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Biomedical subjects

P J Gearhart

Publications and source records attributed to P J Gearhart.

At least 19 recordsLinked to original sources
PubMed2001

Altered nucleotide misinsertion fidelity associated with poliota-dependent replication at the end of a DNA template.

A hallmark of human DNA polymerase iota (poliota) is the asymmetric fidelity of replication at template A and T when the enzyme extends primers annealed to a single-stranded template. Here, we report on the efficiency and accuracy of poliota-dependent replication at a nick, a gap, the very end of a template and from a mispaired primer. Poliota cannot initiate synthesis on a nicked DNA substrate, but fills short gaps efficiently. Surprisingly, poliota's ability to blunt-end a 1 bp recessed terminus is dependent upon the template nucleotide encountered and is highly erroneous. At template G, both C and T are inserted with roughly equal efficiency, whilst at template C, C and A are misinserted 8- and 3-fold more often than the correct base, G. Using substrates containing mispaired primer termini, we show that poliota can extend all 12 mispairs, but with differing efficiencies. Poliota can also extend a tandem mispair, especially when it is located within a short gap. The enzymatic properties of poliota appear consistent with that of a somatic hypermutase and suggest that poliota may be one of the low-fidelity DNA polymerases hypothesized to participate in the hypermutation of immunoglobulin variable genes in vivo.

Animals↗
PubMed2001

Altered spectra of hypermutation in DNA repair-deficient mice.

Affinity maturation of the humoral immune response is based on the ability of immunoglobulin variable genes to undergo a process of rapid and extensive somatic mutation followed by antigenic selection for antibodies with higher affinity. While the behaviour of this somatic hypermutation phenomenon has been well characterized over the last 20 years, the molecular mechanism responsible for inserting mutations has remained shrouded. To better understand this mechanism, we studied the interplay between hypermutation and other DNA associated activities such as DNA repair. There was no effect on the frequency and pattern of hypermutation in mice deficient for nucleotide excision repair, base excision repair and ataxia-telangiectasia mutated gene repair of double strand breaks. However, variable genes from mice lacking some components of mismatch repair had an increased frequency of tandem mutations and had more mutations of G and C nucleotides. These results suggest that the DNA polymerase(s) involved in the hypermutation pathway produces a unique spectra of mutations, which is then altered by mismatch repair and antigenic selection. We, also describe the differential pattern of expression of some nuclear DNA polymerases in hypermutating versus non-hypermutating B lymphocytes. The rapidly dividing germinal centre B cells expressed DNA polymerases alpha, beta, delta, epsilon and zeta, whereas the resting non-germinal centre cells did not express polymerases alpha or epsilon at detectable levels, although they did express polymerases beta, delta and zeta. The lack of expression of polymerase epsilon in the non-germinal centre cells suggests that this enzyme has a critical role in chromosomal replication but does not participate in DNA repair in these cells.

Animals↗
PubMed2001

Analysis of microsatellite instability and hypermutation of immunoglobulin variable genes in Werner syndrome.

Werner syndrome (WS) is a human premature aging syndrome, which is associated with high frequencies of neoplasia and genetic instability. We have examined the occurrence of microsatellite instability, which may result from defective mismatch repair, in lymphoblastoid cell lines derived from nine WS patients. Instability was measured at the D2S123 locus by gel analysis of PCR products. Three WS cell lines had 4-13% altered alleles, compared with 0% in the other six lines. The increased frequency of microsatellite instability could not readily be associated with overt cancer or any other known clinical condition in the three patients. To examine whether the WS defect affected the humoral immune system, we measured the hypermutation of immunoglobulin variable genes in peripheral blood cells from the WS patient who donated the cell line with the highest frequency of microsatellite instability. The frequency and pattern of mutation was similar to that from normal individuals, suggesting that the Werner protein is not involved in generating hypermutation.

Base Sequence↗
PubMed2001

Impact of age on hypermutation of immunoglobulin variable genes in humans.

Chronological aging is associated with an accumulation of DNA mutations that results in cancer formation. The effect of aging on spontaneous mutations in humans is difficult to study because mutations are infrequent in the overall genome and tumors are relatively rare. In contrast, somatic mutations in immunoglobulin variable genes are abundant and can be studied in peripheral blood lymphocytes. To determine if aging alters the frequency and pattern of hypermutation, we sequenced 331 cDNA clones with rearranged V(H)6 genes and compared 452 mutations from young humans to 570 mutations from old humans. There were more mutated clones in the young population compared to the old population. Among the mutated clones, the frequency, location, and types of substitutions were similar between the young and the old groups. However, the ratio of replacement-to-silent mutations was much higher in the complementarity-determining regions of heavy chains from old people, which indicates that their B cells had been selected by antigen. Among individuals, there was variability in the frequency of tandem mutations, which we have observed in mice defective for the PMS2 mismatch repair protein. Microsatellite variability in DNA, which is caused by impaired mismatch repair, was then measured, and there was a strong correlation between the frequency of tandem mutations and microsatellite alterations. The data suggest that individuals vary in their mismatch repair capacity, which can affect the mutational spectra in their antibodies.

