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L A Loeb

Publications and source records attributed to L A Loeb.

At least 127 records · Page 7Linked to original sources
PubMed1991

Frequency and spectrum of c-Ki-ras mutations in human sporadic colon carcinoma, carcinomas arising in ulcerative colitis, and pancreatic adenocarcinoma.

Sporadic colon carcinomas, carcinomas arising in chronic ulcerative colitis, and pancreatic adenocarcinomas have been analyzed for the presence of c-Ki-ras mutations by a combination of histological enrichment, cell sorting, polymerase chain reaction, and direct sequencing. Although 60% (37/61) of sporadic colon carcinomas contained mutations in codon 12, only 1 of 17 specimens of dysplasia or carcinoma from ulcerative colitis patients contained c-Ki-ras mutations, despite a high frequency of aneuploid tumors. In contrast, a higher percentage (16/20 = 80%) of pancreatic adenocarcinomas contained mutations in c-Ki-ras 2, despite a lower frequency of DNA aneuploidy in these neoplasms. Moreover, the spectrum of mutations differed between sporadic colon carcinoma, where the predominant mutation was a G to A transition, and pancreatic carcinomas, which predominantly contained G to C or T transversions. These results suggest that the etiology of ras mutations is different in these three human neoplasms.

Adenocarcinoma↗
PubMed1991

Endogenous mutations and cancer.

We have analyzed two aspects concerning the relationship between mutations and cancer. Firstly, we consider the possibility that spontaneous mutations result from normal cellular processes. Errors in DNA replication, depurination of DNA, and damage to DNA by oxygen free radicals are important potential sources of endogenous mutations. Mutations produced by DNA polymerases in vitro are clustered, and consist predominantly of single-base substitutions. Analysis of termination sites during copying by DNA polymerase-alpha indicates that certain pause sites are associated with misincorporation of single non-complementary nucleotides, and thus pause sites could be associated with mutagenic hotspots. Depurination results predominantly in transversion mutations resultant from the incorporation of deoxyadenosine opposite abasic sites. Mutations resulting from oxygen free radical damage to DNA are predominantly single-base substitutions: C- > T, G- > C, and G- > T being the most frequent. In addition, there is an unusual mutation that results from oxygen free radical damage to DNA, two adjacent cytidines being substituted by two thymidines. The frequency of mutations observed in tumors appears to be much greater than that predicted from the known rates for spontaneous mutagenesis. Recent observations suggest that many human tumors contain four or more independent mutations. If these multiple mutations are each required for tumor development, then the occurrence of malignancies based on normal mutation rates would be exceedingly rare, and it may be necessary to postulate that tumors result from an increase in the rate of endogenous mutagenesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

DNA polymerase alpha-primase complexes from carcinogen-treated Chinese hamster ovary cells.

In order to investigate whether carcinogens induce alterations of the DNA polymerase alpha-primase complex we compared the physiochemical and catalytic properties and the fidelity of DNA synthesis of DNA polymerase alpha-primase complexes from carcinogen-treated and untreated Chinese hamster ovary cells. Complexes were purified by ion exchange or by immunoaffinity chromatography and both DNA-polymerizing activities and those of ancillary enzymes, such as RNA primase and exonuclease, were examined. Further characterization of the complexes included determination of the relative molecular masses, sedimentation coefficients, and diffusion coefficients, and measurements of the KmS for deoxynucleotide triphosphates and DNA templates, which were identical for the preparations from both carcinogen-treated and untreated cells. The fidelity of DNA polymerase alpha-primase complexes measured by the phi X174am3 reversion assay was also similar in carcinogen-treated and untreated cells. Thus, a carcinogen-mediated induction of a DNA polymerase alpha-primase complex with low fidelity was not observed within the detection limits of the phi X174 assay. RNA primase was found to be an ancillary enzyme activity of the DNA polymerase alpha from both carcinogen-treated and untreated cells; however, the RNA primase:DNA polymerase alpha activity ratio was significantly higher in DNA polymerase alpha-primase complexes from carcinogen-treated cells. These complexes also exhibited an at least 3 times greater velocity of synthesis with supercoiled or unprimed single-stranded DNAs as templates. Since the binding sites of DNA polymerase alpha-primase complexes for deoxynucleotide triphosphates and DNA templates were shown to be identical before and after treatment of cells with carcinogens (i.e., identical Km values for different DNA templates and Ki values for specific inhibitors), the increased synthesis catalyzed by the DNA polymerase alpha-primase complex from carcinogen-treated cells might be due to a carcinogen-induced alteration of an accessory protein of the complex.

Animals↗
PubMed1990

Synthesis of DNA by human immunodeficiency virus reverse transcriptase is preferentially blocked at template oligo(deoxyadenosine) tracts.

