Transcriptional pause extension benefits the stand-by rather than catch-up Rho-dependent termination.

Transcriptional pause is essential for all types of termination. In this single-molecule study on bacterial Rho factor-dependent terminators, we confirm that the three Rho-dependent termination routes operate compatibly together in a single terminator, and discover that their termination efficiencies depend on the terminational pauses in unexpected ways. Evidently, the most abundant route is that Rho binds nascent RNA first and catches up with paused RNA polymerase (RNAP) and this catch-up Rho mediates simultaneous releases of transcript RNA and template DNA from RNAP. The fastest route is that the catch-up Rho effects RNA-only release and leads to 1D recycling of RNAP on DNA. The slowest route is that the RNAP-prebound stand-by Rho facilitates only the simultaneous rather than sequential releases. Among the three routes, only the stand-by Rho's termination efficiency positively correlates with pause duration, contrary to a long-standing speculation, invariably in the absence or presence of NusA/NusG factors, competitor RNAs or a crowding agent. Accordingly, the essential terminational pause does not need to be long for the catch-up Rho's terminations, and long pauses benefit only the stand-by Rho's terminations. Furthermore, the Rho-dependent termination of mgtA and ribB riboswitches is controlled mainly by modulation of the stand-by rather than catch-up termination.

Dopamine activity in the nigrostriatal pathway alters cue-induced risky choice patterns in female rats.

Deficits in cost/benefit decision making is a critical risk factor for gambling disorder. Reward-paired cues may play an important role, as these stimuli can enhance risk preference in rats. Despite extensive research implicating the dorsal striatum in the compulsive aspects of addiction, the role of nigrostriatal dopaminergic activity in cue-induced risk preference remains unclear, particularly in females. Accordingly, we examined the effects of manipulating the dopaminergic nigrostriatal pathway on cue-induced risky choice in female rats. TH:Cre rats were trained on the cued version of the rat Gambling Task. This task was designed such that maximal reward is attained by avoiding the high-risk, high-reward options and instead favouring the options associated with lower per-trial gains, as they feature less frequent and shorter time-out penalties. Adding reward-paired audiovisual cues to the task leads to greater risky choice on average. To assess the role of the nigrostriatal pathway, a viral vector carrying either Cre-dependent inhibitory or excitatory DREADD was infused into the substantia nigra. Rats then received clozapine-N-oxide either during task acquisition or after a stable performance baseline was reached. Inhibition of this pathway accelerated the development of risk preference in early sessions and increased risky choice during performance, but long-term inhibition actually improved decision making. Activation of this pathway had minimal effects. These results provide evidence for the involvement of the dopaminergic nigrostriatal pathway in cue-induced risk preference in females, therefore shedding light on its role in cost/benefit decision-making deficits and expanding our knowledge of the female dopaminergic system.

Computational design of a thermolabile uracil-DNA glycosylase of Escherichia coli.

Polymerase chain reaction (PCR) is a powerful tool to diagnose infectious diseases. Uracil DNA glycosylase (UDG) is broadly used to remove carryover contamination in PCR. However, UDG can contribute to false negative results when not inactivated completely, leading to DNA degradation during the amplification step. In this study, we designed novel thermolabile UDG derivatives by supercomputing molecular dynamic simulations and residual network analysis. Based on enzyme activity analysis, thermolability, thermal stability, and biochemical experiments of Escherichia coli-derived UDG and 22 derivatives, we uncovered that the UDG D43A mutant eliminated the false negative problem, demonstrated high efficiency, and offered great benefit for use in PCR diagnosis. We further obtained structural and thermodynamic insights into the role of the D43A mutation, including perturbed protein structure near D43; weakened pairwise interactions of D43 with K42, N46, and R80; and decreased melting temperature and native fraction of the UDG D43A mutant compared with wild-type UDG.