Adult↗
PubMed2001

Emerging links between hypermutation of antibody genes and DNA polymerases.

Substantial antibody variability is created when nucleotide substitutions are introduced into immunoglobulin variable genes by a controlled process of hypermutation. Evidence points to a mechanism involving DNA repair events at sites of targeted breaks. In vertebrate cells, there are many recently identified DNA polymerases that inaccurately copy templates. Some of these are candidates for enzymes that introduce base changes during hypermutation. Recent research has focused on possible roles for DNA polymerases zeta (POLZ), eta (POLH), iota (POLI), and mu (POLM) in the process.

Animals↗
PubMed2001

DNA polymerase eta is an A-T mutator in somatic hypermutation of immunoglobulin variable genes.

To determine whether DNA polymerase eta plays a role in the hypermutation of immunoglobulin variable genes, we examined the frequency and pattern of substitutions in variable VH6 genes from the peripheral blood lymphocytes of three patients with xeroderma pigmentosum variant disease, whose polymerase eta had genetic defects. The frequency of mutation was normal but the types of base changes were different: there was a decrease in mutations at A and T and a concomitant rise in mutations at G and C. We propose that more than one polymerase contributes to hypermutation and that if one is absent, others compensate. The data indicate that polymerase eta is involved in generating errors that occur predominantly at A and T and that another polymerase(s) may preferentially generate errors opposite G and C.

Animals↗
PubMed2001

Third complementarity-determining region of mutated VH immunoglobulin genes contains shorter V, D, J, P, and N components than non-mutated genes.

The third complementarity-determining region (CDR3) of immunoglobulin variable genes for the heavy chain (VH) has been shown to be shorter in length in hypermutated antibodies than in non-hypermutated antibodies. To determine which components of CDR3 contribute to the shorter length, and if there is an effect of age on the length, we analysed 235 cDNA clones from human peripheral blood of VH6 genes rearranged to immunoglobulin M (IgM) constant genes. There was similar use of diversity (D) and joining (JH) gene segments between clones from young and old donors, and there was similar use of D segments among the mutated and non-mutated heavy chains. However, in the mutated heavy chains, there was increased use of shorter JH4 segments and decreased use of longer JH6 segments compared to the non-mutated proteins. The overall length of CDR3 did not change with age within the mutated and non-mutated categories, but was significantly shorter by three amino acids in the mutated clones compared to the non-mutated clones. Analyses of the individual components that comprise CDR3 indicated that they were all shorter in the mutated clones. Thus, there were more nucleotides deleted from the ends of VH, D, and JH gene segments, and fewer P and N nucleotides added. The results suggest that B cells bearing immunoglobulin receptors with shorter CDR3s have been selected for binding to antigen. A smaller CDR3 may allow room in the antibody binding pocket for antigen to interact with CDRs 1 and 2 as well, so that as the VDJ gene undergoes hypermutation, substitutions in all three CDRs can further contribute to the binding energy.

Adult↗
PubMed2000

Disruption of the developmentally regulated Rev3l gene causes embryonic lethality.

The REV3 gene encodes the catalytic subunit of DNA polymerase (pol) zeta, which can replicate past certain types of DNA lesions [1]. Saccharomyces cerevisiae rev3 mutants are viable and have lower rates of spontaneous and DNA-damage-induced mutagenesis [2]. Reduction in the level of Rev31, the presumed catalytic subunit of mammalian pol zeta, decreased damage-induced mutagenesis in human cell lines [3]. To study the function of mammalian Rev31, we inactivated the gene in mice. Two exons containing conserved DNA polymerase motifs were replaced by a cassette encoding G418 resistance and beta-galactosidase, under the control of the Rev3l promoter. Surprisingly, disruption of Rev3l caused mid-gestation embryonic lethality, with the frequency of Rev3l(-/-) embryos declining markedly between 9.5 and 12.5 days post coitum (dpc). Rev3l(-/-) embryos were smaller than their heterozygous littermates and showed retarded development. Tissues in many areas were disorganised, with significantly reduced cell density. Rev3l expression, traced by beta-galactosidase staining, was first detected during early somitogenesis and gradually expanded to other tissues of mesodermal origin, including extraembryonic membranes. Embryonic death coincided with the period of more widely distributed Rev3l expression. The data demonstrate an essential function for murine Rev31 and suggest that bypass of specific types of DNAlesions by pol zeta is essential for cell viability during embryonic development in mammals.

