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Graciela Spivak

Publications and source records attributed to Graciela Spivak.

6 recordsLinked to original sources
PubMed2006

In vivo assays for transcription-coupled repair.

This chapter describes the technologies used in our respective laboratories to study the incidence and repair of lesions induced in specific DNA sequences by ultraviolet light, chemical carcinogens, and products of cellular metabolism. The Southern blot method is suitable for analysis of damage and repair in the individual DNA strands of specific restriction fragments up to 25,000 nucleotides in length, whereas the ligation-mediated polymerase chain reaction approach permits analysis of shorter sequences at the nucleotide level. Both methods have unique advantages and limitations for particular applications.

Animals↗
PubMed2005

UV-sensitive syndrome.

UV-sensitive syndrome (UV(S)S) is a human DNA repair-deficiency disorder with mild clinical manifestations. In contrast to other disorders with photosensitivity, no neurological or developmental abnormalities and no predisposition to cancer have been reported. The cellular and biochemical responses of UV(S)S and Cockayne syndrome (CS) cells to UV light are indistinguishable, and result from defective transcription-coupled repair of photoproducts in expressed genes [G. Spivak, T. Itoh, T. Matsunaga, O. Nikaido, P. Hanawalt, M. Yamaizumi, Ultra violet-sensitive syndrome cells are defective in transcription-coupled repair of cyclobutane pyrimidine dimers, DNA Repair, 1, 2002, 629-643]. The severe neurological and developmental deficiency characteristic of CS may arise from unresolved blockage of transcription at oxidative DNA lesions, which could result in excessive cell death and/or attenuated transcription. We have proposed that individuals with UV(S)S develop normally because they are proficient in repair of oxidative base damage or in transcriptional bypass of these lesions; consistent with this hypothesis, CS-B cells, but not UV(S)S cells, are deficient in host cell reactivation of plasmids containing oxidative base lesions [G. Spivak, P. Hanawalt, Host cell reactivation of plasmids containing oxidative DNA lesions is defective in Cockayne syndrome but normal in UV-sensitive syndrome, 2005, submitted for publication]. In this review, I will summarize the current understanding of the UV-sensitive syndrome and compare it with the Cockayne syndrome.

DNA Repair↗
PubMed2005

Host cell reactivation of plasmids containing oxidative DNA lesions is defective in Cockayne syndrome but normal in UV-sensitive syndrome fibroblasts.

UV-sensitive syndrome (UV(S)S) is a human DNA repair-deficient disease with mild clinical manifestations. No neurological or developmental abnormalities or predisposition to cancer have been reported. In contrast, Cockayne syndrome (CS) patients exhibit severe developmental and neurological defects, in addition to photosensitivity. The cellular and biochemical responses of UV(S)S and CS cells to UV are indistinguishable, and result from defective transcription-coupled repair (TCR) of photoproducts in expressed genes. We propose that UV(S)S patients develop normally because they are proficient in repair of oxidative base damage. Consistent with our model, we show that Cockayne syndrome cells from complementation groups A and B (CS-A, CS-B) are more sensitive to treatment with hydrogen peroxide than wild type or UV(S)S cells. Using a host cell reactivation assay with plasmids containing UV-induced photoproducts, we find that expression of the plasmid-encoded lacZ gene is reduced in the TCR-deficient CS-B and UV(S)S cells. When the plasmids contain the oxidative base lesion thymine glycol, CS-B cells are defective in recovery of expression, whereas UV(S)S cells show levels of expression similar to those in wild type cells. 8-oxoguanine in the plasmids result in similarly defective host cell reactivation in CS-A and CS-B cells; abasic sites or single strand breaks in the plasmids cause similar decreases in expression in all the cell lines examined. Repair of thymine glycols in the lacZ gene was measured in plasmids extracted from transfected cells; removal of the lesions is efficient and without strand bias in all the cell lines tested.

Cell Survival↗
PubMed2005

Nucleotide excision repair activity varies among murine spermatogenic cell types.

Germ cells perform a unique and critical biological function: they propagate the DNA that will be used to direct development of the next generation. Genetic integrity of germ cell DNA is essential for producing healthy and reproductively fit offspring, and yet germ cell DNA is damaged by endogenous and exogenous agents. Nucleotide excision repair (NER) is an important mechanism for coping with a variety of DNA lesions. Little is known about NER activity in spermatogenic cells. We expected that germ cells would be more efficient at DNA repair than somatic cells, and that this efficiency may be reduced with age when the prevalence of spontaneous mutations increases. In the present study, NER was measured in defined spermatogenic cell types, including premeiotic cells (A and B type spermatogonia), meiotic cells (pachytene spermatocytes), and postmeiotic haploid cells (round spermatids) and compared with NER in keratinocytes. Global genome repair and transcription-coupled repair subpathways of NER were examined. All spermatogenic cell types from young mice displayed good repair of (6-4) pyrimidone photoproducts, although the repair rate was slower than in primary keratinocytes. In aged mice, repair of 6-4 pyrimidone photoproducts was depressed in postmeiotic cells. While repair of cyclobutane pyrimidine dimers was not detected in spermatogenic cells or in keratinocytes, the transcribed strands of active genes were repaired with greater efficiency than nontranscribed strands or inactive genes in keratinocytes and in meiotic and postmeiotic cells; spermatogonia displayed low to moderate ability to repair cyclobutane pyrimidine dimers on both DNA strands regardless of transcriptional status. Overall, the data suggest cell type-specific NER activity during murine spermatogenesis, and our results have possible implications for germ cell aging.

Age Factors↗
PubMed2002

Ultraviolet-sensitive syndrome cells are defective in transcription-coupled repair of cyclobutane pyrimidine dimers.

Patients with ultraviolet-sensitive syndrome (UV(S)S) are sensitive to sunlight, but present neither developmental nor neurological deficiencies. Complementation studies with hereditary DNA repair syndromes show that UV(S)S is distinct from all known xeroderma pigmentosum (XP) and Cockayne syndrome (CS) groups. UV(S)S cells exhibit some characteristics typical of CS, including normal global genomic (GGR) repair of UV-photoproducts, poor clonal survival and defective recovery of RNA synthesis after UV exposure. Those observations have led us to suggest that UV(S)S cells, like those from CS, are defective in transcription-coupled repair (TCR) of cyclobutane pyrimidine dimers (CPD). We have now examined the repair of CPD in the transcribed and non-transcribed strands of the active dihydrofolate reductase (DHFR) and p53 genes, and of the silent alpha-fetoprotein (AFP) and mid-size neurofilament (NF-M) genes in normal human cells and in cells belonging to UV(S)S and CS complementation group B. Our results provide compelling evidence that the UV(S)S gene is essential for TCR of CPD and probably other bulky DNA lesions. As a possible distinction between UV(S)S and CS patients, we postulate that the UV(S)S gene may not be required for TCR of oxidative lesions. We have also found that repair of CPD in either DNA strand of the genomic fragments examined, occurs at a slower rate in TCR-deficient cells than in the non-transcribed strands in normal cells; we suggest that in the absence of TCR, global repair complexes have hindered access to lesions in genomic regions that extend beyond individual transcription units.

Cells, Cultured↗