The p90 ribosomal S6 kinase-UBR5 pathway controls Toll-like receptor signaling via miRNA-induced translational inhibition of tumor necrosis factor receptor-associated factor 3.

MicroRNAs (miRNAs) are small, noncoding RNAs that post-transcriptionally regulate gene expression. For example, miRNAs repress gene expression by recruiting the miRNA-induced silencing complex (miRISC), a ribonucleoprotein complex that contains miRNA-engaged Argonaute (Ago) and the scaffold protein GW182. Recently, ubiquitin-protein ligase E3 component N-recognin 5 (UBR5) has been identified as a component of miRISC. UBR5 directly interacts with GW182 proteins and participates in miRNA silencing by recruiting downstream effectors, such as the translation regulator DEAD-box helicase 6 (DDX6) and transducer of ERBB2,1/2,2 (Tob1/2), to the Ago-GW182 complex. However, the regulation of miRISC-associated UBR5 remains largely elusive. In the present study, we showed that UBR5 down-regulates the levels of TNF receptor-associated factor 3 (TRAF3), a key component of Toll-like receptor signaling, via the miRNA pathway. We further demonstrated that p90 ribosomal S6 kinase (p90RSK) is an upstream regulator of UBR5. p90RSK phosphorylates UBR5 at Thr637, Ser1227, and Ser2483, and this phosphorylation is required for the translational repression of TRAF3 mRNA. Phosphorylated UBR5 co-localized with GW182 and Ago2 in cytoplasmic speckles, which implies that miRISC is affected by phospho-UBR5. Collectively, these results indicated that the p90RSK-UBR5 pathway stimulates miRNA-mediated translational repression of TRAF3. Our work has added another layer to the regulation of miRISC.

A physiological significance of the functional interaction between Mus81 and Rad27 in homologous recombination repair.

Fen1 and Mus81-Mms4 are endonucleases involved in the processing of various DNA structural intermediates, and they were shown to have genetic and functional interactions with each other. Here, we show the in vivo significance of the interactions between Mus81 and Rad27 (yeast Fen1). The N-terminal 120 amino-acid (aa) region of Mus81, although entirely dispensable for its catalytic activity, was essential for the abilities of Mus81 to bind to and be stimulated by Rad27. In the absence of SGS1, the mus81Δ120N mutation lacking the N-terminal 120 aa region exhibited synthetic lethality, and the lethality was rescued by deletion of RAD52, a key homologous recombination mediator. These findings, together with the fact that Sgs1 constitutes a redundant pathway with Mus81-Mms4, indicate that the N-terminus-mediated interaction of Mus81 with Rad27 is physiologically important in resolving toxic recombination intermediates. Mutagenic analyses of the N-terminal region identified two distinct motifs, named N21-26 (aa from 21-26) and N108-114 (aa from 108-114) important for the in vitro and in vivo functions of Mus81. Our findings indicate that the N-terminal region of Mus81 acts as a landing pad to interact with Rad27 and that Mus81 and Rad27 work conjointly for efficient removal of various aberrant DNA structures.

A repeat protein-based DNA polymerase inhibitor for an efficient and accurate gene amplification by PCR.

A polymerase chain reaction (PCR) using a thermostable DNA polymerase is the most widely applied method in many areas of research, including life sciences, biotechnology, and medical sciences. However, a conventional PCR incurs an amplification of undesired genes mainly owing to non-specifically annealed primers and the formation of a primer-dimer complex. Herein, we present the development of a Taq DNA polymerase-specific repebody, which is a small-sized protein binder composed of leucine rich repeat (LRR) modules, as a thermolabile inhibitor for a precise and accurate gene amplification by PCR. We selected a repebody that specifically binds to the DNA polymerase through a phage display, and increased its affinity to up to 10 nM through a modular evolution approach. The repebody was shown to effectively inhibit DNA polymerase activity at low temperature and undergo thermal denaturation at high temperature, leading to a rapid and full recovery of the polymerase activity, during the initial denaturation step of the PCR. The performance and utility of the repebody was demonstrated through an accurate and efficient amplification of a target gene without nonspecific gene products in both conventional and real-time PCRs. The repebody is expected to be effectively utilized as a thermolabile inhibitor in a PCR. Biotechnol. Bioeng. 2016;113: 2544-2552. © 2016 Wiley Periodicals, Inc.

