Reference #: YAR-1018-382278
Submit Date: 04/09/2002 14:49:44-0500

DNA Repair and Signal Transduction

ABSTRACT:
Proteins that repair DNA are also involved in other cellular operations, such as RNA transcription or cell cycling. The result is that DNA repair is integrated with other cellular responses to DNA damage. The signaling that is initiated by DNA damage is complex, invoking protein binding domains and scaffolding proteins such as transcription complexes and BRCA-1, as well as kinase cascades, such as those controlled by the family of PIK-related kinases (DNA-PK, ATM, ATR, FRAP). These signaling pathways converge on key proteins that are often tumor suppressor genes, such as p53. These node proteins appear to evaluate the incoming signals and determine the type and degree of cellular response. The responses are then triggered using similar signaling, such as protein binding and phosphorylation. They may promote survival, such as increased DNA repair, or target the cell to apoptosis by invoking caspases and other cell suicide functions, and/or warn surrounding tissue by the induction and release of extracellular signaling molecules such as cytokines. Proteomic data resulting from the genome project suggest that the integration of these pathways is made possible by the combinatorial use of protein binding motifs in signaling and node proteins. Models based on chaos theory may explain how these systems arose and why they are necessarily complex.

Keywords: DNA repair, signal transduction, p53 protein, protein kinase

Reference #: SLE-1017-151917
Submit Date: 03/26/2002 07:55:58-0500

Transcription-coupled and global genome repair differentially influence ultraviolet-B induced acute skin effects, Langerhans cell migration, local and systemic immunosuppression

ABSTRACT:
Exposure to UV-B radiation impairs immune responses by inhibiting especially Th1-mediated immune responses such as CHS and DTH type responses. This immunomodulation is not restricted to the exposed skin, but is also observed at distant sites indicating the existence of mediating/circulating factors that are induced in the exposed skin. Important mechanisms that are shown to be involved in the induction of immunomodulation are urocanic acid isomerisation, neuropeptide release and DNA damage. The latter has been shown to play a role because enhanced nucleotide excision repair (NER) strongly counteracts immunosuppression. To analyse the relation between UV-induced DNA damage and local as well as systemic immunosuppression further four congenic mouse strains carrying different defects in NER were compared: CSB and XPC mice lacking transcription-coupled or global genome NER resp., as well as XPA and TTD/XPD mice carrying complete or partial defects in both NER subpathways resp. The major conclusions of our experiments are 1) transcription-coupled DNA repair is the dominant determinant in protection against acute skin effects and Langerhans cell migration, 2) transcription-coupled DNA repair is the dominant determinant in protection against local immunomodulation, 3) systemic immunomodulation is only affected in XPA and TTD where both NER subpathways are compromised, 4) sunburn appears not related to UV-B induced systemic immunosuppression.

Keywords: UVB radiation, DNA repair, Langerhans cell migration, immunosuppression

Reference #: 093256
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Reference #: TAN-1017-694368
Submit Date: 04/01/2002 14:09:52-0500

Transcription-coupled DNA repair is genomic context dependent

ABSTRACT:
DNA damage is preferentially repaired in the transcribed strand of many active genes. Although the concept of DNA repair coupled with transcription has been widely accepted, its mechanisms remain elusive. We recently reported that in Chinese hamster ovary cells while ultraviolet light-induced cyclobutane pyrimidine dimers (CPDs) are preferentially repaired in the transcribed strand of dihydrofolate reductase gene, CPDs are efficiently repaired in both strands of adenine phosphoribosyltransferase (APRT) locus, in either a transcribed or nontranscribed APRT gene. These results suggested that the transcription dependence of repair may depend on genomic context. To test this hypothesis, we constructed transfectant cell lines containing a single, actively transcribed APRT gene, integrated at different genomic sites. Mapping of CPD repair in the integrated APRT genes in three transfectant cell lines revealed two distinct repair patterns, either preferential repair of CPDs in the transcribed strand, or very poor repair in both strands. Similar kinetics of micrococcal nuclease digestion were seen for all three transfectant APRT gene domains and endogenous APRT locus. Our results suggest that both the efficiency and strand-specificity of repair of an actively transcribed gene are profoundly affected by genomic context, but do not reflect changes in first order nucleosomal structure.

