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R D Kolodner

Publications and source records attributed to R D Kolodner.

At least 73 records · Page 4Linked to original sources
PubMed1995

Somatic mutations in the hMSH2 gene in microsatellite unstable colorectal carcinomas.

Microsatellite instability is frequently seen in tumors from patients with hereditary nonpolyposis colorectal cancer (HNPCC). Germline mutations in the mismatch repair gene hMSH2 account for approximately 50% of these cases. Tumors from sporadic cases also exhibit this microsatellite instability phenotype, although at a lower frequency, and very few somatically derived mutations have so far been reported in such tumors. In this study DNA from 23 primary colorectal carcinomas (four familial and 19 sporadic cases) exhibiting microsatellite instability were screened for mutations in the hMSH2 gene using constant denaturant gel electrophoresis (CDGE). Among the sporadic cases, five (26%) were found to have somatically derived mutations. One tumor revealed two different mutations, possibly leading to a homozygous inactivation of the gene. One of the four familial cases was classified as having HNPCC, and a germline as well as a somatic mutation were found in this tumor. These results demonstrate that a considerable proportion of sporadic colorectal cancers with microsatellite instability, have somatic mutations in the hMSH2 gene.

Adult↗
PubMed1995

Synthetic lethality of sep1 (xrn1) ski2 and sep1 (xrn1) ski3 mutants of Saccharomyces cerevisiae is independent of killer virus and suggests a general role for these genes in translation control.

Strand exchange protein 1 (Sep1) (also referred to as exoribonuclease I [Xrn1]) from Saccharomyces cerevisiae has been implicated in DNA recombination, RNA turnover, karyogamy, and G4 DNA pairing among other disparate cellular processes. Using a genetic approach to study the role of SEP1/XRN1 in mitotic yeast cells, we identified mutations in the genes superkiller 2 (SKI2) and superkiller 3 (SKI3) as synthetically lethal with an sep1 null mutation. The SKI genes are thought to comprise an intracellular antiviral system controlling the expression of killer toxin from double-stranded RNA virus found in many yeast strains. However, the lethality of sep1 ski2 and sep1 ski3 mutants was independent of the L-A and M viruses, suggesting that the SKI genes act in a general cellular process in addition to virus control. We propose that Sep1/Xrn1 and Ski2 both act to block translation on transcripts targeted for degradation. Using a temperature-sensitive allele of SEP1/XRN1, we show that double mutants display a synthetic cell cycle arrest in late G1 at Start.

Base Sequence↗
PubMed1995

Regulation and intracellular localization of Saccharomyces cerevisiae strand exchange protein 1 (Sep1/Xrn1/Kem1), a multifunctional exonuclease.

The Saccharomyces cerevisiae strand exchange protein 1 (Sep1; also referred to as Xrn1, Kem1, Rar5, or Stp beta) catalyzes the formation of hybrid DNA from model substrates in vitro. The protein is also a 5'-to-3' exonuclease active on DNA and RNA. Multiple roles for the in vivo function of Sep1, ranging from DNA recombination and cytoskeleton to RNA turnover, have been proposed. We show that Sep1 is an abundant protein in vegetative S. cerevisiae cells, present at about 80,000 molecules per diploid cell. Protein levels were not changed during the cell cycle or in response to DNA-damaging agents but increased twofold during meiosis. Cell fractionation and indirect immunofluorescence studies indicated that > 90% of Sep1 was cytoplasmic in vegetative cells, and indirect immunofluorescence indicated a cytoplasmic localization in meiotic cells as well. The localization supports the proposal that Sep1 has a role in cytoplasmic RNA metabolism. Anti-Sep1 monoclonal antibodies detected cross-reacting antigens in the fission yeast Schizosccharomyces pombe, in Drosophila melanogaster embryos, in Xenopus laevis, and in a mouse pre-B-cell line.

Animals↗
PubMed1994

Purification and characterization of MSH1, a yeast mitochondrial protein that binds to DNA mismatches.

MSH1 is a homologue of the Escherichia coli MutS gene that is proposed to play an important role in the repair and maintenance of mitochondrial DNA in Saccharomyces cerevisiae (Reenan, R.A., and Kolodner, R. D. (1992) Genetics 132, 985-1985). In this study, we demonstrate that msh1/MSH1 strains accumulate point mutations in mitochondria seven times faster than the wild-type. We also show that the MSH1 protein is targeted to the mitochondria where its mitochondrial-targeting sequence is removed. Purified MSH1 hydrolyzes ATP and recognizes DNA substrates containing nucleotide mismatches and unpaired nucleotides. The hierarchy of binding affinity of MSH1 among various mismatches is similar to that of MutS, suggesting that both proteins share a highly conserved scheme for recognizing mismatches. The specific binding of MSH1 to mismatches is more resistant to NaCl inhibition and has a slower dissociation rate as compared to the nonspecific binding to complementary DNA sequences. Our data support the idea that MSH1 plays a role in eliminating biosynthetic errors and homeologous recombination in mitochondrial genome by recognizing premutagenic DNA mismatches.

