Mark C. Hall

The Escherichia coli MutL Protein Physically Interacts with MutH and Stimulates the MutH-associated Endonuclease Activity

All possible pairwise combinations of UvrD, MutL, MutS, and MutH were tested using the yeast two-hybrid system to identify potential interactions involving mismatch repair proteins. A two-hybrid screen previously identified a physical interaction between MutL and UvrD. Although several other known interactions were not observed, a novel interaction between MutL and MutH was detected. A series of truncations from the NH2 and COOH termini of MutL demonstrated that the COOH-terminal 218 amino acids were sufficient for the two-hybrid interaction with MutH. Removal of a small number of residues from either the NH2 or COOH termini of MutH eliminated the two-hybrid interaction with MutL. Protein affinity chromatography experiments confirmed that MutL, but not MutS, physically associates with MutH. Furthermore, MutL greatly stimulated the d(GATC)-specific endonuclease activity of MutH in the absence of MutS and a mispaired base. Stimulation of the MutH-associated endonuclease activity by MutL was dependent on ATP binding but not ATP hydrolysis. Further stimulation of this reaction by MutS required the presence of a DNA mismatch and a hydrolyzable form of ATP. These results suggest that MutL activates the MutH-associated endonuclease activity through a physical interaction during methyl-directed mismatch repair in Escherichia coli.

Evidence for a physical interaction between the Escherichia coli methyl-directed mismatch repair proteins MutL and UvrD

UvrD (DNA helicase II) is an essential component of two major DNA repair pathways in Escherichia coli: methyl‐directed mismatch repair and UvrABC‐mediated nucleotide excision repair. In addition, it has an undefined role in the RecF recombination pathway and possibly in replication. In an effort to better understand the role of UvrD in these various aspects of DNA metabolism, a yeast two‐hybrid screen was used to search for interacting protein partners. Screening of an E.coli genomic library revealed a potential interaction between UvrD and MutL, a component of the methyl‐directed mismatch repair pathway. The interaction was confirmed by affinity chromatography using purified proteins. Deletion analysis demonstrated that the C‐terminal 218 amino acids (residues 398–615) of MutL were sufficient to produce the two‐hybrid interaction with UvrD. On the other hand, both the N‐ and C‐termini of UvrD were required for interaction with MutL. The implications of this interaction for the mismatch repair mechanism are discussed.

Escherichia coli DNA Helicase II Is Active as a Monomer

Helicases are thought to function as oligomers (generally dimers or hexamers). Here we demonstrate that althoughEscherichia coli DNA helicase II (UvrD) is capable of dimerization as evidenced by a positive interaction in the yeast two-hybrid system, gel filtration chromatography, and equilibrium sedimentation ultracentrifugation (K d = 3.4 μm), the protein is active in vivo andin vitro as a monomer. A mutant lacking the C-terminal 40 amino acids (UvrDΔ40C) failed to dimerize and yet was as active as the wild-type protein in ATP hydrolysis and helicase assays. In addition, the uvrDΔ40C allele fully complemented the loss of helicase II in both methyl-directed mismatch repair and excision repair of pyrimidine dimers. Biochemical inhibition experiments using wild-type UvrD and inactive UvrD point mutants provided further evidence for a functional monomer. This investigation provides the first direct demonstration of an active monomeric helicase, and a model for DNA unwinding by a monomer is presented.

Acm1 Is a Negative Regulator of the Cdh1-Dependent Anaphase-Promoting Complex/Cyclosome in Budding Yeast

