for accumulation of a larger group of DNA damage response factors, such as MDC1, BRCA1, 53BP1, and TopBP1 (2, 7–9), at the DNA damage sites. Translocalization of these proteins to the DNA DSBs facilitates DNA damage checkpoint activation by activating downstream Chk1/Chk2 kinases, which arrest the cell cycle at G₁, S, or G₂ phase (10). In addition, it also enhances the efficiency of DNA damage repair by recruiting and stabilizing the DNA repair machinery at the DNA damage sites (11).Among these important mediators, single-stranded DNA (ssDNA)-binding proteins play important roles during the DNA damage response. For example, following DNA damage, the MRN complex recognizes DNA DSB ends and processes the blunt ends into ssDNA overhangs (12). The replication protein A (RPA) complex, a group of ssDNA-binding proteins, immediately coats these ssDNA overhangs and loads and activates the ATR·ATRIP complex at the DNA damage sites (13). Meanwhile, the RPA complex protects ssDNA from nucleolytic resection and facilitates Rad51 filament formation along ssDNA overhangs, which is a key step for homologous recombination repair (14). Moreover, RPA70 and RPA32 subunits in the complex could recruit several DNA damage response factors to the DNA damage sites that enhance the efficacy of DNA damage repair (15).Besides the RPA complex, several other ssDNA-binding proteins have been identified to participate in the DNA damage response recently. One of them is ssDNA-binding protein 1 (SSB1) (16). Human SSB1 is a 211-amino acid polypeptide with an N-terminal oligosaccharide/oligonucleotide-binding (OB) domain. It has been shown that SSB1 is phosphorylated by ATM and relocates to the DNA damage site following DNA DSBs. Loss of SSB1 impairs DNA damage-induced checkpoint activation and induces genomic instability. Like the RPA complex, SSB1 participates in homologous recombination by facilitating Rad51·ssDNA filament formation and stabilizing Rad51 at the DNA damage sites. Interestingly, SSB1 has a homolog SSB2 that contains an almost identical OB domain at the N terminus. However, the function of SSB2 in the DNA damage response is not clear yet.To examine the molecular mechanism and functional pathway of SSB1 and SSB2 in the DNA damage response, we have searched for functional partners of SSB1 and SSB2 by using protein affinity purification. We have found Integrator3 (INT3) to be a common partner of both SSB1 and SSB2. Like SSB1, following DNA damage, INT3 relocates to the DNA damage sites and regulates ATR activation. Moreover, INT3 not only participates in DNA damage checkpoint activation but also regulates homologous recombination repair. Taken together, we have found a novel mediator in the DNA damage response.     

PTIP Regulates 53BP1 and SMC1 at the DNA Damage Sites

Although PTIP is implicated in the DNA damage response, through interactions with 53BP1, the function of PTIP in the DNA damage response remain elusive. Here, we show that RNF8 controls DNA damage-induced nuclear foci formation of PTIP, which in turn regulates 53BP1 localization to the DNA damage sites. In addition, SMC1, a substrate of ATM, could not be phosphorylated at the DNA damage sites in the absence of PTIP. The PTIP-dependent pathway is important for DNA double strand breaks repair and DNA damage-induced intra-S phase checkpoint activation. Taken together, these results suggest that the role of PTIP in the DNA damage response is downstream of RNF8 and upstream of 53BP1. Thus, PTIP regulates 53BP1-dependent signaling pathway following DNA damage.The DNA damage response pathways are signal transduction pathways with DNA damage sensors, mediators, and effectors, which are essential for maintaining genomic stability (1–3). Following DNA double strand breaks, histone H2AX at the DNA damage sites is rapidly phosphorylated by ATM/ATR/DNAPK (4–10), a family homologous to phosphoinositide 3-kinases (11, 12). Subsequently, phospho-H2AX (γH2AX) provides the platform for accumulation of a larger group of DNA damage response factors, such as MDC1, BRCA1, 53BP1, and the MRE11·RAD50·NBS1 complex (13, 14), at the DNA damage sites. Translocalization of these proteins to the