Human MUS81-EME2 can cleave a variety of DNA structures including intact Holliday junction and nicked duplex.

MUS81 shares a high-degree homology with the catalytic XPF subunit of the XPF-ERCC1 endonuclease complex. It is catalytically active only when complexed with the regulatory subunits Mms4 or Eme1 in budding and fission yeasts, respectively, and EME1 or EME2 in humans. Although Mus81 complexes are implicated in the resolution of recombination intermediates in vivo, recombinant yeast Mus81-Mms4 and human MUS81-EME1 isolated from Escherichia coli fail to cleave intact Holliday junctions (HJs) in vitro. In this study, we show that human recombinant MUS81-EME2 isolated from E. coli cleaves HJs relatively efficiently, compared to MUS81-EME1. Furthermore, MUS81-EME2 catalyzed cleavage of nicked and gapped duplex deoxyribonucleic acids (DNAs), generating double-strand breaks. The presence of a 5' phosphate terminus at nicks and gaps rendered DNA significantly less susceptible to the cleavage by MUS81-EME2 than its absence, raising the possibility that this activity could play a role in channeling damaged DNA duplexes that are not readily repaired into the recombinational repair pathways. Significant differences in substrate specificity observed with unmodified forms of MUS81-EME1 and MUS81-EME2 suggest that they play related but non-overlapping roles in DNA transactions.

Nucleoredoxin promotes adipogenic differentiation through regulation of Wnt/ß-catenin signaling.

Nucleoredoxin (NRX) is a member of the thioredoxin family of proteins that controls redox homeostasis in cell. Redox homeostasis is a well-known regulator of cell differentiation into various tissue types. We found that NRX expression levels were higher in white adipose tissue of obese ob/ob mice and increased in the early adipogenic stage of 3T3-L1 preadipocyte differentiation. Knockdown of NRX decreased differentiation of 3T3-L1 cells, whereas overexpression increased differentiation. Adipose tissue-specific NRX transgenic mice showed increases in adipocyte size as well as number compared with WT mice. We further confirmed that the Wingless/int-1 class (Wnt)/ß-catenin pathway was also involved in NRX-promoted adipogenesis, consistent with a previous report showing NRX regulation of this pathway. Genes involved in lipid metabolism were downregulated, whereas inflammatory genes, including those encoding macrophage markers, were significantly upregulated, likely contributing to the obesity in Adipo-NRX mice. Our results therefore suggest that NRX acts as a novel proadipogenic factor and controls obesity in vivo.

Rad52/Rad59-dependent recombination as a means to rectify faulty Okazaki fragment processing.

The correct removal of 5'-flap structures by Rad27 and Dna2 during Okazaki fragment maturation is crucial for the stable maintenance of genetic materials and cell viability. In this study, we identified RAD52, a key recombination protein, as a multicopy suppressor of dna2-K1080E, a lethal helicase-negative mutant allele of DNA2 in yeasts. In contrast, the overexpression of Rad51, which works conjointly with Rad52 in canonical homologous recombination, failed to suppress the growth defect of the dna2-K1080E mutation, indicating that Rad52 plays a unique and distinct role in Okazaki fragment metabolism. We found that the recombination-defective Rad52-QDDD/AAAA mutant did not rescue dna2-K1080E, suggesting that Rad52-mediated recombination is important for suppression. The Rad52-mediated enzymatic stimulation of Dna2 or Rad27 is not a direct cause of suppression observed in vivo, as both Rad52 and Rad52-QDDD/AAAA proteins stimulated the endonuclease activities of both Dna2 and Rad27 to a similar extent. The recombination mediator activity of Rad52 was dispensable for the suppression, whereas both the DNA annealing activity and its ability to interact with Rad59 were essential. In addition, we found that several cohesion establishment factors, including Rsc2 and Elg1, were required for the Rad52-dependent suppression of dna2-K1080E. Our findings suggest a novel Rad52/Rad59-dependent, but Rad51-independent recombination pathway that could ultimately lead to the removal of faulty flaps in conjunction with cohesion establishment factors.

