Biomedical subjects

J H Petrini

Publications and source records attributed to J H Petrini.

26 records · Page 2Linked to original sources

PubMed1997

The RAD52 epistasis group in mammalian double strand break repair.

The S. cerevisiae RAD52 epistasis group gene products mediate DNA double strand break repair and recombination. These proteins and their modes of action have been extensively characterized. The existence of highly conserved mammalian RAD52 epistasis group homologues suggests that information regarding the functions and mechanisms of double strand break repair proteins in yeast may be applicable to mammalian recombinational DNA repair. Herein, we provide an overview of the S. cerevisiae RAD52 epistasis group and describe the characterization of the five mammalian RAD52 epistasis group homologues identified to date. In the context of their expression patterns and other functional analyses, we discuss potential roles for these proteins in mammalian recombinational DNA repair and specialized recombination events such as V(D)J recombination.

Cell Division↗

PubMed1997

hMre11 and hRad50 nuclear foci are induced during the normal cellular response to DNA double-strand breaks.

We previously identified a conserved multiprotein complex that includes hMre11 and hRad50. In this study, we used immunofluorescence to investigate the role of this complex in DNA double-strand break (DSB) repair. hMre11 and hRad50 form discrete nuclear foci in response to treatment with DSB-inducing agents but not in response to UV irradiation. hMre11 and hRad50 foci colocalize after treatment with ionizing radiation and are distinct from those of the DSB repair protein, hRad51. Our data indicate that an irradiated cell is competent to form either hMre11-hRad50 foci or hRad51 foci, but not both. The multiplicity of hMre11 and hRad50 foci is much higher in the DSB repair-deficient cell line 180BR than in repair-proficient cells. hMre11-hRad50 focus formation is markedly reduced in cells derived from ataxia-telangiectasia patients, whereas hRad51 focus formation is markedly increased. These experiments support genetic evidence from Saccharomyces cerevisiae indicating that Mre11-Rad50 have roles distinct from that of Rad51 in DSB repair. Further, these data indicate that hMre11-hRad50 foci form in response to DNA DSBs and are dependent upon a DNA damage-induced signaling pathway.

Ataxia Telangiectasia↗

PubMed1996

Human Rad50 is physically associated with human Mre11: identification of a conserved multiprotein complex implicated in recombinational DNA repair.

In this report, we describe the identification and molecular characterization of a human RAD50 homolog, hRAD50. hRAD50 was included in a collection of cDNAs which were isolated by a direct cDNA selection strategy focused on the chromosomal interval spanning 5q23 to 5q31. Alterations of the 5q23-q31 interval are frequently observed in myelodysplasia and myeloid leukemia. This strategy was thus undertaken to create a detailed genetic map of that region. Saccharomyces cerevisiae RAD50 (ScRAD50) is one of three yeast RAD52 epistasis group members (ScRAD50, ScMRE11, and ScXRS2) in which mutations eliminate meiotic recombination but confer a hyperrecombinational phenotype in mitotic cells. The yeast Rad50, Mre11, and Xrs2 proteins appear to act in a multiprotein complex, consistent with the observation that the corresponding mutants confer essentially identical phenotypes. In this report, we demonstrate that the human Rad50 and Mre11 proteins are stably associated in a protein complex which may include three other proteins. hRAD50 is expressed in all tissues examined, but mRNA levels are significantly higher in the testis. Other human RAD52 epistasis group homologs exhibit this expression pattern, suggesting the involvement of human RAD52 epistasis group proteins in meiotic recombination. Human RAD52 epistasis group proteins are highly conserved and act in protein complexes that are analogous to those of their yeast counterparts. These findings indicate that the function of the RAD52 epistasis group is conserved in human cells.

Amino Acid Sequence↗

PubMed1995

Isolation and characterization of the human MRE11 homologue.

Mutation of the Saccharomyces cerevisiae RAD52 epistasis group gene, MRE11, blocks meiotic recombination, confers profound sensitivity to double-strand break damage, and has a hyperrecombinational phenotype in mitotic cells. We isolated a highly conserved human MRE11 homologue using a two-hybrid screen for DNA ligase I-interacting proteins. Human MRE11 shares approximately 50% identity with its yeast counterpart over the N-terminal half of the protein. MRE11 is expressed at the highest levels in proliferating tissues, but is also observed in other tissues. The MRE11 locus maps to human chromosome 11q21 in a region frequently associated with cancer-related chromosomal abnormalities. A MRE11-related locus was found on chromosome 7q11.2-q11.3.

Amino Acid Sequence↗

PubMed1995

DNA ligase I mediates essential functions in mammalian cells.