DNA double strand breaks (DSBs)³ facilitates DNA damage checkpoint activation and enhances the efficiency of DNA damage repair (14, 15).Recently, PTIP (Pax2 transactivation domain-interacting protein, or Paxip) has been identified as a DNA damage response protein and is required for cell survival when exposed to ionizing radiation (IR) (1, 16–18). PTIP is a 1069-amino acid nuclear protein and has been originally identified in a yeast two-hybrid screening as a partner of Pax2 (19). Genetic deletion of the PTIP gene in mice leads to early embryonic lethality at embryonic day 8.5, suggesting that PTIP is essential for early embryonic development (20). Structurally, PTIP contains six tandem BRCT (BRCA1 carboxyl-terminal) domains (16–18, 21). The BRCT domain is a phospho-group binding domain that mediates protein-protein interactions (17, 22, 23). Interestingly, the BRCT domain has been found in a large number of proteins involved in the cellular response to DNA damages, such as BRCA1, MDC1, and 53BP1 (7, 24–29). Like other BRCT domain-containing proteins, upon exposure to IR, PTIP forms nuclear foci at the DSBs, which is dependent on its BRCT domains (16–18). By protein affinity purification, PTIP has been found in two large complexes. One includes the histone H3K4 methyltransferase ALR and its associated cofactors, the other contains DNA damage response proteins, including 53BP1 and SMC1 (30, 31). Further experiments have revealed that DNA damage enhances the interaction between PTIP and 53BP1 (18, 31).To elucidate the DNA damage response pathways, we have examined the upstream and downstream partners of PTIP. Here, we report that PTIP is downstream of RNF8 and upstream of 53BP1 in response to DNA damage. Moreover, PTIP and 53BP1 are required for the phospho-ATM association with the chromatin, which phosphorylates SMC1 at the DSBs. This PTIP-dependent pathway is involved in DSBs repair.     

Bloom Syndrome Helicase Stimulates RAD51 DNA Strand Exchange Activity through a Novel Mechanism

Loss or inactivation of BLM, a helicase of the RecQ family, causes Bloom syndrome, a genetic disorder with a strong predisposition to cancer. Although the precise function of BLM remains unknown, genetic data has implicated BLM in the process of genetic recombination and DNA repair. Previously, we demonstrated that BLM can disrupt the RAD51-single-stranded DNA filament that promotes the initial steps of homologous recombination. However, this disruption occurs only if RAD51 is present in an inactive ADP-bound form. Here, we investigate interactions of BLM with the active ATP-bound form of the RAD51-single-stranded DNA filament. Surprisingly, we found that BLM stimulates DNA strand exchange activity of RAD51. In contrast to the helicase activity of BLM, this stimulation does not require ATP hydrolysis. These data suggest a novel BLM function that is stimulation of the RAD51 DNA pairing. Our results demonstrate the important role of the RAD51 nucleoprotein filament conformation in stimulation of DNA pairing by BLM.Mutations of BLM helicase cause Bloom syndrome (BS),² a rare autosomal disorder, which is associated with stunted growth, facial sun sensitivity, immunodeficiency, fertility defects, and a greatly elevated incidence of many types of cancer occurring at an early age (1). BLM belongs to the highly conserved family of RecQ helicases that are required for the maintenance of genome integrity in all organisms (2, 3). There are five RecQ helicases in humans; mutations in three of them, WRN, RECQ4, and BLM, have been associated with the genetic abnormalities known as Werner, Rothmund-Thomson, and Bloom syndrome, respectively (4, 5). The cells from BS patients display genomic instability; the hallmark of BS is an increase in the frequency of sister chromatid and interhomolog exchanges (1, 6). Because homologous recombination (HR) is responsible for chromosomal exchanges, it is thought that BLM helicase functions in regulating HR (7–9). Also, BLM helicase is required for faithful chromosome segregation (10) and repair of stalled replication forks (11, 12), the processes that are linked to HR (13–15). BLM was found to interact physically