Ligation of newly replicated DNA controls the timing of DNA mismatch repair.

Mismatch repair (MMR) safeguards genome stability through recognition and excision of DNA replication errors. How eukaryotic MMR targets the newly replicated strand in vivo has not been established. MMR reactions reconstituted in vitro are directed to the strand containing a preexisting nick or gap, suggesting that strand discontinuities could act as discrimination signals.

Identification of MLH2/hPMS1 dominant mutations that prevent DNA mismatch repair function.

Inactivating mutations affecting key mismatch repair (MMR) components lead to microsatellite instability (MSI) and cancer. However, a number of patients with MSI-tumors do not present alterations in classical MMR genes. Here we discovered that specific missense mutations in the MutL homolog MLH2, which is dispensable for MMR, confer a dominant mutator phenotype in S.

Extensive 5'-surveillance guards against non-canonical NAD-caps of nuclear mRNAs in yeast.

The ubiquitous redox coenzyme nicotinamide adenine dinucleotide (NAD) acts as a non-canonical cap structure on prokaryotic and eukaryotic ribonucleic acids. Here we find that in budding yeast, NAD-RNAs are abundant (>1400 species), short (<170 nt), and mostly correspond to mRNA 5'-ends. The modification percentage of transcripts is low (<5%).

Inactivation of folylpolyglutamate synthetase Met7 results in genome instability driven by an increased dUTP/dTTP ratio.

The accumulation of mutations is frequently associated with alterations in gene function leading to the onset of diseases, including cancer. Aiming to find novel genes that contribute to the stability of the genome, we screened the Saccharomyces cerevisiae deletion collection for increased mutator phenotypes. Among the identified genes, we discovered MET7, which encodes folylpolyglutamate synthetase (FPGS), an enzyme that facilitates several folate-dependent reactions including the synthesis of purines, thymidylate (dTMP) and DNA methylation.

Alterations in cellular metabolism triggered by or inactivation cause imbalanced dNTP pools and increased mutagenesis.

Eukaryotic DNA replication fidelity relies on the concerted action of DNA polymerase nucleotide selectivity, proofreading activity, and DNA mismatch repair (MMR). Nucleotide selectivity and proofreading are affected by the balance and concentration of deoxyribonucleotide (dNTP) pools, which are strictly regulated by ribonucleotide reductase (RNR). Mutations preventing DNA polymerase proofreading activity or MMR function cause mutator phenotypes and consequently increased cancer susceptibility.

Visualization of mismatch repair complexes using fluorescence microscopy.

DNA mismatch repair (MMR) is a surveillance mechanism present in most living organisms, which repairs errors introduced by DNA polymerases. Importantly, loss of MMR function due to inactivating mutations and/or epigenetic silencing results in the accumulation of mutations and as consequence increased cancer susceptibility, as observed in Lynch syndrome patients. During the past decades important progress has been made in the MMR field resulting in the identification and characterization of essential MMR components, culminating in the in vitro reconstitution of 5' and 3' nick-directed MMR.

New insights into the mechanism of DNA mismatch repair.

The genome of all organisms is constantly being challenged by endogenous and exogenous sources of DNA damage. Errors like base:base mismatches or small insertions and deletions, primarily introduced by DNA polymerases during DNA replication are repaired by an evolutionary conserved DNA mismatch repair (MMR) system. The MMR system, together with the DNA replication machinery, promote repair by an excision and resynthesis mechanism during or after DNA replication, increasing replication fidelity by up-to-three orders of magnitude.

PCNA and Msh2-Msh6 activate an Mlh1-Pms1 endonuclease pathway required for Exo1-independent mismatch repair.

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.

Mlh2 is an accessory factor for DNA mismatch repair in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the essential mismatch repair (MMR) endonuclease Mlh1-Pms1 forms foci promoted by Msh2-Msh6 or Msh2-Msh3 in response to mispaired bases. Here we analyzed the Mlh1-Mlh2 complex, whose role in MMR has been unclear. Mlh1-Mlh2 formed foci that often colocalized with and had a longer lifetime than Mlh1-Pms1 foci.

Dominant mutations in S. cerevisiae PMS1 identify the Mlh1-Pms1 endonuclease active site and an exonuclease 1-independent mismatch repair pathway.

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.

Checkpoint kinases regulate a global network of transcription factors in response to DNA damage.

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.

Mismatch repair, but not heteroduplex rejection, is temporally coupled to DNA replication.

In eukaryotes, it is unknown whether mismatch repair (MMR) is temporally coupled to DNA replication and how strand-specific MMR is directed. 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 phase of the cell cycle. 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.

Visualization of eukaryotic DNA mismatch repair reveals distinct recognition and repair intermediates.

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 recruitment of the Mlh1-Pms1 complex. Here, we visualized functional fluorescent versions of Msh2-Msh6 and Mlh1-Pms1 in living cells.

SRD5A3 is required for converting polyprenol to dolichol and is mutated in a congenital glycosylation disorder.

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 (CDGs). We describe a new type of CDG caused by mutations in the steroid 5alpha-reductase type 3 (SRD5A3) gene. Patients have mental retardation and ophthalmologic and cerebellar defects.

Cdc28/Cdk1 positively and negatively affects genome stability in S. cerevisiae.

We studied the function of the cyclin-dependent kinase Cdc28 (Cdk1) in the DNA damage response and maintenance of genome stability using Saccharomyces cerevisiae. Reduced Cdc28 activity sensitizes cells to chronic DNA damage, but Cdc28 is not required for cell viability upon acute exposure to DNA-damaging agents. Cdc28 is also not required for activation of the DNA damage and replication checkpoints.

Generation of active protein phosphatase 2A is coupled to holoenzyme assembly.

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.

Identification of a subunit of a novel Kleisin-beta/SMC complex as a potential substrate of protein phosphatase 2A.

Protein phosphatase 2A (PP2A) holoenzymes consist of a catalytic C subunit, a scaffolding A subunit, and one of several regulatory B subunits that recruit the AC dimer to substrates. PP2A is required for chromosome segregation, but PP2A's substrates in this process remain unknown. To identify PP2A substrates, we carried out a two-hybrid screen with the regulatory B/PR55 subunit.