ABSTRACT Cdh1 is a coactivator of the anaphase-promoting complex/cyclosome (APC/C) and contributes to mitotic exit and G1 maintenance by facilitating the polyubiquitination and subsequent proteolysis of specific substrates. Here, we report that budding yeast Cdh1 is a component of a cell cycle-regulated complex that includes the 14-3-3 homologs Bmh1 and Bmh2 and a previously uncharacterized protein, which we name Acm1 (APC/CCdh1modulator 1). Association of Cdh1 with Bmh1 and Bmh2 requires Acm1, and the Acm1 protein is cell cycle regulated, appearing late in G1 and disappearing in late M. In acm1Δ strains, Cdh1 localization to the bud neck and association with two substrates, Clb2 and Hsl1, were strongly enhanced. Several lines of evidence suggest that Acm1 can suppress APC/CCdh1-mediated proteolysis of mitotic cyclins. First, overexpression of Acm1 fully restored viability to cells expressing toxic levels of Cdh1 or a constitutively active Cdh1 mutant lacking inhibitory phosphorylation sites. Second, overexpression of Acm1 was toxic in sic1Δ cells. Third, ACM1 deletion exacerbated a low-penetrance elongated-bud phenotype caused by modest overexpression of Cdh1. This bud elongation was independent of the morphogenesis checkpoint, and the combination of acm1Δ and hsl1Δ resulted in a dramatic enhancement of bud elongation and G2/M delay. Effects on bud elongation were attenuated when Cdh1 was replaced with a mutant lacking the C-terminal IR dipeptide, suggesting that APC/C-dependent proteolysis is required for this phenotype. We propose that Acm1 and Bmh1/Bmh2 constitute a specialized inhibitor of APC/CCdh1.

Unique D Box and KEN Box Sequences Limit Ubiquitination of Acm1 and Promote Pseudosubstrate Inhibition of the Anaphase-promoting Complex

The anaphase-promoting complex (APC) regulates cell division in eukaryotes by targeting specific proteins for destruction. APC substrates generally contain one or more short degron sequences that help mediate their recognition and poly-ubiquitination by the APC. The most common and well characterized degrons are the destruction box (D box) and the KEN box. The budding yeast Acm1 protein, an inhibitor of Cdh1-activated APC (APCCdh1) also contains several conserved D and KEN boxes, and here we report that two of these located in the central region of Acm1 constitute a pseudosubstrate sequence required for APCCdh1 inhibition. Acm1 interacted with and inhibited substrate binding to the WD40 repeat domain of Cdh1. Combined mutation of the central D and KEN boxes strongly reduced both binding to the Cdh1 WD40 domain and APCCdh1 inhibition. Despite this, the double mutant, but not wild-type Acm1, was poly-ubiquitinated by APCCdh1 in vitro. Thus, unlike substrates in which D and KEN boxes promote ubiquitination, these same elements in the central region of Acm1 prevent ubiquitination. We propose that this unique property of the Acm1 degron sequences results from an unusually high affinity interaction with the substrate receptor site on the WD40 domain of Cdh1 that may serve both to promote APC inhibition and protect Acm1 from destruction.

Cdc28 and Cdc14 Control Stability of the Anaphase-promoting Complex Inhibitor Acm1

The anaphase-promoting complex (APC) regulates the eukaryotic cell cycle by targeting specific proteins for proteasomal degradation. Its activity must be strictly controlled to ensure proper cell cycle progression. The co-activator proteins Cdc20 and Cdh1 are required for APC activity and are important regulatory targets. Recently, budding yeast Acm1 was identified as a Cdh1 binding partner and APCCdh1 inhibitor. Acm1 disappears in late mitosis when APCCdh1 becomes active and contains conserved degron-like sequences common to APC substrates, suggesting it could be both an inhibitor and substrate. Surprisingly, we found that Acm1 proteolysis is independent of APC. A major determinant of Acm1 stability is phosphorylation at consensus cyclin-dependent kinase sites. Acm1 is a substrate of Cdc28 cyclin-dependent kinase and Cdc14 phosphatase both in vivo and in vitro. Mutation of Cdc28 phosphorylation sites or conditional inactivation of Cdc28 destabilizes Acm1. In contrast, inactivation of Cdc14 prevents Acm1 dephosphorylation and proteolysis. Cdc28 stabilizes Acm1 in part by promoting binding of the 14-3-3 proteins Bmh1 and Bmh2. We conclude that the opposing actions of Cdc28 and Cdc14 are primary factors limiting Acm1 to the interval from G1/S to late mitosis and are capable of establishing APC-independent expression patterns similar to APC substrates.

Mutation of a highly conserved arginine in motif IV of Escherichia coli DNA helicase II results in an ATP-binding defect.