Single-molecule sorting reveals how ubiquitylation affects substrate recognition and activities of FBH1 helicase.

DNA repair helicases function in the cell to separate DNA duplexes or remodel nucleoprotein complexes. These functions are influenced by sensing and signaling; the cellular pool of a DNA helicase may contain subpopulations of enzymes carrying different post-translational modifications and performing distinct biochemical functions. Here, we report a novel experimental strategy, single-molecule sorting, which overcomes difficulties associated with comprehensive analysis of heterologously modified pool of proteins. This methodology was applied to visualize human DNA helicase F-box-containing DNA helicase (FBH1) acting on the DNA structures resembling a stalled or collapsed replication fork and its interactions with RAD51 nucleoprotein filament. Individual helicase molecules isolated from human cells with their native post-translational modifications were analyzed using total internal reflection fluorescence microscopy. Separation of the activity trajectories originated from ubiquitylated and non-ubiquitylated FBH1 molecules revealed that ubiquitylation affects FBH1 interaction with the RAD51 nucleoprotein filament, but not its translocase and helicase activities.

The N-terminal 45-kDa domain of Dna2 endonuclease/helicase targets the enzyme to secondary structure DNA.

The removal of initiating primers from the 5'-ends of each Okazaki fragment, required for the generation of contiguous daughter strands, can be catalyzed by the combined action of DNA polymerase δ and Fen1. When the flaps generated by displacement of DNA synthesis activity of polymerase δ become long enough to bind replication protein A or form hairpin structures, the helicase/endonuclease enzyme, Dna2, becomes critical because of its ability to remove replication protein A-coated or secondary structure flaps. In this study, we show that the N-terminal 45-kDa domain of Dna2 binds hairpin structures, allowing the enzyme to target secondary structure flap DNA. We found that this activity was essential for the efficient removal of hairpin flaps by the endonuclease activity of Dna2 with the aid of its helicase activity. Thus, the efficient removal of hairpin structure flaps requires the coordinated action of all three functional domains of Dna2. We also found that deletion of the N-terminal 45-kDa domain of Dna2 led to a partial loss of the intra-S-phase checkpoint function and an increased rate of homologous recombination in yeast. We discuss the potential roles of the N-terminal domain of Dna2 in the maintenance of genomic stability.

Biochemical studies of the Saccharomyces cerevisiae Mph1 helicase on junction-containing DNA structures.

Saccharomyces cerevisiae Mph1 is a 3-5' DNA helicase, required for the maintenance of genome integrity. In order to understand the ATPase/helicase role of Mph1 in genome stability, we characterized its helicase activity with a variety of DNA substrates, focusing on its action on junction structures containing three or four DNA strands. Consistent with its 3' to 5' directionality, Mph1 displaced 3'-flap substrates in double-fixed or equilibrating flap substrates. Surprisingly, Mph1 displaced the 5'-flap strand more efficiently than the 3' flap strand from double-flap substrates, which is not expected for a 3-5' DNA helicase. For this to occur, Mph1 required a threshold size (>5 nt) of 5' single-stranded DNA flap. Based on the unique substrate requirements of Mph1 defined in this study, we propose that the helicase/ATPase activity of Mph1 play roles in converting multiple-stranded DNA structures into structures cleavable by processing enzymes such as Fen1. We also found that the helicase activity of Mph1 was used to cause structural alterations required for restoration of replication forks stalled due to damaged template. The helicase properties of Mph1 reported here could explain how it resolves D-loop structure, and are in keeping with a model proposed for the error-free damage avoidance pathway.