The genome of human immunodeficiency virus (HIV) and especially the envelope gene are mutated with unusually high frequency during in vivo replication. Recent studies indicate that HIV reverse transcriptase (RT) is unusually error prone and that the number of generated mutations is disproportionately high within repetitive base sequences. To study the ability of recombinant and wild-type HIV RT to traverse specific homo-oligomeric stretches, we used bacteriophage M13 DNA templates that contain different oligo(purine) and oligo(pyrimidine) inserted tracts. The progress of HIV RT along these templates was potently inhibited from further progression only at a (dA)16 insert. Comparison with other polymerases indicates that the almost complete blockage of polymerization beyond an oligo(dA) insert is unique to HIV RT and Moloney murine leukemia virus RT, which has high sequence homology with HIV RT. The extent of termination of HIV RT at the oligo(dA) run is not affected by alterations in the concentration of KCl, Mg2+, dNTP, or by a decrease in pH. Obstruction of HIV RT opposite the oligo(dA) insert is not alleviated by moving the primer position further upstream from the oligo(dA) insert. Lastly, HIV RT purified directly from virions is also specifically arrested at an oligo(dA) tract. Competition experiments indicate that the concentration of active HIV RT in the presence of M13(dA)16 DNA is similar to that observed in the presence of M13(dG)16 DNA. In addition, preincubation of M13(dA)16 DNA with HIV RT does not subsequently inhibit avian myeloblastosis virus RT from successfully traversing the (dA)16 insert. Therefore, it appears that the blockage of chain elongation of HIV RT at the (dA)16 insert is not the result of trapping the enzyme at this site.

Avian Myeloblastosis Virus↗
PubMed1990

Metal binding to DNA polymerase I, its large fragment, and two 3',5'-exonuclease mutants of the large fragment.

DNA polymerase I (Pol I) is an enzyme of DNA replication and repair containing three active sites, each requiring divalent metal ions such as Mg2+ or Mn2+ for activity. As determined by EPR and by 1/T1 measurements of water protons, whole Pol I binds Mn2+ at one tight site (KD = 2.5 microM) and approximately 20 weak sites (KD = 600 microM). All bound metal ions retain one or more water ligands as reflected in enhanced paramagnetic effects of Mn2+ on 1/T1 of water protons. The cloned large fragment of Pol I, which lacks the 5',3'-exonuclease domain, retains the tight metal binding site with little or no change in its affinity for Mn2+, but has lost approximately 12 weak sites (n = 8, KD = 1000 microM). The presence of stoichiometric TMP creates a second tight Mn2+ binding site or tightens a weak site 100-fold. dGTP together with TMP creates a third tight Mn2+ binding site or tightens a weak site 166-fold. The D424A (the Asp424 to Ala) 3',5'-exonuclease deficient mutant of the large fragment retains a weakened tight site (KD = 68 microM) and has lost one weak site (n = 7, KD = 3500 microM) in comparison with the wild-type large fragment, and no effect of TMP on metal binding is detected. The D355A, E357A (the Asp355 to Ala, Glu357 to Ala double mutant of the large fragment of Pol I) 3',5'-exonuclease-deficient double mutant has lost the tight metal binding site and four weak metal binding sites. The binding of dGTP to the polymerase active site of the D355A,E357A double mutant creates one tight Mn2+ binding site with a dissociation constant (KD = 3.6 microM), comparable with that found on the wild-type enzyme, which retains one fast exchanging water ligand. Mg2+ competes at this site with a KD of 100 microM. It is concluded that the single tightly bound Mn2+ on Pol I and a weakly bound Mn2+ which is tightened 100-fold by TMP are at the 3',5'-exonuclease active site and are essential for 3',5'-exonuclease activity, but not for polymerase activity. Additional weak Mn2+ binding sites are detected on the 3',5'-exonuclease domain, which may be activating, and on the polymerase domain, which may be inhibitory. The essential divalent metal activator of the polymerase reaction requires the presence of the dNTP substrate for tight metal binding indicating that the bound substrate coordinates the metal.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1990

Hydrolysis of 3'-terminal mispairs in vitro by the 3'----5' exonuclease of DNA polymerase delta permits subsequent extension by DNA polymerase alpha.

