Tuan Anh Nguyen

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.

Human Replication Factor C Stimulates Flap Endonuclease 1

Flap endonuclease 1 (FEN1) is the enzyme responsible for specifically removing the flap structure produced during DNA replication, repair, and recombination. Here we report that the human replication factor C (RFC) complex stimulates the nuclease activity of human FEN1 in an ATP-independent manner. Although proliferating cell nuclear antigen is also known to stimulate FEN1, less RFC was required for comparable FEN1 stimulation. Kinetic analyses indicate that the mechanism by which RFC stimulates FEN1 is distinct from that by proliferating cell nuclear antigen. Heat-denatured RFC or its subunit retained, fully or partially, the ability to stimulate FEN1. Via systematic deletion analyses, we have defined three specific regions of RFC4 capable of stimulating FEN1. The region of RFC4 with the highest activity spans amino acids 170–194 and contains RFC box VII. Four amino acid residues (i.e. Tyr-182, Glu-188, Pro-189, and Ser-192) are especially important for FEN1 stimulatory activity. Thus, RFC, via several stimulatory motifs per molecule, potently activates FEN1. This function makes RFC a critical partner with FEN1 for the processing of eukaryotic Okazaki fragments.

Human MUS81 complexes stimulate flap endonuclease 1

The yeast heterodimeric Mus81–Mms4 complex possesses a structure‐specific endonuclease activity that is critical for the restart of stalled replication forks and removal of toxic recombination intermediates. Previously, we reported that Mus81–Mms4 and Rad27 (yeast FEN1, another structure‐specific endonuclease) showed mutual stimulation of nuclease activity. In this study, we investigated the interactions between human FEN1 and MUS81–EME1 or MUS81–EME2, the human homologs of the yeast Mus81–Mms4 complex. We found that both MUS81–EME1 and MUS81–EME2 increased the activity of FEN1, but FEN1 did not stimulate the activity of MUS81–EME1/EME2. The MUS81 subunit alone and its N‐terminal half were able to bind to FEN1 and stimulate its endonuclease activity. A truncated FEN1 fragment lacking the C‐terminal region that retained catalytic activity was not stimulated by MUS81. Michaelis–Menten kinetic analysis revealed that MUS81 increased the interaction between FEN1 and its substrates, resulting in increased turnover. We also showed that, after DNA damage in human cells, FEN1 co‐localizes with MUS81. These findings indicate that the human proteins and yeast homologs act similarly, except that the human FEN1 does not stimulate the nuclease activities of MUS81–EME1 or MUS81–EME2. Thus, the mammalian MUS81 complexes and FEN1 collaborate to remove the various flap structures that arise during many DNA transactions, including Okazaki fragment processing.

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 dna2Δ405N 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 Dna2Δ405N, 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 vts1Δ dna2Δ405N and vts1Δdna2-7 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.

Analysis of subunit assembly and function of the Saccharomyces cerevisiae RNase H2 complex

RNase H2 of Saccharomyces cerevisiae consists of three essential subunits (Rnh201, Rnh202 and Rnh203) and plays a critical role in the removal of RNA incorporated in duplex DNA. In the present study, we purified individual subunits and heterodimeric subcomplexes to examine the assembly and biochemical function of subunits of RNase H2 in vitro. Reconstitution experiments revealed that Rnh202 and Rnh203 first form a subcomplex, followed by the recruitment of Rnh201 to complete complex formation. Rnh201 alone or in combination with Rnh203 showed neither substrate‐binding, nor catalytic activity, indicating that both activities of Rnh201 are latent until it becomes an integral part of the complex. However, Rnh202 by itself showed substrate‐binding activity. RNase H2 containing mutant Rnh202 defective in substrate binding had decreased substrate‐binding activity, indicating that Rnh202 contributes directly to substrate binding. Reconstitution of RNase H2 complexes with various mutant subunits allowed us to assess the influence of conserved amino acid residues in either Rnh201 or Rnh202 on substrate‐binding and catalytic activities. We found that the substrate‐binding activities of both Rnh201 and Rnh202 were critical for cleavage of the phosphodiester bond present between DNA and RNA in RNase H2 substrates.

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.

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

Background: The significance of physical/genetic interactions between Dna2 and Fen1 is poorly understood. Results: Fen1 stimulated the endonuclease activity of both Dna2 and Fen1 itself. Conclusion: Fen1 is a bona fide trans-autostimulatory enzyme, and this activity resides within the C-terminal 16 amino acids of Rad27. Significance: Our finding provides a novel insight into how Dna2 and Rad27 jointly contribute to processing of Okazaki fragments. 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.

SRSF3 recruits DROSHA to the basal junction of primary microRNAs

The Microprocessor complex, consisting of an RNase III DROSHA and the DGCR8 dimer, cleaves primary microRNA transcripts (pri-miRNAs) to initiate microRNA (miRNA) maturation. Pri-miRNAs are stem-loop RNAs, and ∼79% of them contain at least one of the three major and conserved RNA motifs, UG, UGU, and CNNC. We recently demonstrated that the basal UG and apical UGU motifs of pri-miRNAs interact with DROSHA and DGCR8, respectively. They help orient Microprocessor on pri-miRNA in a proper direction in which DROSHA and DGCR8 localize to the basal and apical pri-miRNA junctions, respectively. In addition, CNNC, located at ∼17 nucleotides (nt) from the Microprocessor cleavage site, interacts with SRSF3 (SRp20) to stimulate Microprocessor to process pri-miRNAs. The mechanism underlying this stimulation, however, is unknown. In this study, we discovered that SRSF3 recruits DROSHA to the basal junction in a CNNC-dependent manner, thereby enhancing Microprocessor activity. Furthermore, by generating various pri-miRNA substrates containing CNNC at different locations, we demonstrated that such stimulation only occurs when CNNC is located at ∼17 nt from the Microprocessor cleavage site. Our findings reveal the molecular mechanism of SRSF3 in pri-miRNA processing and support the previously proposed explanation for the highly conserved position of CNNC in SRSF3-enhanced pri-miRNA processing.

Microprocessor depends on hemin to recognize the apical loop of primary microRNA

Abstract Microprocessor, which consists of a ribonuclease III DROSHA and its cofactor DGCR8, initiates microRNA (miRNA) maturation by cleaving primary miRNA transcripts (pri-miRNAs). We recently demonstrated that the DGCR8 dimer recognizes the apical elements of pri-miRNAs, including the UGU motif, to accurately locate and orient Microprocessor on pri-miRNAs. However, the mechanism underlying the selective RNA binding remains unknown. In this study, we find that hemin, a ferric ion-containing porphyrin, enhances the specific interaction between the apical UGU motif and the DGCR8 dimer, allowing Microprocessor to achieve high efficiency and fidelity of pri-miRNA processing in vitro. Furthermore, by generating a DGCR8 mutant cell line and carrying out rescue experiments, we discover that hemin preferentially stimulates the expression of miRNAs possessing the UGU motif, thereby conferring differential regulation of miRNA maturation. Our findings reveal the molecular action mechanism of hemin in pri-miRNA processing and establish a novel function of hemin in inducing specific RNA-protein interaction.