Animals↗
PubMed1999

Less repair of pyrimidine dimers and single-strand breaks in genes by scid cells.

Severe combined immunodeficient (Scid) mice have a mutation in the catalytic subunit of the DNA binding protein kinase that is involved in repair of double-strand breaks in DNA. To determine if the protein also influences repair of single-strand breaks, we examined the ability of Scid cells to repair lesions introduced by ultraviolet light and gamma-ray irradiation. DNA repair was measured both in total genomic DNA and in specific genes from murine Scid and wildtype fibroblast cell lines. The removal of pyrimidine dimers and repair of strand breaks in genes was measured using quantitative Southern blot analyses. After ultraviolet irradiation, there was no significant difference in the repair of photoproducts in bulk DNA between Scid and wildtype cells, as measured by cellular survival and unscheduled DNA synthesis. However, deficient repair was evident in genes, where Scid cells had 25-50% less repair in the c-myc and dihydrofolate reductase genes. After gamma-irradiation, Scid fibroblasts had 20-35% less repair of DNA breaks in immunoglobulin kappa and heavy constant genes than wildtype cells. The data suggest that intact DNA-PK enzyme is needed for the efficient operation of cellular repair of pyrimidine dimers and single-strand breaks in genes, as well as in its established role in rejoining double-strand breaks.

Animals↗
PubMed1999

Hypermutation in Ig V genes from mice deficient in the MLH1 mismatch repair protein.

During somatic hypermutation of Ig V genes, mismatched nucleotide substitutions become candidates for removal by the DNA mismatch repair pathway. Previous studies have shown that V genes from mice deficient for the MSH2 and PMS2 mismatch repair proteins have frequencies of mutation that are comparable with those from wild-type (wt) mice; however, the pattern of mutation is altered. Because the absence of MSH2 and PMS2 produced different mutational spectra, we examined the role of another protein involved in mismatch repair, MLH1, on the frequency and pattern of hypermutation. MLH1-deficient mice were immunized with oxazolone Ag, and splenic B cells were analyzed for mutations in their V kappa Ox1 light chain genes. Although the frequency of mutation in MLH1-deficient mice was twofold lower than in wt mice, the pattern of mutation in Mlh1-/- clones was similar to wt clones. These findings suggest that the MLH1 protein has no direct effect on the mutational spectrum.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Homogeneous rate of degradation of nuclear DNA during apoptosis.

DNA fragmentation during apoptosis is characterized by endonucleolytic cleavage of chromosomal DNA into an oligonucleosomal ladder. To determine if actively transcribed genes are more susceptible to cleavage during apoptosis than non-transcribed genes, the rate of fragmentation of differentially expressed genes was measured in B-lymphocyte hybridoma cells. Five genes were studied based on their transcriptional activity and/or nuclear localization, and mitochondrial DNA was assayed as a negative control for apoptotic fragmentation. Apoptosis was induced in the hybridoma cells by ultraviolet light, and DNA was prepared at multiple time points after ultraviolet irradiation. Degradation into an oligonucleosomal ladder appeared as early as 2 h after treatment, showing that fragmentation is rapidly activated in hybridoma cells. The DNA was then digested with restriction enzymes, separated by gel electrophoresis and hybridized with the gene-specific probes for Southern blot analyses. Loss of gene-specific signals was measured by quantitation of autoradiographs. The results show all of the nuclear genes were degraded at the same rate regardless of their transcriptional status or nuclear localization. The data suggest that once the cell activates its destruction program, nuclear DNA is rapidly degraded in a homogeneous manner.

Animals↗
PubMed1998

Characterization of an aFGF gene expression vector with therapeutic potential.

BACKGROUND: Topical application of growth factors to wounds has proven to be suboptimal in achieving epithelial growth and accelerating healing. We propose transfection of fibroblasts with a gene for acidic fibroblast growth factor (aFGF) which will allow continuous, local delivery of the growth factor to wounds, ulcerative lesions, or healing tissues. METHODS: We utilized a pMEXneo vector containing the human aFGF gene with a secretory signal sequence from the hst/KS3 gene to obtain continuous secretion of therapeutic doses of aFGF. NIH 3T3 fibroblasts were transfected using a liposomal transfection reagent and grown in selective media. RESULTS: Dot blot hybridization with labeled complementary DNA probes revealed the presence of plasmid DNA in transfected but not wild type fibroblasts. Intracellular concentrations of aFGF remained low in transfected cells; however, the media contained high levels (32 +/- 7 nM) of aFGF as measured by ELISA. Concentrations of aFGF capable of stimulating cell proliferation were maintained for several weeks. CONCLUSIONS: The aFGF cDNA was transcribed and translated into a functional polypeptide that is secreted from NIH 3T3 cells at physiologically significant concentrations. Stable transfection with a eukaryotic vector which induces secretion of aFGF at levels promoting cell growth holds promise for clinical application in wounds or healing tissue. Transfection could be achieved by topical or endoscopic injection of this type of vector.