with RAD51, a key protein of HR (16) that catalyzes the central steps in HR including the search for homology and the exchange of strands between homologous ssDNA and dsDNA sequences (17). In cells, BLM forms nuclear foci, a subset of which co-localize with RAD51. Interestingly, the extent of RAD51 and BLM co-localization increases in response to ionizing radiation, indicating a possible role of BLM in the repair of DNA double-strand breaks (16).Biochemical studies suggest that BLM may perform several different functions in HR. BLM was shown to promote the dissociation of HR intermediates (D-loops) (18–20), branch migration of Holliday junctions (21), and dissolution of double Holliday junctions acting in a complex with TopoIIIα and BLAP75 (22–24). BLM may also facilitate DNA synthesis during the repair process by unwinding the DNA template in front of the replication fork (25). In addition, BLM and its yeast homolog Sgs1 may play a role at the initial steps of DNA double-strand break repair by participating in exonucleolitic resection of the DNA ends to generate DNA molecules with the 3′-ssDNA tails, a substrate for RAD51 binding (26–29).In vivo, the process of HR is tightly regulated by various mechanisms (30). Whereas some proteins promote HR (14, 31), others inhibit this process, thereby preventing its untimely initiation (32, 33). Disruption of the Rad51-ssDNA nucleoprotein filament appears to be an especially important mechanism of controlling HR. This filament disruption activity was demonstrated for the yeast Srs2 helicase (34, 35) and human RECQ5 helicase (36). Recently, we found that BLM can also catalyze disruption of the RAD51-ssDNA filament (25). This disruption only occurs if the filament is present in an inactive ADP-bound form, e.g. in the presence of Mg²⁺. Conversion of RAD51 into an active ATP-bound form, e.g. in the presence of Ca²⁺ (37), renders the filament resistant to BLM disruption (25). In this study, we analyze the interactions of BLM with an active ATP-bound RAD51-ssDNA filament. Surprisingly, we found that BLM stimulates the DNA strand exchange activity of RAD51. Thus, depending on the conformational state of the RAD51 nucleoprotein filament, BLM may either inhibit or stimulate the DNA strand exchange activity of RAD51. Our analysis demonstrated that, in contrast to several known stimulatory proteins that act by promoting formation of the RAD51-ssDNA filament, BLM stimulates the DNA strand exchange activity of RAD51 at a later stage, during synapsis. Stimulation appears to be independent of the ATPase activity of BLM. We suggest that this stimulation of RAD51 may represent a novel function of BLM in homologous recombination.     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Proliferating Cell Nuclear Antigen Is Protected from Degradation by Forming a Complex with MutT Homolog2

Proliferating cell nuclear antigen (PCNA) has been demonstrated to interact with multiple proteins involved in several metabolic pathways such as DNA replication and repair. However, there have been fewer reports about whether these PCNA-binding proteins influence stability of PCNA. Here, we observed a physical interaction between PCNA and MutT homolog2 (MTH2), a new member of the MutT-related proteins that hydrolyzes 8-oxo-7,8-dihydrodeoxyguanosine triphosphate (8-oxo-dGTP). In several unstressed human cancer cell lines and in normal human fibroblast cells, PCNA and MTH2 formed a complex and their mutual binding fragments were confirmed. It was intriguing that PCNA and MTH2 were dissociated dependent on acetylation of PCNA, which in turn induced degradation of PCNA in response to UV irradiation, but not in response to other forms of DNA-damaging stress. To further explore the link between dissociation of PCNA-MTH2 and degradation of PCNA, RNAi against MTH2 was performed to mimic the dissociated status of PCNA to evaluate changes in the half-life of PCNA. Knockdown of MTH2 significantly promoted degradation of PCNA, suggesting that the physiological interaction of PCNA-MTH2 may confer protection from degradation for PCNA, whereas UV irradiation accelerates PCNA degradation by inducing dissociation of