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R D Kolodner

Publications and source records attributed to R D Kolodner.

At least 37 records · Page 2Linked to original sources
PubMed2000

Sensitivity and specificity of clinical criteria for hereditary non-polyposis colorectal cancer associated mutations in MSH2 and MLH1.

BACKGROUND AND AIMS: There are multiple criteria for the clinical diagnosis of hereditary non-polyposis colorectal cancer (HNPCC). The value of several of the newer proposed diagnostic criteria in identifying subjects with mutations in HNPCC associated mismatch repair genes has not been evaluated, and the performance of the different criteria have not been formally compared with one another. METHODS: We classified 70 families with suspected hereditary colorectal cancer (excluding familial adenomatous polyposis) by several existing clinical criteria for HNPCC, including the Amsterdam criteria, the Modified Amsterdam criteria, the Amsterdam II criteria, and the Bethesda criteria. The results of analysis of the mismatch repair genes MSH2 and MLH1 by full gene sequencing were available for a proband with colorectal neoplasia in each family. The sensitivity and specificity of each of the clinical criteria for the presence of MSH2 and MLH1 mutations were calculated. RESULTS: Of the 70 families, 28 families fulfilled the Amsterdam criteria, 39 fulfilled the Modified Amsterdam Criteria, 34 fulfilled the Amsterdam II criteria, and 56 fulfilled at least one of the seven Bethesda Guidelines for the identification of HNPCC patients. The sensitivity and specificity of the Amsterdam criteria were 61% (95% CI 43-79) and 67% (95% CI 50-85). The sensitivity of the Modified Amsterdam and Amsterdam II criteria were 72% (95% CI 58-86) and 78% (95% CI 64-92), respectively. Overall, the most sensitive criteria for identifying families with pathogenic mutations were the Bethesda criteria, with a sensitivity of 94% (95% CI 88-100); the specificity of these criteria was 25% (95% CI 14-36). Use of the first three criteria of the Bethesda guidelines only was associated with a sensitivity of 94% and a specificity of 49% (95% CI 34-64). CONCLUSIONS: The Amsterdam criteria for HNPCC are neither sufficiently sensitive nor specific for use as a sole criterion for determining which families should undergo testing for MSH2 and MLH1 mutations. The Modified Amsterdam and the Amsterdam II criteria increase sensitivity, but still miss many families with mutations. The most sensitive clinical criteria for identifying subjects with pathogenic MSH2 and MLH1 mutations were the Bethesda Guidelines; a streamlined version of the Bethesda Guidelines may be more specific and easier to use in clinical practice.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Germ-line msh6 mutations in colorectal cancer families.

Hereditary nonpolyposis colorectal carcinoma (HNPCC) is due primarily to inherited mutations in two mismatch repair genes, MSH2 and MLH1, whereas germ-line mutations in other mismatch repair genes are rare. We examined the frequency of germ-line msh6 mutations in a population-based series of 140 colorectal cancer patients, including 45 sporadic cases, 91 familial non-HNPCC cases, and 4 HNPCC cases. Among the 91 population-based familial non-HNPCC cases, germ-line msh6 mutations were found in 6 patients (7.1% of probands analyzed; median age at diagnosis, 61 years). These mutations included a splice site mutation, a frameshift mutation, two missense mutations that were demonstrated to be loss of function mutations, and two missense mutations for which functional studies were not possible. In contrast, germ-line msh6 mutations were not found in any of the 45 sporadic cases and the 4 HNPCC cases in the population-based series or in the second series of 58 clinic-based, primarily HNPCC families. Our data suggest that germ-line msh6 mutations predispose individuals to primarily late-onset, familial colorectal carcinomas that do not fulfill classic criteria for HNPCC.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Biochemical characterization of the interaction between the Saccharomyces cerevisiae MSH2-MSH6 complex and mispaired bases in DNA.

The interaction of the Saccharomyces cerevisiae MSH2-MSH6 complex with mispaired bases was analyzed using gel mobility shift assays and surface plasmon resonance methods. Under equilibrium binding conditions, MSH2-MSH6 bound to homoduplex DNA with a K(d) of 3.9 nM and bound oligonucleotide duplexes containing T:G, +1, +2, +4, and +10 insertion/deletion loop (IDL) mispairs with K(d) values of 0.20, 0.25, 11, 3.2, and 0.55 nM, respectively. Competition binding experiments using 65 different substrates revealed a 10-fold range in mispair discrimination. In general, base-base mispairs and a +1 insertion/deletion mispair were recognized better than intermediate sized insertion/deletion mispairs of 2-8 bases. Larger IDL mispairs (>8 bases) were recognized almost as well as the +1 IDL mispair. Recognition of mispairs by MSH2-MSH6 was influenced by sequence context, with the 6-nucleotide region surrounding the mispair being primarily responsible for influencing mispair recognition. Effects of sequences as far away as 15 nucleotides were also observed. Differential effects of ATP on the stability of MSH2-MSH6-mispair complexes suggested that base-base mispairs and the smaller IDL mispairs were recognized by a different binding mode than larger IDL mispairs, consistent with genetic experiments indicating that MSH2-MSH6 functions primarily in the repair of base-base and small IDL mispairs.