3T3 Cells↗
PubMed1998

Altered spectra of hypermutation in antibodies from mice deficient for the DNA mismatch repair protein PMS2.

Mutations are introduced into rearranged Ig variable genes at a frequency of 10(-2) mutations per base pair by an unknown mechanism. Assuming that DNA repair pathways generate or remove mutations, the frequency and pattern of mutation will be different in variable genes from mice defective in repair. Therefore, hypermutation was studied in mice deficient for either the DNA nucleotide excision repair gene Xpa or the mismatch repair gene Pms2. High levels of mutation were found in variable genes from XPA-deficient and PMS2-deficient mice, indicating that neither nucleotide excision repair nor mismatch repair pathways generate hypermutation. However, variable genes from PMS2-deficient mice had significantly more adjacent base substitutions than genes from wild-type or XPA-deficient mice. By using a biochemical assay, we confirmed that tandem mispairs were repaired by wild-type cells but not by Pms2(-/-) human or murine cells. The data indicate that tandem substitutions are produced by the hypermutation mechanism and then processed by a PMS2-dependent pathway.

Adenosine Triphosphatases↗
PubMed1998

Increased hypermutation at G and C nucleotides in immunoglobulin variable genes from mice deficient in the MSH2 mismatch repair protein.

Rearranged immunoglobulin variable genes are extensively mutated after stimulation of B lymphocytes by antigen. Mutations are likely generated by an error-prone DNA polymerase, and the mismatch repair pathway may process the mispairs. To examine the role of the MSH2 mismatch repair protein in hypermutation, Msh2-/- mice were immunized with oxazolone, and B cells were analyzed for mutation in their VkappaOx1 light chain genes. The frequency of mutation in the repair-deficient mice was similar to that in Msh2+/+ mice, showing that MSH2-dependent mismatch repair does not cause hypermutation. However, there was a striking bias for mutations to occur at germline G and C nucleotides. The results suggest that the hypermutation pathway frequently mutates G.C pairs, and a MSH2-dependent pathway preferentially corrects mismatches at G and C.

Animals↗
PubMed1998

Dual enigma of somatic hypermutation of immunoglobulin variable genes: targeting and mechanism.

The immunoglobulin loci are uniquely unstable regions of the genome which undergo as much mutation and selection in a matter of days as a species can undergo in generations of evolution. We have studied the mutational pattern and targeting of this unusual hypermutation process over the past 16 years. The pattern of somatic mutations in rearranged variable (V) genes differs from the pattern of meiotic mutations, indicating that a different mechanism generates hypermutation than generates spontaneous mutation. Hypermutations begin on the 5' end of rearranged V genes downstream of the transcription initiation site and continue through the V exon and into the 3'-flanking region before tapering off. Mutations are located randomly throughout the DNA sequence and exhibit strand bias. The targeting of mutations to the region in and around the rearranged V gene appears to require interactions between the promoter and downstream intronic DNA sequences. The same mechanism that initiates hypermutation around V genes may also produce double-strand breaks that catalyze homologous recombination between rearranged V genes on two chromosomal alleles. With this data we have built a model of hypermutation which predicts that V-region DNA is destabilized at the nuclear matrix during transcription and undergoes strand breaks.

Animals↗
PubMed1997

Insertion of 2 kb of bacteriophage DNA between an immunoglobulin promoter and leader exon stops somatic hypermutation in a kappa transgene.

Somatic hypermutation in rearranged immunoglobulin variable genes occurs in a 2kb region of DNA that is delimited on the 5' side by the promoter and on the 3' side by intron DNA. To identify sequence features that activate the mutation mechanism, we increased the distance between the promoter and the leader region to test whether the spacing of these elements was important. The promoter was separated from the leader sequence by inserting a 2 kb fragment of noncoding bacteriophage lambda DNA between the TATA box and ATG initiator codon in a kappa transgene. Mice from three founder lines were immunized, RNA and DNA were isolated from spleen and Peyer's patch B cells, and transcription of the transgene was confirmed. The frequency of mutation in endogenous heavy chain genes was high, indicating that some B cells underwent hypermutation. However, no hypermutation was found in the transgenic bacteriophage or variable region sequences. Hypermutation did occur in another kappa transgene that had a deletion of the VJ coding sequence, showing that the basic construct is functional and that the VJ exon is not necessary for the mutation mechanism. It is likely that the bacteriophage sequence is a potential substrate for mutation because other heterologous sequences have been shown to undergo mutation if placed downstream of the leader exon. The results suggest that the promoter should be contiguous with the leader exon for the mutation mechanism to function.

Animals↗