Adenosine Triphosphatases↗
PubMed1994

The effect of DNA mismatches on the ATPase activity of MSH1, a protein in yeast mitochondria that recognizes DNA mismatches.

MSH1 is a DNA-binding protein in yeast mitochondria that recognizes nucleotide mismatches in DNA and plays a role in mitochondrial mutation avoidance (Chi, W., and Kolodner, R. D. (1994) J. Biol. Chem. 269, 29984-29992). MSH1 exhibits an ATPase activity that hydrolyses approximately 1 ATP molecule/min in the absence of DNA. In this study, p3 show that DNA alters the pH dependence of the MSH1 ATPase and stimulates ATP hydrolysis at neutral pH. Using heteroduplex DNA containing mismatches with various affinity, we show an inverse correlation between the extent of ATPase stimulation by DNA and the binding affinity of MSH1 for the DNA effector. We also show that the presence of ATP increases the mismatch specificity of MSH1-DNA binding. Taken together, the observed interaction between the ATPase and the mismatch-binding activities suggests that MSH1 binds to ATP and mismatches with positive cooperativity. This interaction may provide a system for elucidating the role of ATP in mismatch recognition and repair.

Adenosine Triphosphatases↗
PubMed1994

Protein interactions in genetic recombination in Escherichia coli. Interactions involving RecO and RecR overcome the inhibition of RecA by single-stranded DNA-binding protein.

RecA promotes homologous pairing of single-stranded DNA (ssDNA) with double-stranded DNA (dsDNA). This reaction occurs inefficiently if the ssDNA substrate is preincubated with Escherichia coli ssDNA-binding protein (SSB). However, RecO and RecR can act together as accessory factors for RecA to overcome this inhibition by SSB (Umezu, K., Chi, N.-W., and Kolodner, R. D. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 3875-3879). To elucidate the mechanism that underlies this process, we examined protein-protein interactions between RecA, RecF, RecO, RecR, and SSB, and characterized the structure and activity of the ssDNA complexes formed with different combinations of these proteins. We obtained the following results. (i) RecO physically interacts with both RecR and SSB. The interaction between RecO and SSB is stronger than the RecO-RecR interaction. (ii) RecO and RecR do not remove SSB from SSB.ssDNA complexes, but instead bind to these complexes. The resulting RecO.RecR.SSB.ssDNA complexes were more active in RecA-mediated joint molecule formation than were SSB.ssDNA complexes. (iii) RecA can nucleate on the RecO.RecR.SSB.ssDNA complexes more efficiently than on SSB.ssDNA complexes. (iv) When RecA presynaptic filaments were formed in the presence of SSB, RecO, and RecR, the protein-DNA complexes obtained contained 70% of the amount of RecA required to saturate ssDNA. These complexes, however, can mediate joint molecule formation and strand exchange as efficiently as presynaptic filaments which are fully saturated with RecA. Based on these results, we propose dual roles for RecO and RecR in joint molecule formation. First, RecO and RecR bind to SSB.ssDNA complexes and modify their structure to allow RecA to nucleate on them efficiently. Second, RecO and RecR are retained in RecA presynaptic filaments and play a role in the subsequent homologous pairing process promoted by RecA.

Bacterial Proteins↗
PubMed1994

MLH1, PMS1, and MSH2 interactions during the initiation of DNA mismatch repair in yeast.

The discovery that mutations in DNA mismatch repair genes can cause hereditary nonpolyposis colorectal cancer has stimulated interest in understanding the mechanism of DNA mismatch repair in eukaryotes. In the yeast Saccharomyces cerevisiae, DNA mismatch repair requires the MSH2, MLH1, and PMS1 proteins. Experiments revealed that the yeast MLH1 and PMS1 proteins physically associate, possibly forming a heterodimer, and that MLH1 and PMS1 act in concert to bind a MSH2-heteroduplex complex containing a G-T mismatch. Thus, MSH2, MLH1, and PMS1 are likely to form a ternary complex during the initiation of eukaryotic DNA mismatch repair.

Adaptor Proteins, Signal Transducing↗
PubMed1994

Homologous pairing and strand exchange promoted by the Escherichia coli RecT protein.