Adenosine Diphosphate↗
PubMed1999

Interpretation of genetic test results for hereditary nonpolyposis colorectal cancer: implications for clinical predisposition testing.

CONTEXT: Genetic testing for cancer predisposition is evolving from purely research applications to affecting clinical management. OBJECTIVE: To determine how often genetic test results for hereditary nonpolyposis colorectal cancer (HNPCC) can be definitively interpreted and used to guide clinical management. DESIGN: Case-series study conducted in 1996 to 1998 in which a complete sequence analysis of hMSH2 and hMLH1 coding sequence and flanking intronic regions was performed. Mutations were categorized as protein truncating and missense. In the case of missense alterations, additional analyses were performed in an effort to assess pathogenicity. SETTING AND PARTICIPANTS: Families were identified by self-referral or health care provider referral to a cancer genetics program. Participants and kindreds were classified into 1 of 4 categories: (1) Amsterdam criteria for HNPCC, (2) modified Amsterdam criteria for HNPCC, (3) young age at onset, or (4) HNPCC-variant. In addition, each proband was classified according to the Bethesda guidelines for identification of individuals with HNPCC. MAIN OUTCOME MEASURE: Alterations of hMSH2 and hMLH1 genes. RESULTS: Twenty-seven alterations of hMSH2 and hMLH1 were found in 24 of 70 families (34.3%). Of these, deleterious mutations that could be used with confidence in clinical management were identified in 25.7% (18/70) of families. The rates of definitive results for families fulfilling Amsterdam criteria, modified Amsterdam criteria, young age at onset, HNPCC-variant, and Bethesda guidelines were 27 (39.3%), 13 (18.2%), 12 (16.7%), 11 (15.8%), and 21 (30.4%), respectively. The prevalence of missense mutations, genetic heterogeneity of the syndrome, and limited availability of validated functional assays present a challenge in the interpretation of genetic test results of HNPCC families. CONCLUSIONS: The identification of pathogenic mutations in a significant subset of families for whom the results may have marked clinical importance makes genetic testing an important option for HNPCC and HNPCC-like kindreds. However, for the majority of individuals in whom sequence analysis of hMSH2 and hMLH1 does not give a definitive result, intensive follow-up is still warranted.

Adaptor Proteins, Signal Transducing↗
PubMed1999

'Saccharomyces cerevisiae MSH2/6 complex interacts with Holliday junctions and facilitates their cleavage by phage resolution enzymes.

Genetic and biochemical studies have indicated that mismatch repair proteins can interact with recombination intermediates. In this study, gel shift assays and electron microscopic analysis were used to show that the Saccharomyces cerevisiae MSH2/6 complex binds to Holliday junctions and has an affinity and specificity for them that is at least as high as it has as for mispaired bases. Under equilibrium binding conditions, the MSH2/6 complex had a Kd of binding to Holliday junctions of 0.5 nM. The MSH2/6 complex enhanced the cleavage of Holliday junctions by T4 endonuclease VII and T7 endonuclease I. This is consistent with the view that the MSH2/6 complex can function in both mismatch repair and the resolution of recombination intermediates as predicted by genetic studies.

Base Sequence↗
PubMed1999

Eukaryotic DNA mismatch repair.

Eukaryotic mismatch repair (MMR) has been shown to require two different heterodimeric complexes of MutS-related proteins: MSH2-MSH3 and MSH2-MSH6. These two complexes have different mispair recognition properties and different abilities to support MMR. Alternative models have been proposed for how these MSH complexes function in MMR. Two different heterodimeric complexes of MutL-related proteins, MLH1-PMS1 (human PMS2) and MLH1-MLH3 (human PMS1) also function in MMR and appear to interact with other MMR proteins including the MSH complexes and replication factors. A number of other proteins have been implicated in MMR, including DNA polymerase delta, RPA (replication protein A), PCNA (proliferating cell nuclear antigen), RFC (replication factor C), Exonuclease 1, FEN1 (RAD27) and the DNA polymerase delta and epsilon associated exonucleases. MMR proteins have also been shown to function in other types of repair and recombination that appear distinct from MMR. MMR proteins function in these processes in conjunction with components of nucleotide excision repair (NER) and, possibly, recombination.