Keywords: transcription-coupled repair, cyclobutane pyrimidine dimer, APRT, genomic context

Reference #: KOW-1017-691100
Submit Date: 04/01/2002 10:21:01-0500

An interesting region within the putative nuclease domain of E. coli endonuclease V

ABSTRACT:
In Escherichia coli, endonuclease V is the major enzyme that recognizes hypoxanthine and xanthine. In addition, the enzyme also recognizes a number of structurally unrelated DNA lesions such as AP sites, single base mismatches, hairpins, unpaired loops, and pseudo-Y and flap DNA structures. The flap endonuclease activity of endonuclease V is similar to those of E. coli Pol I and the eukaryotic flap endonuclease; however, E. coli endonuclease V is unable to remove the RNA-DNA flap. Instead, endonuclease V acted as a 5-3 RNAse, degrading the RNA-DNA flap as monoribonucleotides. Endonuclease V homologs were cloned and purified from Archaeglobus fulgidus and Thermatoga maritima. Comparative enzymatic study showed that a 15-amino acids region, called the E-T domain might be important for the recognition of DNA lesions other than hypoxanthine. Site specific mutagenesis was performed to investigate the importance of this E-T domain. D103A mutant endonuclease V was found to be inactive. However, D98A mutant protein, which contained a mutation at a semi-conserved aspartic acid within the E-T domain, lost the base mismatch and flap endonuclease activities, but not the hypoxanthine endonuclease activity. These data suggest that the 15-amino acids region contains essential amino acid residues that are crucial for the recognition of DNA lesions other than hypoxanthine.

Keywords: endonuclease V, hypoxanthine, xanthine, flap endonuclease

Reference #: BOI-1018-696776
Submit Date: 04/13/2002 06:08:33-0500

Base Excision Repair of Oxidative DNA Damage in Mammalian Cells: The Early Steps

ABSTRACT:
The generation of reactive oxygen species in the cell provokes, among other lesions, the formation of 7,8-dihydro-8-oxoguanine (8-oxoG) in DNA. Due to mispairing with adenine during replication, 8-oxoG is highly mutagenic. In mammalian cells, 8-oxoG is primarily removed by the base excision repair (BER) process. The early steps in the course of the BER pathway includes; i) the recognition and the removal of the 8-oxoG lesion by a DNA N-glycosylase / AP lyase (OGG1p) and ii) the cleavage of the resulting AP site by an AP endonuclease (APE1p). Early steps in the course of BER of 8-oxoG were investigated in vitro using reconstituted protein systems and in vivo using a plasmid assay. Our in vitro results show that the 8-oxoG DNA N-glycosylase activity of OGG1p is stimulated by APE1p. The stimulation of OGG1p does not require a physical interaction between the proteins. Another protein, XRCC1p also has a stimulatory effect on the early steps of BER. The results show that the XRCC1p physically interacts with APE1 and stimulates its AP endonuclease activity. These in vitro data suggest that XRCC1p coordinates the early steps of BER as well as the late steps. We have also investigated the repair of 8-oxoG in wild type and ogg1-/- MEF (mouse embryo fibroblasts) immortalized cell lines. We measured the repair of a single 8-oxoG.C base pair located on a transcribed sequence (TS) or a non transcribed sequence (NTS) in non replicating plasmids. The results show that OGG1p is required for the repair of 8-oxoG in NTS. In contrast, 8-oxoG is efficiently repaired in TS in ogg1-/- MEF indicating an OGG1p-independent transcription coupled repair (TCR) pathway for 8-oxoG in murine cells. The involvement of other proteins such as XPAp, CSBp, PARP-1p and DNA polymerase beta in 8-oxoG repair is also investigated. Current models of short-patch and long-patch BER will be discussed.

Keywords: DNA Repair, Oxidative DNA Damage, DNA glycosylases, AP endonucleases