RecT protein of Escherichia coli promotes the formation of joint molecules between homologous linear double-stranded M13mp19 replicative-form bacteriophage DNA and circular single-stranded M13mp19 DNA in the presence of exonuclease VIII, the recE gene product. The joint molecules were formed by a mechanism involving the pairing of the complementary strand of the linear double-stranded DNA substrate with the circular single-stranded DNA substrate coupled with the displacement of the noncomplementary strand. When the homologous linear double-stranded DNA substrate had homologous 3' or 5' single-stranded tails, then RecT promoted homologous pairing and strand exchange in the absence of exonuclease VIII. Histone H1 could substitute for RecT protein; however, joint molecules formed in the presence of histone H1 did not undergo strand exchange. These results indicate that under the reaction conditions used, the observed strand exchange reaction is promoted by RecT and is not the result of spontaneous branch migration. These results are consistent with the observation that expression of RecE (exonuclease VIII) and RecT substitutes for RecA in some recombination reactions in E. coli.

Bacterial Proteins↗
PubMed1994

The activity of the Saccharomyces cerevisiae strand exchange protein 1 intrinsic exonuclease during joint molecule formation.

Strand exchange protein 1 (Sep1) from Saccharomyces cerevisiae catalyzes the formation of heteroduplex DNA from single-stranded and homologous linear duplex DNA. The initial pairing reaction requires limited exonucleolytic digestion of the double-stranded DNA (dsDNA) by the intrinsic 5' to 3' exonuclease of Sep1 or by an exogenous exonuclease. Subsequent strand exchange proceeds without the need for exonuclease activity. Sep1 degrades linear dsDNA at a rate of 20 nucleotides/min with an average processivity of 45 nucleotides. During strand exchange reactions joint molecules are first observed after 1-2 min, suggesting that only limited digestion is necessary for pairing. The linear dsDNA was found to pair with single-stranded DNA (ssDNA) when it was resected by only 22 nucleotides, and linear dsDNA digested by more than 22 nucleotides was observed only in joint molecules. Approximately 20 nucleotides were also the minimum extent of digestion that could support pairing. In the absence of exonuclease activity, Sep1-promoted pairing requires dsDNA molecules with single-stranded tails homologous to the circular ssDNA. These results suggest that Sep1-promoted strand exchange requires a single-strand annealing event prior to the strand displacement phase of the reaction.

Bacteriophage M13↗
PubMed1994

Characterization of the interaction of Saccharomyces cerevisiae strand exchange protein 1 with DNA.

We have analyzed in greater detail the interaction of strand exchange protein 1 (Sep1) from Saccharomyces cerevisiae with DNA. The binding site size of Sep1 on single-stranded DNA (ssDNA) was determined to be 70 nucleotides per protein monomer using a fluorescence assay and 100 nucleotides using an exonuclease titration technique. The amount of Sep1 required for maximum aggregation of ssDNA was the amount needed to saturate the DNA. When double-stranded DNA (dsDNA) and ssDNA were both present, the duplex DNA was efficiently aggregated only at protein concentrations above that required for saturation of ssDNA. Strand exchange reactions with blunt-ended linear dsDNA and homologous ssDNA substrates required saturation of the ssDNA with Sep1 since free Sep1 is needed for exonuclease activity to initiate pairing with the dsDNA substrate. Preincubation of Sep1 with resected duplex DNA before adding ssDNA allowed joint molecule formation to occur at protein concentrations at least 10-fold below that required for saturation of the ssDNA. However, preincubation of Sep1 with ssDNA before the addition of resected duplex DNA required saturating amounts of Sep1 for joint molecule formation to occur. These results suggest that pairing requires Sep1 on both the ssDNA and the resected ends of the dsDNA.

Base Sequence↗
PubMed1994

Structure of the human MSH2 locus and analysis of two Muir-Torre kindreds for msh2 mutations.

Hereditary nonpolyposis colorectal carcinoma (HNPCC) is a major cancer susceptibility syndrome known to be caused by inheritance of mutations in genes such as hMSH2 and hMLH1, which encode components of a DNA mismatch repair system. The MSH2 genomic locus has been cloned and shown to cover approximately 73 kb of genomic DNA and to contain 16 exons. The sequence of all the intron-exon junctions has been determined and used to develop methods for analyzing each MSH2 exon for mutations. These methods have been used to analyze two large HNPCC kindreds exhibiting features of the Muir-Torre syndrome and demonstrate that cancer susceptibility is due to the inheritance of a frameshift mutation in the MSH2 gene in one family and a nonsense mutation in the MSH2 gene in the other family.

Amino Acid Sequence↗
PubMed1994

Mismatch repair and cancer susceptibility.

Mismatch-repair systems have been identified in organisms ranging from Escherichia coli to humans. They can repair almost all DNA base pair mismatches as well as small insertion/deletion mismatches. Molecular and biochemical analyses have shown that the core components of eukaryotic mismatch-repair systems are highly homologous to their bacterial counterparts. In humans, defects in four mismatch-repair genes have been linked both to hereditary non-polyposis colorectal cancer and to spontaneous cancers that exhibit rearrangements in DNA containing simple repeat sequences.