Purified DNA polymerase alpha, the major replicating enzyme found in mammalian cells, lacks an associated 3'----5' proofreading exonuclease that, in bacteria, contributes significantly to the accuracy of DNA replication. Calf thymus DNA polymerase alpha cannot remove mispaired 3'-termini, nor can it extend them efficiently. We designed a biochemical assay to search in cell extracts for a putative proofreading exonuclease that might function in concert with DNA polymerase alpha in vivo but dissociates from it during purification. Using this assay, we purified a 3'----5' exonuclease from calf thymus that preferentially hydrolyzes mispaired 3'-termini, permitting subsequent extension of the correctly paired 3'-terminus by DNA polymerase alpha. This exonuclease copurifies with a DNA polymerase activity that is biochemically distinct from DNA polymerase alpha and exhibits characteristics described for a second replicative DNA polymerase, DNA polymerase delta. In related studies, we showed that the 3'----5' exonuclease of authentic DNA polymerase delta, like the purified exonuclease, removes terminal mispairs, allowing extension by DNA polymerase alpha. These data suggest that a single proofreading exonuclease could be shared by DNA polymerases alpha and delta, functioning at the site of DNA replication in mammalian cells.

Animals↗
PubMed1990

Errors in DNA synthesis: a source of spontaneous mutations.

Spontaneous mutations in somatic cells may engender several pathologic processes, including cancer. The sources of these mutations remain to be established. We present a conceptual framework in which to analyze the sources of spontaneous mutations and focus here on 3 endogenous processes that have the potential to generate spontaneous sequence alterations in DNA. These are: replication errors, depurination of DNA, and damage to DNA by the generation of active-oxygen species. Each of these processes occurs more frequently than the rate of mutagenesis in somatic cells, but are repaired by different and overlapping mechanisms. Model systems are being developed to determine the spectrum of mutations produced by each of these processes in vitro. A comparison of these spectra with the overall spectrum of spontaneous mutations in somatic cells may help to determine the contribution of each of these processes to spontaneous mutation.

Animals↗
PubMed1989

Sequence specificity of pausing by DNA polymerases.

We have constructed recombinant M13 DNA templates containing stretches of oligo (purines) and oligo (pyrimidines). Each of these inserts hinders the advancement of the large fragment of E. coli Pol I during DNA synthesis. The pattern of blockage is independent of changes in KCl or Mg2+ concentrations and pausing is moderately alleviated at lower pH. Blockage is not affected by either the concentration of template or by the position of the DNA primer. The pattern of pause sites is similar for calf thymus DNA polymerase-alpha, implying that replicative barriers are determined by the structure of the DNA at its growing point. There is a lack of correlation between the position of pause sites with different inserts and known alternate DNA structures. Thus, the homo-oligomeric inserts may possess a different structure when complexed with DNA polymerase. This concept accounts for the appearance of unique new upstream and downstream pause sites that result from the insertion of each oligonucleotide.

Base Sequence↗
PubMed1989

Mutants generated by the insertion of random oligonucleotides into the active site of the beta-lactamase gene.

We have remodeled the gene coding for beta-lactamase by replacing DNA at the active site with random nucleotide sequences. The oligonucleotide replacement (Phe66XXXSer70XXLys73) preserves the codon for the active serine-70 but also contains 15 base pairs of chemically synthesized random sequences that code for 2.5 x 10(6) amino acid substitutions. From a population of Escherichia coli infected with plasmids containing these random inserts, we have selected seven new active-site mutants that render E. coli resistant to carbenicillin and a series of related analogues. Each of the new mutants contains multiple nucleotide substitutions that code for different amino acids surrounding serine-70. Each of the mutants exhibits a temperature-sensitive beta-lactamase activity. This technique offers the possibility of constructing alternative active sites in enzymes on the basis of biological selection for functional variants.

Amino Acid Sequence↗
PubMed1989

Analysis of c-Ki-ras mutations in human colon carcinoma by cell sorting, polymerase chain reaction, and DNA sequencing.

We have analyzed colon carcinomas by a combination of histological enrichment, cell sorting, polymerase chain reaction, and direct sequencing of the c-Ki-ras-2 gene. DNA was chemically extracted from 50-microns sections of paraffin-embedded colon carcinomas and amplified in vitro, and mutations were documented directly by DNA sequencing. Enrichment for tumor cells was obtained histologically and by sorting nuclei on the basis of DNA content differences. Mutations in codon 12 were present in both aneuploid and diploid subpopulations of sorted carcinomas, suggesting that these mutations precede ploidy alterations in the progression of these neoplasms. We have demonstrated the feasibility of utilizing DNA from tissues treated with different fixatives, including methyl carnoys, formalin, and Hollande's solution. This procedure allows one to retrospectively reconstruct the temporal relationship between the occurrence of mutations and sequential morphological changes during tumorigenic progression.

Base Sequence↗
PubMed1989

Differential extension of 3' mispairs is a major contribution to the high fidelity of calf thymus DNA polymerase-alpha.