Animals↗
PubMed1999

MSH2 and MSH6 are required for removal of adenine misincorporated opposite 8-oxo-guanine in S. cerevisiae.

Oxidation of G in DNA yields 8-oxo-G (GO), a mutagenic lesion that leads to misincorporation of A opposite GO. In E. coli, GO in GO:C base pairs is removed by MutM, and A in GO:A mispairs is removed by MutY. In S. cerevisiae, mutations in MSH2 or MSH6 caused a synergistic increase in mutation rate in combination with mutations in OGG1, which encodes a MutM homolog, resulting in a 140- to 218-fold increase in the G:C-to-T:A transversion rate. Consistent with this, MSH2-MSH6 complex bound to GO:A mispairs and GO:C base pairs with high affinity and specificity. These data indicate that in S. cerevisiae, MSH2-MSH6-dependent mismatch repair is the major mechanism by which misincorporation of A opposite GO is corrected.

Adenine↗
PubMed1999

Gross chromosomal rearrangements in Saccharomyces cerevisiae replication and recombination defective mutants.

Cancer progression is often associated with the accumulation of gross chromosomal rearrangements (GCRs), such as translocations, deletion of a chromosome arm, interstitial deletions or inversions. In many instances, GCRs inactivate tumour-suppressor genes or generate novel fusion proteins that initiate carcinogenesis. The mechanism underlying GCR formation appears to involve interactions between DNA sequences of little or no homology. We previously demonstrated that mutations in the gene encoding the largest subunit of the Saccharomyces cerevisiae single-stranded DNA binding protein (RFA1) increase microhomology-mediated GCR formation. To further our understanding of GCR formation, we have developed a novel mutator assay in S. cerevisiae that allows specific detection of such events. In this assay, the rate of GCR formation was increased 600-5, 000-fold by mutations in RFA1, RAD27, MRE11, XRS2 and RAD50, but was minimally affected by mutations in RAD51, RAD54, RAD57, YKU70, YKU80, LIG4 and POL30. Genetic analysis of these mutants suggested that at least three distinct pathways can suppress GCRs: two that suppress microhomology-mediated GCRs (RFA1 and RAD27) and one that suppresses non-homology-mediated GCRs (RAD50/MRE11/XRS2).

Alleles↗
PubMed1999

Saccharomyces cerevisiae pol30 (proliferating cell nuclear antigen) mutations impair replication fidelity and mismatch repair.

To understand the role of POL30 in mutation suppression, 11 Saccharomyces cerevisiae pol30 mutator mutants were characterized. These mutants were grouped based on their mutagenic defects. Many pol30 mutants harbor multiple mutagenic defects and were placed in more than one group. Group A mutations (pol30-52, -104, -108, and -126) caused defects in mismatch repair (MMR). These mutants exhibited mutation rates and spectra reminiscent of MMR-defective mutants and were defective in an in vivo MMR assay. The mutation rates of group A mutants were enhanced by a msh2 or a msh6 mutation, indicating that MMR deficiency is not the only mutagenic defect present. Group B mutants (pol30-45, -103, -105, -126, and -114) exhibited increased accumulation of either deletions alone or a combination of deletions and duplications (4 to 60 bp). All deletion and duplication breakpoints were flanked by 3 to 7 bp of imperfect direct repeats. Genetic analysis of one representative group B mutant, pol30-126, suggested polymerase slippage as the likely mutagenic mechanism. Group C mutants (pol30-100, -103, -105, -108, and -114) accumulated base substitutions and exhibited synergistic increases in mutation rate when combined with msh6 mutations, suggesting increased DNA polymerase misincorporation as a mutagenic defect. The synthetic lethality between a group A mutant, pol30-104, and rad52 was almost completely suppressed by the inactivation of MSH2. Moreover, pol30-104 caused a hyperrecombination phenotype that was partially suppressed by a msh2 mutation. These results suggest that pol30-104 strains accumulate DNA breaks in a MSH2-dependent manner.

Amino Acid Transport Systems↗
PubMed1999

Mononucleotide microsatellite instability and germline MSH6 mutation analysis in early onset colorectal cancer.