DNA Repair↗
PubMed1994

Intron splice acceptor site sequence variation in the hereditary non-polyposis colorectal cancer gene hMSH2.

Common but weakly penetrant mutations of certain genes may confer an increased susceptibility to colorectal cancer and account for a proportion of 'sporadic' cases. We analysed DNA from 111 colorectal cancer cases and 114 controls for a specific candidate sequence variation in the hereditary non-polyposis colorectal cancer gene hMSH2. The variant sequence was found in a quarter of individuals, and there was no difference between cancer cases and controls, according to age of development of cancer or presence of family history. It thus appears that this particular sequence variation is a polymorphism rather than a mutation which increases cancer susceptibility.

Adolescent↗
PubMed1994

Interaction between mismatch repair and genetic recombination in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae encodes a set of genes that show strong amino acid sequence similarity to MutS and MutL, proteins required for mismatch repair in Escherichia coli. We examined the role of MSH2 and PMS1, yeast homologs of mutS and mutL, respectively, in the repair of base pair mismatches formed during meiotic recombination. By using specifically marked HIS4 and ARG4 alleles, we showed that msh2 mutants displayed a severe defect in the repair of all base pair mismatches as well as 1-, 2- and 4-bp insertion/deletion mispairs. The msh2 and pms1 phenotypes were indistinguishable, suggesting that the wild-type gene products act in the same repair pathway. A comparison of gene conversion events in wild-type and msh2 mutants indicated that mismatch repair plays an important role in genetic recombination. (1) Tetrad analysis at five different loci revealed that, in msh2 mutants, the majority of aberrant segregants displayed a sectored phenotype, consistent with a failure to repair mismatches created during heteroduplex formation. In wild type, base pair mismatches were almost exclusively repaired toward conversion rather than restoration. (2) In msh2 strains 10-19% of the aberrant tetrads were Ab4:4. (3) Polarity gradients at HIS4 and ARG4 were nearly abolished in msh2 mutants. The frequency of gene conversion at the 3' end of these genes was increased and was nearly the frequency observed at the 5' end. (4) Co-conversion studies were consistent with mismatch repair acting to regulate heteroduplex DNA tract length. We favor a model proposing that recombination events occur through the formation and resolution of heteroduplex intermediates and that mismatch repair proteins specifically interact with recombination enzymes to regulate the length of symmetric heteroduplex DNA.

DNA Repair↗
PubMed1993

Biochemical interaction of the Escherichia coli RecF, RecO, and RecR proteins with RecA protein and single-stranded DNA binding protein.

The Escherichia coli RecF, RecO, and RecR proteins were analyzed for their effect on RecA-mediated pairing of single-stranded circular DNA and homologous linear duplex DNA substrates. As shown by other workers, joint molecule formation by RecA was inhibited by E. coli single-stranded DNA binding protein (SSB) when it was added to single-stranded DNA before RecA. This inhibitory effect was overcome by the addition of RecO and RecR or RecF, RecO, and RecR. Both the rate and extent of joint molecule formation were restored to the maximal level observed when SSB was added after RecA. RecF, RecO, and RecR proteins had no effect on the conversion of joint molecules to final products and only appeared to stimulate an early step in the pairing reaction. The stimulatory effect of RecF, RecO, and RecR was not seen without SSB or when SSB was added after RecA. RecF protein by itself inhibited reactions in mixtures containing RecA and SSB, and this inhibition was overcome by the addition of RecO and RecR. These data suggest that RecO and RecR, and possibly RecF, help RecA overcome inhibition by SSB and utilize SSB-single-stranded-DNA complexes as substrates.

Bacterial Proteins↗
PubMed1993

Identification and characterization of the Escherichia coli RecT protein, a protein encoded by the recE region that promotes renaturation of homologous single-stranded DNA.

Recombination of plasmid DNAs and recombination of bacteriophage lambda red mutants in recB recC sbcA Escherichia coli mutants, in which the recE region is expressed, do not require recA. The recE gene is known to encode exonuclease VIII (exoVIII), which is an ATP-independent exonuclease involved in the RecE pathway of recombination. A 33,000-molecular-weight (MW) protein was observed to be coexpressed with both exoVIII and a truncated version of exoVIII, pRac3 exo, when they were overproduced under the control of strong promoters. We have purified this 33,000-MW protein (p33) and demonstrated by protein sequence analysis that it is encoded by the same coding sequence that encodes the C-terminal 33,000-MW portion of exoVIII. p33 is expressed independently of exoVIII but is probably translated from the same mRNA. p33 was found to bind to single-stranded DNA and also to promote the renaturation of complementary single-stranded DNA. It appears that p33 is functionally analogous to the bacteriophage lambda beta protein, which may explain why RecE pathway recombination does not require recA.

Amino Acid Sequence↗