- Email: [email protected]
P V.2 Comparative sequence analysis of human and rodent DNA repair genes
S34
S-V: Nucleotide excision repair and transcription
10 V A.41
Processing of struclurally different DNA lesions by nucleotide esetslen repair
Leon H.F. Mullenders, Anneke van Hoffen, Michiel van Oosterw ijk, Maaike Vreesw ijlc, Harry Vrieling, A.T. Natarajan, Albert A. van Zeeland. Leiden
revertant of xeroderma pigmentosum group A. We discuss the implications of our findings in the context of these eukaryotic analogs . Keyword(s): SOS response ; excision repair; Uvr proteins
University. Leiden, The Netherlands Two nucleotide excision repair (NER) subpathways have been identified: one dealing with lesions in the transcribed strand of active genes (transcriptioncoupled repair, TCR), the other performing repair of bulk chromat in (global genome repair, GGR) . For most types of DNA lesions, the relative contr ibution of both pathways to repair is not known. One way to elucidate the mechanisms of the NER is to study the repair of different lesions in cell lines which are only capable to remove DNA lesions by one of the two pathways . Repair of UV-induced photo lesions (pyrim idine dimers (CPO) and 6-4 photoproducts (6-4PP» and NA-AAF induced adducts (dO-eS-AF and dGC8·AAF) was analysed at the gene level in normal human (TCR and OGR), xeroderma pigmentosum group e (only TeR) and Cockayne's syndrome cells (only GOR). These bulky lesion' are targets for TCR but in normal cells the contribution of TCR to repair of 6-4PP and dG-C8 -AAF in active genes is minor. These lesions which induce strong DNA helix distortions, are predominantly repaired by GOR and poss ibly are not removed by TCR due to severe inhibition of transcription initiation. Repair of dG-CS-AF in active genes is not strand specific in normal cells and the kinetics of repair in normal and CS cells are the same although es cells are sensitive to NA-AAF. In contrast to normal human cells, CS cells are unable to recover NA-AAF inhibited RNA synthesis. Therefore, it is questionable whether the absence of recovery of the transcription process in CS cells in the presence of DNA damage is due to a defect in TCR. Rather, TCR might be absent because of a prolonged inhibition of transcription initiation by DNA damage. We propose that upon DNA damage induction, transc ription factor TAIH becomes involved in repair and that CS gene products are involved in the conversion of TFIIH back to transcription initiation. In this view es prote ins act as repair-transcription uncoupling factors . Results further supporting this model will be presented .
Ip v.21
Comparative sequence analysis of human and rodent DNA repair genes
lane Lamerdin, Paula McCready , Aaron Adamson, Stephanie Stilwagen, Nan Lilt, Larry Thompson, Anthony Carrano. Lawrence Livermore Narional
Laboratory. Biology and Biotechnology Research Program. Livermore CA. USA We have engaged in comparative DNA sequenc ing of genom ic regions conta ining DNA repair genes . Roughly 904 kb of sequence has been targeted to seven different human and/or mouse DNA repair genes (XRCCI, XRCC2, XRCC3, XRCC9, ERCC2 , ERCC4, and RAD23A). Comparative analyses have been performed for human and mouse XRCCI and ERCC2 , and are underway for XRCC2 and ERCC4. Such comparative analyses allow us to determine the level of conservation and genomic structure of the genes of interest, as well as identify non-cod ing conserved clements of putative regulatory importance. The comparative analysis of the XRCCI locus in mouse and human identified 9 non-cod ing conserved clements w ith sequence identities ranging from 65-78%. As XRCCI is highly expressed in testis relative to other tissues, and Xrccl-l- m ice arc embryonic lethals, we hypothesize that these elements may be important for the temporal Or tissue-specific regulation of this gene . Such non-cod ing conserved elements were not observed with in 20 kb of upstream sequence determ ined for the ERCC2 gene. Preliminary comparison of the coding regions from mouse and human ERCC4 and XRCC2 suggests that they are less well conserve
Keyword(s): repair; bulky lesions; Cockayne's syndrome Keyword(s) : DNA repair
POSTERS A
IP y.ll
The SOS response of E. collis required Cor global nucleotide excision repair of cyclobutane pyrimidine dlmen but not 6-4 photoproducts
Ip V.31
Exdslon repair patcb lengths are similar for transcrIption coupled repair and global genome repair In UV-Irr8dlated buman cells
David 1. Crowley, Philip C. Hanawall. Department of Biological Sciences,