The fidelity of DNA polymerase-alpha-primase from calf thymus has been analyzed by measuring mutagenesis in vitro and by site-specific nucleotide misinsertion and mispair extension. Using the phi X174 am3 DNA reversion assay errors are detected at the amber3 site only when both dATP and dCTP are significantly biased during in vitro copying reactions. Analysis of these products on DNA sequencing gels reveals pause sites due to the slow extension of mispaired 3' termini. Measurements of misinsertion rates opposite template A show that the rates of dAMP or dCMP misinsertion are similar and occur 40-50 times more rapidly than dGMP misinsertion. The rate of extension from an A:C mispair is 100- and 400-fold greater than from an A:A mispair and an A:G mispair, respectively. Nucleotide misinsertions to generate all 12 possible mispairs have been measured kinetically on phi X174 DNA templates that contain either A, C, G, or T at position 587. Misinsertion frequencies range from 1/4000 to 1/10(6) depending on the mispairs generated. Extension from all 12 different mispairs was examined by starting with oligonucleotide primers that contain different 3'-terminal mispairs. Rates of extension from mispairs are 10(3) to 10(6) times slower than from correctly paired bases. Extension frequencies were purine:pyrimidine greater than pyrimidine:pyrimidine greater than purine:purine. Lack of extension of misincorporated bases suggests the involvement of exonucleolytic proofreading to enable continued DNA synthesis and to guarantee the high fidelity of eucaryotic DNA replication.

Animals↗
PubMed1989

Extension of mismatched 3' termini of DNA is a major determinant of the infidelity of human immunodeficiency virus type 1 reverse transcriptase.

The unusually high error rate of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) suggests that polymerization errors by this enzyme contribute to the genetic variability of the AIDS virus. We have analyzed the mechanism for HIV-1 RT infidelity by studying two distinct steps that might lead to base substitution mutations: nucleotide misinsertions and elongation from 3'-terminal DNA mispairs. Our results indicate that the capacity of HIV-1 RT to polymerize nucleotides onto mispaired termini is a major factor in the production of mutations by this enzyme. When a noncomplementary dAMP was inserted opposite a template adenine by HIV-1 RT, the nascent 3'-terminal A.A mispair was readily extended by subsequent incorporation of the next complementary nucleotide. The frequencies of nucleotide addition onto 3'-terminal A-A, A-C, and A-G mispairs were determined by quantitating the amount of extended primers with a gel electrophoresis assay and by measuring mutagenesis after hybridization of mismatched primers opposite an amber mutation in bacteriophage phi X174 DNA. The mispair extension frequencies are approximately 50-fold higher by HIV-1 RT than by the mammalian replicative enzyme DNA polymerase alpha.

Base Composition↗
PubMed1989

Mutations in the KRAS2 oncogene during progressive stages of human colon carcinoma.

A series of colon carcinomas, adenomas, and adjacent tissues were analyzed for ploidy alterations and mutations in KRAS2. To increase the sensitivity for identifying mutations, we used histological enrichment, cell sorting, and DNA amplification by the polymerase-catalyzed chain reaction followed by direct DNA sequence analysis. Of the 40 carcinomas analyzed, 27 contained aneuploid cells and 26 contained mutations at the first position of codon 12 of KRAS2. Of the 12 adenomas studied, 4 contained aneuploid cells and 9 contained the same mutation at codon 12. In both adenomas and carcinomas, mutations were identified in both diploid and aneuploid cells. In some cases, regions of histologically benign mucosa adjacent to the carcinoma contained mutations. These combined results suggest that mutations in KRAS2 occur early in the development of human colon carcinoma, before change in ploidy, and that these mutations exist in diploid cells from which an aneuploid subpopulation arises. Furthermore, mutations may exist in histologically normal mucosa in regions adjacent to carcinoma, suggesting that a field of genetically abnormal mucosa may surround these tumors.

Adenoma↗
PubMed1989

Proofreading by the epsilon subunit of Escherichia coli DNA polymerase III increases the fidelity of calf thymus DNA polymerase alpha.

Addition of the 3'----5' proofreading exonuclease, epsilon subunit of Escherichia coli DNA polymerase III, to DNA polymerase alpha from calf thymus has been studied. Alone, calf thymus DNA polymerase alpha terminates in vitro DNA synthesis upon insertion of noncomplementary nucleotides. Upon addition of the epsilon subunit, DNA polymerase alpha elongates the newly synthesized DNA as a result of hydrolysis of the 3'-terminal mispair. The fidelity of DNA polymerase alpha in vitro is increased 7-fold by addition of the exonuclease. The functional interaction between DNA polymerase alpha and the epsilon subunit is independent of any detectable physical association. This suggests that a mechanism for proofreading could exist in mammalian cells involving sequential catalysis by DNA polymerase alpha excision of errors by a separate 3'----5' exonuclease, and further elongation onto correctly base-paired 3' termini by DNA polymerase alpha.

Animals↗