A site-directed mutation in motif IV ofEscherichia coli DNA helicase II (UvrD) was generated to examine the functional significance of this region. The highly conserved arginine at position 284 was replaced with alanine to construct UvrD-R284A. The ability of the mutant allele to function in methyl-directed mismatch repair and UvrABC-mediated nucleotide excision repair was examined by genetic complementation assays. The R284A substitution abolished function in both DNA repair pathways. To identify the biochemical defects responsible for the loss of biological function, UvrD-R284A was purified to apparent homogeneity, and its biochemical properties were compared with wild-type UvrD. UvrD-R284A failed to unwind a 92-base pair duplex region and was severely compromised in unwinding a 20-base pair duplex region. TheK m of UvrD-R284A for ATP was significantly greater than 3 mm compared with 80 μm for UvrD. A large decrease in ATP binding was confirmed using a nitrocellulose filter binding assay. These data suggested that the R284A mutation severely reduced the affinity of helicase II for ATP. The reduced unwinding activity and loss of biological function of UvrD-R284A was probably the result of decreased affinity for ATP. These results implicate motif IV of superfamily I helicases in nucleotide binding and represent the first characterization of a helicase mutation outside motifs I and II that severely impacted the K m for ATP.

Multi-Kinase Phosphorylation of the APC/C Activator Cdh1 Revealed by Mass Spectrometry

by activating a large ubiquitin ligase called the anaphase-promoting complex, or cyclosome (APC/C). At the end of G1, APC/CCdh1 is inhibited by cyclin-dependent kinase (CDK) phosphorylation of Cdh1. The specific Cdh1 phosphorylation sites used to regulate APC/CCdh1 activity have not been directly identified. Here, we used a mass spectrometric approach to identify the in vivo phosphorylation sites on yeast Cdh1. Surprisingly, in addition to several expected CDK phosphorylation sites, we discovered numerous non-CDK phosphorylation sites. In total, at least 19 serine and threonine residues on Cdh1 are phosphorylated in vivo. Seventeen of these sites are located in the N-terminal half of Cdh1, outside the highly conserved WD40 repeats. The pattern of phosphorylation was the same when Cdh1 was purified from yeast cultures arrested in S, early M and late M. Mutation of CDK consensus sequences eliminated detectable phosphorylation at many of the non-CDK sites. In contrast, mutation of non-CDK sites had no significant effect on CDK phosphorylation. We conclude that phosphorylation of CDK sites promotes the subsequent recognition of Cdh1 by at least one additional kinase. The function of non-CDK phosphorylation may differ from CDK phosphorylation because mutation of non-CDK sites did not result in constitutive activation of APC and consequent cell cycle arrest. These results suggest that phosphoregulation of APC/CCdh1 activity is much more complex than previously thought.

Time-Dependent Activation of Phox2a by the Cyclic AMP Pathway Modulates Onset and Duration of p27Kip1 Transcription

ABSTRACT In noradrenergic progenitors, Phox2a mediates cell cycle exit and neuronal differentiation by inducing p27Kip1 transcription in response to activation of the cyclic AMP (cAMP) pathway. The mechanism of cAMP-mediated activation of Phox2a is unknown. We identified a cluster of phosphoserine-proline sites in Phox2a by mass spectrometry. Ser206 appeared to be the most prominent phosphorylation site. A phospho-Ser206 Phox2a antibody detected dephosphorylation of Phox2a that was dependent on activation of the cAMP pathway, which occurred prior to neuronal differentiation of noradrenergic CAD cells. Employing serine-to-alanine and serine-to-aspartic acid Phox2a substitution mutants expressed in inducible CAD cell lines, we demonstrated that the transcriptional activity of Phox2a is regulated by two sequential cAMP-dependent events: first, cAMP signaling promotes dephosphorylation of Phox2a in at least one site, Ser206, thereby allowing Phox2a to bind DNA and initiate p27Kip1 transcription; second, following dephosphorylation of the phosphoserine cluster (Ser202 and Ser208), Phox2a becomes phosphorylated by protein kinase A (PKA) on Ser153, which prevents association of Phox2a with DNA and terminates p27Kip1 transcription. This represents a novel mechanism by which the same stimulus, cAMP signaling, first activates Phox2a by dephosphorylation of Ser206 and then, after a built-in delay, inactivates Phox2a via PKA-dependent phosphorylation of Ser153, thereby modulating onset and duration of p27Kip1 transcription.