Stanford University. Sianford, CA, USA Nucleotide excision repair (NER) is a ubiquitous process responsible for the removal of a variety of lesions from damaged DNA. In Escherichia coli, NER is known to involve the UvrA, UvrB, and UvrC gene products . These prote ins are present at different basal levels in undamaged cells but both the uvrA and uvrB genes arc rapidly induced at an early stage of the SOS response. The SOS response is mediated by the action of RecA and LexA and includes many other genes that become up-regulated following DNA damag ing treatments such as exposure to ultraviolet light (UV) . We find that induction of the SOS response is necessary for efficient removal of the major UV induced les ion, the cyclobutane pyrimidine dimer (CPO), from the overall genome . Constitutive expression of the SOS response results in a more rapid removal of CPOs from the genome than in wild type cells. Elimination of the SOS response either genetically or by treatment with the transcription inhibitor rifampicin has no effect on the removal of the 6-4 photoproducts (6-4PP) from the DNA, but greatly impairs CPO repair. These results provide evidence that the CPO, in contrast to the 6-4Pp, is a poor substrate for the UvrAB recognition complex and thus requires induced levels of Uvr proteins for effect ive genome repair. Similar phenomena have been described in some Saccharomyces cereoisiae NER mutants and in a partial
Krista Bowman, Allen Smith, Philip Hanawalt SranjiJrd University, Sianford.
CA. USA Nucleot ide excision repair (NER), including the two subpathways of transcription-coupled repair (rCR) and global genome repair (GOR), functions primarily to remove potentially lethal and/or mutagenic DNA lesions from living organisms. The functions of most of the prote ins involved in NER have recently been established from measurements of excision and repair synthesis using reconstituted repair systems in vitro. The XPC prote in, deficient in patients suffering from xeroderma pigmentoswn, complemetation group C (XPe), is required for GGR and for repair of purified ONA substrates in vitro, but not for TCR . Thus , the precise role of the XPC prote in remains unclear. We have used the S-bromouracil buoyant denSity shift method to measure excision repair patch lengths in both UV-irrad iated repair-proficient human cells and XPe primary fibroblasts. The patch size was found to be about 30 nucleotidcs for both cell types, correlating With tbe size of the DNA fragments excised in vitro by the dual incisions of the structure-specific nucleases XPG and ERCCI -XPF. These results suggest that the XPC protein is not required to target the excision nucleases to sites of DNA cleavage in transcribed strands of expressed genes or to protect the newly incised DNA from funher processing by exonucleases. Models for the possible roles of the XPC protein in GGR will be discussed. Keyword(s) : XPC; repair patch length
S-V: Nucleotide excision repair and transcription
10 V A.41
Processing of struclurally different DNA lesions by nucleotide esetslen repair
Leon H.F. Mullenders, Anneke van Hoffen, Michiel van Oosterw ijk, Maaike Vreesw ijlc, Harry Vrieling, A.T. Natarajan, Albert A. van Zeeland. Leiden
revertant of xeroderma pigmentosum group A. We discuss the implications of our findings in the context of these eukaryotic analogs . Keyword(s): SOS response ; excision repair; Uvr proteins
University. Leiden, The Netherlands Two nucleotide excision repair (NER) subpathways have been identified: one dealing with lesions in the transcribed strand of active genes (transcriptioncoupled repair, TCR), the other performing repair of bulk chromat in (global genome repair, GGR) . For most types of DNA lesions, the relative contr ibution of both pathways to repair is not known. One way to elucidate the mechanisms of the NER is to study the repair of different lesions in cell lines which are only capable to remove DNA lesions by one of the two pathways . Repair of UV-induced photo lesions (pyrim idine dimers (CPO) and 6-4 photoproducts (6-4PP» and NA-AAF induced adducts (dO-eS-AF and dGC8·AAF) was analysed at the gene level in normal human (TCR and OGR), xeroderma pigmentosum group e (only TeR) and Cockayne's syndrome cells (only GOR). These bulky lesion' are targets for TCR but in normal cells the contribution of TCR to repair of 6-4PP and dG-C8 -AAF in active genes is minor. These lesions which induce strong DNA helix distortions, are predominantly repaired by GOR and poss ibly are not removed by TCR due to severe inhibition of transcription initiation. Repair of dG-CS-AF in active genes is not strand specific in normal cells and the kinetics of repair in normal and CS cells are the same although es cells are sensitive to NA-AAF. In contrast to normal human cells, CS cells are unable to recover NA-AAF inhibited RNA synthesis. Therefore, it is questionable whether the absence of recovery of the transcription process in CS cells in the presence of DNA damage is due to a defect in TCR. Rather, TCR might be absent because of a prolonged inhibition of transcription initiation by DNA damage. We propose that upon DNA damage induction, transc ription factor TAIH becomes involved in repair and that CS gene products are involved in the conversion of TFIIH back to transcription initiation. In this view es prote ins act as repair-transcription uncoupling factors . Results further supporting this model will be presented .
