Summary DNA Mismatch Repair (MMR) increases replication fidelity by eliminating mispaired bases resulting from replication errors. In Saccharomyces cerevisiae mispairs are primarily detected by the Msh2-Msh6 complex and corrected following subsequent recruitment of the Mlh1-Pms1 complex. Here, we visualized functional fluorescent versions of Msh2-Msh6 and Mlh1-Pms1 in living cells. Msh2-Msh6 formed foci in S-phase that colocalized with replication factories; this localized pool accounted for 10–15% of MMR in wild-type cells but was essential for MMR in the absence of the exonuclease Exo1. Mlh1-Pms1 also formed foci that, while requiring Msh2-Msh6 for their formation, rarely colocalized with Msh2-Msh6. Mlh1-Pms1 foci increased when the number of mispaired bases was increased; in contrast, Msh2-Msh6 foci were unaffected. These results suggest that (I) mispair recognition can occur via either a replication factory-targeted or a second distinct pool of Msh2-Msh6, and (II) superstoichiometric Mlh1-Pms1 assembly triggered by mispair-bound Msh2-Msh6 defines sites of active MMR.

SUMMARY N-linked glycosylation is the most frequent modification of secreted and membrane-bound proteins in eukaryotic cells, disruption of which is the basis of the Congenital Disorders of Glycosylation (CDG). We describe a new type of CDG caused by mutations in the steroid 5α-reductase type 3 (SRD5A3) gene. Patients have mental retardation, ophthalmologic and cerebellar defects. We found that SRD5A3 is necessary for the reduction of the alpha-isoprene unit of polyprenols to form dolichols, required for synthesis of dolichol-linked monosaccharides and the oligosaccharide precursor used for N-glycosylation. The presence of residual dolichol in cells depleted for this enzyme suggests the existence of an unexpected alternative pathway for dolichol de novo biosynthesis. Our results thus suggest that SRD5A3 is likely to be the long-sought polyprenol reductase and reveal the genetic basis of one of the earliest steps in protein N-linked glycosylation.

Protein phosphatase 2A (PP2A) is an essential intracellular serine/threonine phosphatase containing a catalytic subunit that possesses the potential to dephosphorylate promiscuously tyrosine-phosphorylated substrates in vitro. How PP2A acquires its intracellular specificity and activity for serine/threonine-phosphorylated substrates is unknown. Here we report a novel and phylogenetically conserved mechanism to generate active phospho-serine/threonine-specific PP2A in vivo. Phosphotyrosyl phosphatase activator (PTPA), a protein of so far unknown intracellular function, is required for the biogenesis of active and specific PP2A. Deletion of the yeast PTPA homologs generated a PP2A catalytic subunit with a conformation different from the wild-type enzyme, as indicated by its altered substrate specificity, reduced protein stability, and metal dependence. Complementation and RNA-interference experiments showed that PTPA fulfills an essential function conserved from yeast to man. Protein phosphorylation is a posttranslational modification, mostly reversible, that is used in cells for the regulation of multiple processes. Analyses of eukaryotic genomes reveal that the genes coding for protein kinases, the enzymes catalyzing the phosphorylation reaction, outnumber by two-to threefold genes for protein phosphatases, the enzymes catalyzing dephosphorylation (Zolnierowicz 2000). Protein phosphatases counterbalance the activity of the large number of substrate-specific kinases by the combinatorial assembly of holoenzymes with different substrate specificity. Holoenzymes of a certain protein phosphatase family consist of a common catalytic subunit associated with different regulatory subunits that determine substrate targeting and modulate catalytic activity. Hence, the catalytic subunits of the major protein phosphatase families are produced in abundance. For instance, the catalytic subunit (C subunit) of protein phosphatase 2A (PP2A), comprises, dependent on the cell type, 0.3%-1% of total cellular protein (Virshup 2000).Based on its specificity for phosphorylated serine/ threonine residues, the PP2A C subunit belongs to the family of eukaryotic protein-serine/threonine phosphatases (PSTPs). PSTPs possess a catalytic core structure that is distinct from the core of protein tyrosine phosphatases (PTPs) and dual specificity phosphatases (DSPs). In consequence of the structural differences, the different protein phosphatase families use distinct catalytic mechanisms for the hydrolysis of the phosphoester bond. In contrast to PTPs (and the DSP subfamily) PSTPs are metallo-phosphoesterases that require metals in the active site for catalysis and for their structural integrity. When isolated from eukaryotic sources, the PSTP family members protein phosphatase 1 (PP1) and PP2A ("native" PP1 or PP2A) do not require the addition of metal ions for their activity. However, PP1 and PP2A convert into metal-dependent enzymes during long-term storage or on treatment with the phosphatase inhibitors ATP, pyrophosphate (PPi), or NaF (Burche...