DNA replication, repair, and recombination are essential processes in mammalian cells. Hence, the application of gene targeting to the study of these DNA metabolic pathways requires the creation of nonnull mutations. We have developed a method for introducing partially defective mutants in murine embryonic stem cells that circumvents the problem of cellular lethality of targeted mutations at essential loci. Using this approach, we have determined that mammalian DNA ligase I is essential for cell viability. Thus, DNA ligases II and III are not redundant with DNA ligase I for the function(s) associated with cell proliferation. Partial complementation of the lethal DNA ligase I null mutation allowed the creation of deficient embryonic stem cell lines. We found that a wild-type DNA ligase I cDNA, as well as a variant DNA ligase I cDNA, was able to rescue the lethality of the homozygous null mutation, whereas an N-terminal deletion mutant consisting of the minimal DNA ligase I catalytic domain was not. This observation demonstrates that sequences outside the DNA ligase I catalytic domain are essential for DNA ligase I function in vivo.

Animals↗

PubMed1994

Normal V(D)J coding junction formation in DNA ligase I deficiency syndromes.

Bloom syndrome and a clinically related syndrome represented by the cell line 46BR have been associated with reduction in DNA ligase I activity. In these syndromes, DNA ligase I deficiency severely impairs the development and function of the immune system. We undertook analysis of DNA ligase I-deficient cells to determine whether the observed immune deficiency is attributable to a perturbation in the process of V(D)J recombination. V(D)J recombination in Bloom syndrome cell lines and 46BR was examined by a transient transfection assay. No effect on the fidelity of coding and signal junction formation in DNA ligase I-deficient cells was observed. The frequency of V(D)J recombination in DNA ligase I-deficient cells was also examined using recombination substrates modified to function in human cells. Similar recombination frequencies were observed in normal and DNA ligase I-deficient cells, demonstrating that the efficiency of the V(D)J recombination process is unaffected by alterations in DNA ligase I activity. Rearranged immunoglobulin loci from Bloom syndrome cell lines and patient material were molecularly cloned by an inverse polymerase chain reaction strategy which should be applicable to a variety of human immunodeficiency syndromes and were indistinguishable from those found in normal bone marrow samples. Our data argue that the immune system defects associated with DNA ligase I deficiency do not result from perturbation of the V(D)J recombination pathway.

Base Sequence↗

PubMed1991

A wild-type DNA ligase I gene is expressed in Bloom's syndrome cells.

Alteration of DNA ligase I activity is a consistent biochemical feature of Bloom's syndrome (BS) cells. DNA ligase I activity in BS cells either is reduced and abnormally thermolabile or is present in an anomalously dimeric form. To assess the role of DNA ligase function in the etiology of BS, we have cloned the DNA ligase I cDNA from normal human cells by a PCR strategy using degenerate oligonucleotide primers based on conserved regions of the Saccharomyces cerevisiae and Schizosaccharomyces pombe DNA ligase genes. Human DNA ligase I cDNAs from normal and BS cells complemented a S. cerevisiae DNA ligase mutation, and protein extracts prepared from S. cerevisiae transformants expressing normal and BS cDNA contained comparable levels of DNA ligase I activity. DNA sequencing and Northern blot analysis of DNA ligase I expression in two BS human fibroblast lines representing each of the two aberrant DNA ligase I molecular phenotypes demonstrated that this gene was unchanged in BS cells. Thus, another factor may be responsible for the observed reduction in DNA ligase I activity associated with this chromosomal breakage syndrome.

Amino Acid Sequence↗

PubMed1990

T-cell receptor gene rearrangements in functional T-cell clones from severe combined immune deficient (scid) mice: reversion of the scid phenotype in individual lymphocyte progenitors.

The severe combined immune deficient (scid) mouse mutant is characterized by a general absence of functional B and T lymphocytes. This deficiency appears to result from a defect in the variable-diversity-joining (VDJ) recombinase system, which is responsible for the assembly of V, D, and J gene segments that code for immunoglobulin and T-cell receptor (TCR) V regions. Most rearranged immunoglobulin or TCR genes in transformed scid lymphocytes contain abnormal J-associated deletions and are nonfunctional. A few functional lymphocyte clones do arise, however, in some young adult scid mice and in virtually all old scid mice; this phenomenon is referred to as leakiness. Alloreactive, CD3+ T-cell clones were isolated from leaky scid mice and the status of their TCR beta and gamma loci was examined in an effort to assess the nature of the recombinase activity that gives rise to functional scid lymphocytes. The recombination junctions of six gamma and two beta alleles were sequenced, representing four alloreactive T-cell clones. All of the junctions were indistinguishable from those seen in normal cells. These results cannot be attributed to selection by antigen because other rearranged TCR genes account for the TCR molecules expressed by these T-cell clones. We conclude that reversion of the scid phenotype can occur in rare lymphocyte progenitors and may account for most functional lymphocyte clones in leaky scid mice.

Animals↗