PCNA-MTH2. Moreover, secondary to degradation of PCNA, UV-induced inhibition of DNA synthesis or cell cycle progression was enhanced. Collectively, our data demonstrate for the first time that PCNA is protected by this newly identified partner molecule MTH2, which is related to DNA synthesis and cell cycle progression.Proliferating cell nuclear antigen (PCNA)³ is a member of the DNA sliding clamp family and consists of a ring-shaped trimeric complex (1–3). Three PCNA monomers, each comprising two similar domains, are joined in a head-to-tail arrangement to form a closed ring (4, 5). Because of this unique structure, PCNA encircles the DNA double helix and slides freely along it. PCNA was originally characterized as a DNA polymerase processivity factor and it increases the processivity of DNA synthesis by interacting with polymerase δ (6, 7). Subsequent studies revealed that PCNA plays an important role in DNA replication (8, 9). For example, PCNA not only functions as a protein binding platform to interact with the DNA polymerases, flap endonuclease-1 (Fen1) or DNA ligase I (10–12), but also coordinates complicated processes in DNA replication (2, 13). In addition, PCNA also plays a role in DNA damage repair (14–17) and cell cycle control (18–20).Because PCNA is essential for DNA synthesis both in DNA replication and repair, a dynamic balance between PCNA synthesis and degradation is critical for maintaining normal DNA synthesis. Up-regulation of PCNA accelerates DNA synthesis and promotes cell proliferation, such that PCNA is regarded as a general proliferation marker in tumor development. On the other hand, degradation of PCNA leads to inhibition of DNA synthesis (9, 21). In this case, in response to inhibition of DNA synthesis by PCNA degradation, both cell proliferation and DNA repair are inhibited, and cells are thus subject to death.In Escherichia coli, MutT protein encoded by the mutT gene has 8-oxo-dGTPase activity, and hydrolyzes 8-oxo-dGTP to 8-oxo-dGMP, which is nonutilizable for DNA synthesis, thus preventing misincorporation of 8-oxo-dGTP into DNA (22). 8-Oxo-dGTP is a product of dGTP oxidation and can be inserted into opposite dA or dC residues of template DNA at almost equal efficiencies. As a result, G:C to T:A or T:A to G:C transversion mutations occur (22–24). In a mutT-deficient strain, the rate of spontaneous occurrence of A:T to C:G transversion increases by 1000-fold compared with that of cells with wild type mutT (25–27). Therefore, MutT protein is required for preventing mutations and maintaining high fidelity of DNA replication (28). In addition, RibA is a backup enzyme for MutT in E. coli and also plays a role in maintaining high fidelity of DNA replication (29). The MutT homologue MTH1 is the first MutT-related protein found in mammalian cells (30). The spontaneous mutation frequency in MTH1-deficient cells showed an increase of ∼2-fold as compared with that in wild type MTH1 cells (31). Comparing the mutation frequency in mutT-deficient E. coli cells with that in MTH1-deficient mammalian cells suggests that there must be other proteins responsible for preventing occurrence of high numbers of oxidative damage induced mutations in mammalian cells. By searching the GenBank^TM EST data base, our research group and others (32) have cloned a new member of MutT-related protein, MTH2. The increased mutation frequency in mutT-deficient cells was significantly reduced by overexpression of MTH2 cDNA (32). Therefore, MTH2 may help to ensure cells achieve accurate DNA synthesis. However, aside from the activity of 8-oxo-dGTPase, the exact mechanism by which MTH2 influences DNA synthesis has not been explored.The functions of both PCNA and MTH2 partially overlap in DNA synthesis, thus warranting exploration of whether MTH2 works together with PCNA to regulate DNA replication or repair. In this study, we found that MTH2 directly interacts with PCNA, and this interaction enhances PCNA stability. However, when cells were exposed to UV light, the interaction of MTH2 and PCNA was disrupted, and PCNA degradation was accelerated. Consequently, DNA synthesis was reduced, and cell cycling was arrested.     