Germline mutations in the MSH2 and MLH1 mismatch repair genes account for most cases of hereditary non-polyposis colon cancer syndrome (HNPCC). In addition, germline MSH2 and MLH1 mutations have been detected in patients with non-HNPCC early onset colorectal cancer. Germline MSH6 mutations appear to be rare in classical HNPCC families, but their frequency in young colorectal cancer cases has not been studied previously. In a population based study of early onset colorectal cancer (<50 years) investigated for tumour microsatellite instability (MSI), we identified a subgroup of tumours with MSI for mono- but not dinucleotide repeat markers (m-MSI+ group). In contrast to tumours with classical MSI for dinucleotide markers (d-MSI+), the m-MSI+ group cancers were mainly left sided (6/7). As MSH6 mutations in yeast and human cell lines are associated with weak (and preferential mononucleotide) MSI, the complete MSH6 gene coding region was sequenced in blood DNA from the five m-MSI+ cases available for analysis. A germline nonsense mutation was identified in an isolated case of early onset colorectal cancer (age 43 years). These results support previous findings that germline MSH6 mutations may not be associated with classical MSI and suggest a role for germline MSH6 mutations in isolated early onset colorectal cancer.

Adult↗
PubMed1998

Physical interaction between components of DNA mismatch repair and nucleotide excision repair.

Nucleotide excision repair (NER) and DNA mismatch repair are required for some common processes although the biochemical basis for this requirement is unknown. Saccharomyces cerevisiae RAD14 was identified in a two-hybrid screen using MSH2 as "bait," and pairwise interactions between MSH2 and RAD1, RAD2, RAD3, RAD10, RAD14, and RAD25 subsequently were demonstrated by two-hybrid analysis. MSH2 coimmunoprecipitated specifically with epitope-tagged versions of RAD2, RAD10, RAD14, and RAD25. MSH2 and RAD10 were found to interact in msh3 msh6 and mlh1 pms1 double mutants, suggesting a direct interaction with MSH2. Mutations in MSH2 increased the UV sensitivity of NER-deficient yeast strains, and msh2 mutations were epistatic to the mutator phenotype observed in NER-deficient strains. These data suggest that MSH2 and possibly other components of DNA mismatch repair exist in a complex with NER proteins, providing a biochemical and genetical basis for these proteins to function in common processes.

Base Pair Mismatch↗
PubMed1998

Identification of a human gene encoding a homologue of Saccharomyces cerevisiae EXO1, an exonuclease implicated in mismatch repair and recombination.

The EXO1 gene was identified in Saccharomyces cerevisiae as a gene encoding an exonuclease that interacts with MSH2 and functions in mismatch repair and genetic recombination. To understand the role of EXO1 in higher eukaryotes, we identified the human EXO1 gene. The hEXO1 predicted amino acid sequence shares 26.6% identity with the S. cerevisiae EXO1 amino acid sequence. The human and S. cerevisiae proteins showed a similar ability to complement the mutator phenotype of S. cerevisiae rad27 mutants indicating that the two proteins are functionally similar. There appear to be two forms of hEXO1 that differ by the COOH-terminal 1 and 44 amino acids, respectively, and these appear to result from alternative RNA splicing. The hEXO1 gene consists of 14 exons and is transcribed to yield a 3-kb mRNA. Radiation hybrid and fluorescence in situ hybridization mapping studies indicate that the human gene is located at 1q42.2-qter. Northern blot analysis demonstrates that hEXO1 is expressed in high levels in testis; elevated expression was also observed in thymus and colon and to a lesser extent in small intestine, placenta, spleen, and ovary.

Amino Acid Sequence↗
PubMed1998

The Saccharomyces cerevisiae MLH3 gene functions in MSH3-dependent suppression of frameshift mutations.

The Saccharomyces cerevisiae genome encodes four MutL homologs. Of these, MLH1 and PMS1 are known to act in the MSH2-dependent pathway that repairs DNA mismatches. We have investigated the role of MLH3 in mismatch repair. Mutations in MLH3 increased the rate of reversion of the hom3-10 allele by increasing the rate of deletion of a single T in a run of 7 Ts. Combination of mutations in MLH3 and MSH6 caused a synergistic increase in the hom3-10 reversion rate, whereas the hom3-10 reversion rate in an mlh3 msh3 double mutant was the same as in the respective single mutants. Similar results were observed when the accumulation of mutations at frameshift hot spots in the LYS2 gene was analyzed, although mutation of MLH3 did not cause the same extent of affect at every LYS2 frameshift hot spot. MLH3 interacted with MLH1 in a two-hybrid system. These data are consistent with the idea that a proportion of the repair of specific insertion/deletion mispairs by the MSH3-dependent mismatch repair pathway uses a heterodimeric MLH1-MLH3 complex in place of the MLH1-PMS1 complex.