Summary Genetic evidence has implicated multiple pathways in eukaryotic DNA mismatch repair (MMR) downstream of mispair recognition and Mlh1-Pms1 recruitment, including Exonuclease 1 (Exo1) dependent and independent pathways. We identified 14 mutations in POL30, which encodes PCNA in Saccharomyces cerevisiae, specific to Exo1-independent MMR. The mutations identified affected amino acids at three distinct sites on the PCNA structure. Multiple mutant PCNA proteins had defects either in trimerization and Msh2-Msh6 binding or in activation of the Mlh1-Pms1 endonuclease that initiates excision during MMR. The latter class of mutations led to hyper-accumulation of repair intermediate Mlh1-Pms1 foci and were enhanced by an msh6 mutation that disrupted the Msh2-Msh6 interaction with PCNA. These results reveal a central role for PCNA in the Exo1-independent MMR pathway and suggest that Msh2-Msh6 localizes PCNA to repair sites after mispair recognition to activate the Mlh1-Pms1 endonuclease for initiating Exo1-dependent repair or for driving progressive excision in Exo1-independent repair.

In eukaryotes, it is unknown if mismatch repair (MMR) is temporally coupled to DNA replication and how strand-specific MMR is directed. Here we fused Saccharomyces cerevisiae MSH6 with cyclins to restrict the availability of the Msh2-Msh6 mismatch recognition complex to either S-phase or G2/M. The Msh6-S cyclin fusion was proficient for suppressing mutations at three loci that replicate at mid-S-phase, whereas the Msh6-G2/M cyclin fusion was defective. However, the Msh6-G2/M cyclin fusion was functional for MMR at a very late-replicating region of the genome. In contrast, the heteroduplex rejection function of MMR during recombination was partially functional during both S-phase and G2/M. These results indicate a temporal coupling of MMR, but not heteroduplex rejection, to DNA replication.

Protein phosphatase 2A (PP2A) is a prime example of the multisubunit architecture of protein serine/threonine phosphatases. Until substrate-specific PP2A holoenzymes assemble, a constitutively active, but nonspecific, catalytic C subunit would constitute a risk to the cell. While it has been assumed that the severe proliferation impairment of yeast lacking the structural PP2A subunit, TPD3, is due to the unrestricted activity of the C subunit, we recently obtained evidence for the existence of the C subunit in a low-activity conformation that requires the RRD/PTPA proteins for the switch into the active conformation. To study whether and how maturation of the C subunit is coupled with holoenzyme assembly, we analyzed PP2A biogenesis in yeast. Here we show that the generation of the catalytically active C subunit depends on the physical and functional interaction between RRD2 and the structural subunit, TPD3. The phenotype of the tpd3Δ strain is therefore caused by impaired, rather than increased, PP2A activity. TPD3/RRD2-dependent C subunit maturation is under the surveillance of the PP2A methylesterase, PPE1, which upon malfunction of PP2A biogenesis, prevents premature generation of the active C subunit and holoenzyme assembly by counteracting the untimely methylation of the C subunit. We propose a novel model of PP2A biogenesis in which a tightly controlled activation cascade protects cells from untargeted activity of the free catalytic PP2A subunit.

SUMMARY DNA damage activates checkpoint kinases that induce several downstream events, including widespread changes in transcription. However, the specific connections between the checkpoint kinases and downstream transcription factors (TFs) are not well understood. Here, we integrate kinase mutant expression profiles, transcriptional regulatory interactions, and phosphoproteomics to map kinases and downstream TFs to transcriptional regulatory networks. Specifically, we investigate the role of the Saccharomyces cerevisiae checkpoint kinases (Mec1, Tel1, Chk1, Rad53, and Dun1) in the transcriptional response to DNA damage caused by methyl methanesulfonate. The result is a global kinase-TF regulatory network in which Mec1 and Tel1 signal through Rad53 to synergistically regulate the expression of more than 600 genes. This network involves at least nine TFs, many of which have Rad53-dependent phosphorylation sites, as regulators of checkpoint-kinase-dependent genes. We also identify a major DNA damage-induced transcriptional network that regulates stress response genes independently of the checkpoint kinases.

Lynch syndrome (hereditary nonpolypsis colorectal cancer or HNPCC) is a common cancer predisposition syndrome. Predisposition to cancer in this syndrome results from increased accumulation of mutations due to defective mismatch repair (MMR) caused by a mutation in one of the mismatch repair genes MLH1, MSH2, MSH6 or PMS2/scPMS1. To better understand the function of Mlh1-Pms1 in MMR, we used Saccharomyces cerevisiae to identify six pms1 mutations (pms1-G683E, pms1-C817R, pms1-C848S, pms1-H850R, pms1-H703A and pms1-E707A) that were weakly dominant in wild-type cells, which surprisingly caused a strong MMR defect when present on low copy plasmids in an exo1Δ mutant. Molecular modeling showed these mutations caused amino acid substitutions in the metal coordination pocket of the Pms1 endonuclease active site and biochemical studies showed that they inactivated the endonuclease activity. This model of Mlh1-Pms1 suggested that the Mlh1-FERC motif contributes to the endonuclease active site. Consistent with this, the mlh1-E767stp mutation caused both MMR and endonuclease defects similar to those caused by the dominant pms1 mutations whereas mutations affecting the predicted metal coordinating residue Mlh1-C769 had no effect. These studies establish that the Mlh1-Pms1 endonuclease is required for MMR in a previously uncharacterized Exo1-independent MMR pathway.