The trans-autostimulatory activity of Rad27 suppresses dna2 defects in Okazaki fragment processing.

Dna2 and Rad27 (yeast Fen1) are the two endonucleases critical for Okazaki fragment processing during lagging strand DNA synthesis that have been shown to interact genetically and physically. In this study, we addressed the functional consequences of these interactions by examining whether purified Rad27 of Saccharomyces cerevisiae affects the enzymatic activity of Dna2 and vice versa. For this purpose, we constructed Rad27DA (catalytically defective enzyme with an Asp to Ala substitution at amino acid 179) and found that it significantly stimulated the endonuclease activity of wild type Dna2, but failed to do so with Dna2Δ405N that lacks the N-terminal 405 amino acids. This was an unexpected finding because dna2Δ405N cells were still partially suppressed by overexpression of rad27DA in vivo. Further analyses revealed that Rad27 is a trans-autostimulatory enzyme, providing an explanation why overexpression of Rad27, regardless of its catalytic activity, suppressed dna2 mutants as long as an endogenous wild type Rad27 is available. We found that the C-terminal 16-amino acid fragment of Rad27, a highly polybasic region due to the presence of multiple positively charged lysine and arginine residues, was sufficient and necessary for the stimulation of both Rad27 and Dna2. Our findings provide further insight into how Dna2 and Rad27 jointly affect the processing of Okazaki fragments in eukaryotes.

Dna2 on the road to Okazaki fragment processing and genome stability in eukaryotes.

DNA replication is a primary mechanism for maintaining genome integrity, but it serves this purpose best by cooperating with other proteins involved in DNA repair and recombination. Unlike leading strand synthesis, lagging strand synthesis has a greater risk of faulty replication for several reasons: First, a significant part of DNA is synthesized by polymerase alpha, which lacks a proofreading function. Second, a great number of Okazaki fragments are synthesized, processed and ligated per cell division. Third, the principal mechanism of Okazaki fragment processing is via generation of flaps, which have the potential to form a variety of structures in their sequence context. Finally, many proteins for the lagging strand interact with factors involved in repair and recombination. Thus, lagging strand DNA synthesis could be the best example of a converging place of both replication and repair proteins. To achieve the risky task with extraordinary fidelity, Okazaki fragment processing may depend on multiple layers of redundant, but connected pathways. An essential Dna2 endonuclease/helicase plays a pivotal role in processing common structural intermediates that occur during diverse DNA metabolisms (e.g. lagging strand synthesis and telomere maintenance). Many roles of Dna2 suggest that the preemptive removal of long or structured flaps ultimately contributes to genome maintenance in eukaryotes. In this review, we describe the function of Dna2 in Okazaki fragment processing, and discuss its role in the maintenance of genome integrity with an emphasis on its functional interactions with other factors required for genome maintenance.

Physical and functional interactions of Caenorhabditis elegans WRN-1 helicase with RPA-1.

The Caenorhabditis elegans Werner syndrome protein, WRN-1, a member of the RecQ helicase family, has a 3'-5' DNA helicase activity. Worms with defective wrn-1 exhibit premature aging phenotypes and an increased level of genome instability. In response to DNA damage, WRN-1 participates in the initial stages of checkpoint activation in concert with C. elegans replication protein A (RPA-1). WRN-1 helicase is stimulated by RPA-1 on long DNA duplex substrates. However, the mechanism by which RPA-1 stimulates DNA unwinding and the function of the WRN-1-RPA-1 interaction are not clearly understood. We have found that WRN-1 physically interacts with two RPA-1 subunits, CeRPA73 and CeRPA32; however, full-length WRN-1 helicase activity is stimulated by only the CeRPA73 subunit, while the WRN-1(162-1056) fragment that harbors the helicase activity requires both the CeRPA73 and CeRPA32 subunits for the stimulation. We also found that the CeRPA73(1-464) fragment can stimulate WRN-1 helicase activity and that residues 335-464 of CeRPA73 are important for physical interaction with WRN-1. Because CeRPA73 and the CeRPA73(1-464) fragment are able to bind single-stranded DNA (ssDNA), the stimulation of WRN-1 helicase by RPA-1 is most likely due to the ssDNA binding activity of CeRPA73 and the direct interaction of WRN-1 and CeRPA73.