Base Sequence↗
PubMed1998

Cloning and characterization of the human and Caenorhabditis elegans homologs of the Saccharomyces cerevisiae MSH5 gene.

In Saccharomyces cerevisiae the MSH5 gene encoding a MutS homolog was identified as a gene required for meiotic crossing over. To understand the role of MSH5 in higher eukaryotes, we have identified both the human and the Caenorhabditis elegans MSH5 genes. The human and C. elegans MSH5 predicted amino acid sequences share, respectively, 25.3 and 22.0% identity with the S. cerevisiae MSH5 amino acid sequence. The human MSH5 gene consists of 25 exons and spans at least 12 kb of genomic DNA, while the C. elegans gene comprises 17 exons distributed over at least 5.8 kb. Radiation hybrid mapping studies indicate that the human gene is located at 6p22.3-p21.3. Northern blot analysis demonstrates that human MSH5 is expressed to some extent in all tissues, but that particularly high levels of expression occur in testis, thymus, and other tissues of the immune system. Two-hybrid interaction analysis demonstrates that the human MSH4 and MSH5 proteins interact as observed for S. cerevisiae MSH4 and MSH5.

Amino Acid Sequence↗
PubMed1998

Mutational analysis of exoribonuclease I from Saccharomyces cerevisiae.

Exoribonuclease I from yeast is a 175 kDa protein that is responsible for the majority of cytoplasmic mRNA degradation. Alignment of the Xrn1p sequence with homologs from yeast as well as from higher eukaryotes suggests that the protein is composed of several domains: two acidic N-terminal domains which likely contain the exonuclease, a basic middle domainand a basic C-terminal domain. Deletion analysisdemonstrated that the C-terminus is dispensable for most in vivo and in vitro functions but confers a dominant negative growth inhibition when expressed at high levels. This growth inhibition is not due to the exonuclease function of the protein. To identify specific residues responsible for in vivo function, a screen was carried out for non-complementing missense mutations. Fourteen single point mutations were identified that altered highly conserved amino acids within the first N-terminal domain of Xrn1p. All of the mutations reduced exonuclease activity measured in vivo and in vitro using affinity-purified proteins. The mutants fell into two phenotypic classes, those that reduced or abolished exonuclease activity without qualitatively changing the products of RNA degradation and those that gave rise to novel degradation intermediates on certain RNAs.

Amino Acid Sequence↗
PubMed1998

Saccharomyces Ku70, mre11/rad50 and RPA proteins regulate adaptation to G2/M arrest after DNA damage.

Saccharomyces cells suffering a single unrepairable double-strand break (DSB) exhibit a long, but transient arrest at G2/M. hdf1 cells, lacking Ku70p, fail to escape from this RAD9/RAD17-dependent checkpoint. The effect of hdf1 results from its accelerated 5' to 3' degradation of the broken chromosome. Permanent arrest in hdf1 cells is suppressed by rad50 or mre11 deletions that retard this degradation. Wild-type HDF1 cells also become permanently arrested when they experience two unrepairable DSBs. Both DSB-induced arrest conditions are suppressed by a mutation in the single-strand binding protein, RPA. We suggest that escape from the DNA damage-induced G2/M checkpoint depends on the extent of ssDNA created at broken chromosome ends. RPA appears to play a key intermediate step in this adaptation.

Adaptation, Physiological↗
PubMed1998

Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma.

Inactivation of the genes involved in DNA mismatch repair is associated with microsatellite instability (MSI) in colorectal cancer. We report that hypermethylation of the 5' CpG island of hMLH1 is found in the majority of sporadic primary colorectal cancers with MSI, and that this methylation was often, but not invariably, associated with loss of hMLH1 protein expression. Such methylation also occurred, but was less common, in MSI- tumors, as well as in MSI+ tumors with known mutations of a mismatch repair gene (MMR). No hypermethylation of hMSH2 was found. Hypermethylation of colorectal cancer cell lines with MSI also was frequently observed, and in such cases, reversal of the methylation with 5-aza-2'-deoxycytidine not only resulted in reexpression of hMLH1 protein, but also in restoration of the MMR capacity in MMR-deficient cell lines. Our results suggest that microsatellite instability in sporadic colorectal cancer often results from epigenetic inactivation of hMLH1 in association with DNA methylation.

Adaptor Proteins, Signal Transducing↗