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 Cdc7 protein kinase is required throughout the yeast S phase to activate origins (1, 2). The S phase cyclin-dependent kinase also activates yeast origins of replication (3–5). It has been proposed that Dbf4 activates Cdc7 kinase in S phase, and that Dbf4 interaction with Cdc7 is essential for Cdc7 kinase activity (6). However, it is not known how Dbf4-Cdc7 (DDK)² acts during S phase to trigger the initiation of DNA replication. DDK has homologs in other eukaryotic species, and the role of Cdc7 in activation of replication origins during S phase may be conserved (7–10).The Mcm2-7 complex functions with Cdc45 and GINS to unwind DNA at a replication fork (11–15). A mutation of MCM5 (mcm5-bob1) bypasses the cellular requirements for DBF4 and CDC7 (16), suggesting a critical physiologic interaction between Dbf4-Cdc7 and Mcm proteins. DDK phosphorylates Mcm2 in vitro with proteins purified from budding yeast (17, 18) or human cells (19). Furthermore, there are mutants of MCM2 that show synthetic lethality with DBF4 mutants (6, 17), suggesting a biologically relevant interaction between DBF4 and MCM2. Nevertheless, the physiologic role of DDK phosphorylation of Mcm2 is a matter of dispute. In human cells, replacement of MCM2 DDK-phosphoacceptor residues with alanines inhibits DNA replication, suggesting that Dbf4-Cdc7 phosphorylation of Mcm2 in humans is important for DNA replication (20). In contrast, mutation of putative DDK phosphorylation sites at the N terminus of Schizosaccharomyces pombe Mcm2 results in viable cells, suggesting that phosphorylation of S. pombe Mcm2 by DDK is not critical for cell growth (10).In budding yeast, Cdc7 is present at high levels in G₁ and S phase, whereas Dbf4 levels peak in S phase (18, 21, 22). Furthermore, budding yeast DDK binds to chromatin during S phase (6), and it has been shown that Dbf4 is required for Cdc7 binding to chromatin in budding yeast (23, 24), fission yeast (25), and Xenopus (9). Human and fission yeast Cdc7 are inert on their own (7, 8), but Dbf4-Cdc7 is active in phosphorylating Mcm proteins in budding yeast (6, 26), fission yeast (7), and human (8, 10). Based on these data, it has been proposed that Dbf4 activates Cdc7 kinase in S phase and that Dbf4 interaction with Cdc7 is essential for Cdc7 kinase activity (6, 9, 18, 21–24). However, a mechanistic analysis of how Dbf4 activates Cdc7 has not yet been accomplished. For example, the multimeric state of the active Dbf4-Cdc7 complex is currently disputed. A heterodimer of fission yeast Cdc7 (Hsk1) in complex with fission yeast Dbf4 (Dfp1) can phosphorylate Mcm2 (7). However, in budding yeast, oligomers of Cdc7 exist in the cell (27), and Dbf4-Cdc7 exists as oligomers of 180 and 300 kDa (27).DDK phosphorylates the N termini of human Mcm2 (19, 20, 28), human Mcm4 (10), budding yeast Mcm4 (26), and fission yeast Mcm6 (10). Although the sequences of the Mcm N termini are poorly conserved, the DDK sites identified in each study have neighboring acidic residues. The residues of budding yeast Mcm2 that are phosphorylated by DDK have not yet been identified.In this study, we find that budding yeast Cdc7 is weakly active as a multimer in phosphorylating Mcm2. However, a low molecular weight form of Dbf4-Cdc7, likely a heterodimer, has a higher specific activity for phosphorylation of Mcm2. Dbf4 or DDK, but not Cdc7, binds tightly to Mcm2, suggesting that Dbf4 recruits Cdc7 to Mcm2. DDK phosphorylates two serine residues of Mcm2, Ser-164 and Ser-170, in an acidic region of the protein. Mutation of Ser-170 is lethal to yeast cells, but this phenotype is rescued by the DDK bypass mutant mcm5-bob1. We conclude that DDK phosphorylation of Ser-170 of Mcm2 is required for budding yeast growth.