Acetylation of malate dehydrogenase 1 promotes adipogenic differentiation via activating its enzymatic activity.

Acetylation is one of the most crucial post-translational modifications that affect protein function. Protein lysine acetylation is catalyzed by acetyltransferases, and acetyl-CoA functions as the source of the acetyl group. Additionally, acetyl-CoA plays critical roles in maintaining the balance between carbohydrate metabolism and fatty acid synthesis. Here, we sought to determine whether lysine acetylation is an important process for adipocyte differentiation. Based on an analysis of the acetylome during adipogenesis, various proteins displaying significant quantitative changes were identified by LC-MS/MS. Of these identified proteins, we focused on malate dehydrogenase 1 (MDH1). The acetylation level of MDH1 was increased up to 6-fold at the late stage of adipogenesis. Moreover, overexpression of MDH1 in 3T3-L1 preadipocytes induced a significant increase in the number of cells undergoing adipogenesis. The introduction of mutations to putative lysine acetylation sites showed a significant loss of the ability of cells to undergo adipogenic differentiation. Furthermore, the acetylation of MDH1 dramatically enhanced its enzymatic activity and subsequently increased the intracellular levels of NADPH. These results clearly suggest that adipogenic differentiation may be regulated by the acetylation of MDH1 and that the acetylation of MDH1 is one of the cross-talk mechanisms between adipogenesis and the intracellular energy level.

TRIM32 protein sensitizes cells to tumor necrosis factor (TNFα)-induced apoptosis via its RING domain-dependent E3 ligase activity against X-linked inhibitor of apoptosis (XIAP).

TRIM32, which belongs to the tripartite motif (TRIM) protein family, has the RING finger, B-box, and coiled-coil domain structures common to this protein family, along with an additional NHL domain at the C terminus. TRIM32 reportedly functions as an E3 ligase for actin, a protein inhibitor of activated STAT y (PIASy), dysbindin, and c-Myc, and it has been associated with diseases such as muscular dystrophy and epithelial carcinogenesis. Here, we identify a new substrate of TRIM32 and propose a mechanism through which TRIM32 might regulate apoptosis. Our overexpression and knockdown experiments demonstrate that TRIM32 sensitizes cells to TNFα-induced apoptosis. The RING domain is necessary for this pro-apoptotic function of TRM32 as well as being responsible for its E3 ligase activity. TRIM32 colocalizes and directly interacts with X-linked inhibitor of apoptosis (XIAP), a well known cancer therapeutic target, through its coiled-coil and NHL domains. TRIM32 overexpression enhances XIAP ubiquitination and subsequent proteasome-mediated degradation, whereas TRIM32 knockdown has the opposite effect, indicating that XIAP is a substrate of TRIM32. In vitro reconstitution assay reveals that XIAP is directly ubiquitinated by TRIM32. Our novel results collectively suggest that TRIM32 sensitizes TNFα-induced apoptosis by antagonizing XIAP, an anti-apoptotic downstream effector of TNFα signaling. This function may be associated with TRIM32-mediated tumor suppressive mechanism.

Soluble CD93 induces differentiation of monocytes and enhances TLR responses.

The cell surface protein CD93 is known to be involved in the regulation of phagocytosis and cell adhesion. Although typically membrane-bound, a soluble form of CD93 (sCD93) has recently been identified. Currently, however, the role of sCD93 in monocyte function is unknown. In the current study, we analyzed the functional effects of sCD93 on THP-1 monocytic cells and human primary monocytes. Various forms of recombinant human sCD93 were used to investigate the effects of this molecule on both human primary monocytes and a monocytic cell line, THP-1. We found that sCD93 induced differentiation of monocytes to macrophage-like cells, as evidenced by activated cell adhesion and increased phagocytic activities. In addition, this differentiation resulted in an enhanced response to TLR stimulation in terms of differentiation marker expression and proinflammatory cytokine production. Furthermore, sCD93 enhanced LPS-stimulated TNF-α production even prior to monocyte differentiation. To investigate a possible role for sCD93 in the pathogenesis of chronic inflammatory diseases, we assessed the concentration of sCD93 in synovial fluid from patients with rheumatoid arthritis and found it to be significantly increased compared with synovial fluid from patients with osteoarthritis. Together, these data revealed a function for sCD93 that may have implications in inflammation and inflammatory diseases including rheumatoid arthritis.

Genetic and functional interactions between Mus81-Mms4 and Rad27.

The two endonucleases, Rad27 (yeast Fen1) and Dna2, jointly participate in the processing of Okazaki fragments in yeasts. Mus81-Mms4 is a structure-specific endonuclease that can resolve stalled replication forks as well as toxic recombination intermediates. In this study, we show that Mus81-Mms4 can suppress dna2 mutational defects by virtue of its functional and physical interaction with Rad27. Mus81-Mms4 stimulated Rad27 activity significantly, accounting for its ability to restore the growth defects caused by the dna2 mutation. Interestingly, Rad27 stimulated the rate of Mus81-Mms4 catalyzed cleavage of various substrates, including regressed replication fork substrates. The ability of Rad27 to stimulate Mus81-Mms4 did not depend on the catalytic activity of Rad27, but required the C-terminal 64 amino acid fragment of Rad27. This indicates that the stimulation was mediated by a specific protein-protein interaction between the two proteins. Our in vitro data indicate that Mus81-Mms4 and Rad27 act together during DNA replication and resolve various structures that can impede normal DNA replication. This conclusion was further strengthened by the fact that rad27 mus81 or rad27 mms4 double mutants were synergistically lethal. We discuss the significance of the interactions between Rad27, Dna2 and Mus81-Mms4 in context of DNA replication.

Involvement of Vts1, a structure-specific RNA-binding protein, in Okazaki fragment processing in yeast.

The non-essential VTS1 gene of Saccharomyces cerevisiae is highly conserved in eukaryotes and encodes a sequence- and structure-specific RNA-binding protein. The Vts1 protein has been implicated in post-transcriptional regulation of a specific set of mRNAs that contains its-binding site at their 3'-untranslated region. In this study, we identified VTS1 as a multi-copy suppressor of dna2-K1080E, a lethal mutant allele of DNA2 that lacks DNA helicase activity. The suppression was allele-specific, since overexpression of Vts1 did not suppress the temperature-dependent growth defects of dna2Delta405N devoid of the N-terminal 405-amino-acid residues. Purified recombinant Vts1 stimulated the endonuclease activity of wild-type Dna2, but not the endonuclease activity of Dna2Delta405N, indicating that the activation requires the N-terminal domain of Dna2. Stimulation of Dna2 endonuclease activity by Vts1 appeared to be the direct cause of suppression, since the multi-copy expression of Dna2-K1080E suppressed the lethality observed with its single-copy expression. We found that vts1Delta dna2Delta405N and vts1Deltadna2-7 double mutant cells displayed synergistic growth defects, in support of a functional interaction between two genes. Our results provide both in vivo and in vitro evidence that Vts1 is involved in lagging strand synthesis by modulating the Dna2 endonuclease activity that plays an essential role in Okazaki fragment processing.