Ip v.21
Comparative sequence analysis of human and rodent DNA repair genes
lane Lamerdin, Paula McCready , Aaron Adamson, Stephanie Stilwagen, Nan Lilt, Larry Thompson, Anthony Carrano. Lawrence Livermore Narional
Laboratory. Biology and Biotechnology Research Program. Livermore CA. USA We have engaged in comparative DNA sequenc ing of genom ic regions conta ining DNA repair genes . Roughly 904 kb of sequence has been targeted to seven different human and/or mouse DNA repair genes (XRCCI, XRCC2, XRCC3, XRCC9, ERCC2 , ERCC4, and RAD23A). Comparative analyses have been performed for human and mouse XRCCI and ERCC2 , and are underway for XRCC2 and ERCC4. Such comparative analyses allow us to determine the level of conservation and genomic structure of the genes of interest, as well as identify non-cod ing conserved clements of putative regulatory importance. The comparative analysis of the XRCCI locus in mouse and human identified 9 non-cod ing conserved clements w ith sequence identities ranging from 65-78%. As XRCCI is highly expressed in testis relative to other tissues, and Xrccl-l- m ice arc embryonic lethals, we hypothesize that these elements may be important for the temporal Or tissue-specific regulation of this gene . Such non-cod ing conserved elements were not observed with in 20 kb of upstream sequence determ ined for the ERCC2 gene. Preliminary comparison of the coding regions from mouse and human ERCC4 and XRCC2 suggests that they are less well conserve
Keyword(s): repair; bulky lesions; Cockayne's syndrome Keyword(s) : DNA repair
POSTERS A
IP y.ll
The SOS response of E. collis required Cor global nucleotide excision repair of cyclobutane pyrimidine dlmen but not 6-4 photoproducts
Ip V.31
Exdslon repair patcb lengths are similar for transcrIption coupled repair and global genome repair In UV-Irr8dlated buman cells
David 1. Crowley, Philip C. Hanawall. Department of Biological Sciences,
Stanford University. Sianford, CA, USA Nucleotide excision repair (NER) is a ubiquitous process responsible for the removal of a variety of lesions from damaged DNA. In Escherichia coli, NER is known to involve the UvrA, UvrB, and UvrC gene products . These prote ins are present at different basal levels in undamaged cells but both the uvrA and uvrB genes arc rapidly induced at an early stage of the SOS response. The SOS response is mediated by the action of RecA and LexA and includes many other genes that become up-regulated following DNA damag ing treatments such as exposure to ultraviolet light (UV) . We find that induction of the SOS response is necessary for efficient removal of the major UV induced les ion, the cyclobutane pyrimidine dimer (CPO), from the overall genome . Constitutive expression of the SOS response results in a more rapid removal of CPOs from the genome than in wild type cells. Elimination of the SOS response either genetically or by treatment with the transcription inhibitor rifampicin has no effect on the removal of the 6-4 photoproducts (6-4PP) from the DNA, but greatly impairs CPO repair. These results provide evidence that the CPO, in contrast to the 6-4Pp, is a poor substrate for the UvrAB recognition complex and thus requires induced levels of Uvr proteins for effect ive genome repair. Similar phenomena have been described in some Saccharomyces cereoisiae NER mutants and in a partial
Krista Bowman, Allen Smith, Philip Hanawalt SranjiJrd University, Sianford.
CA. USA Nucleot ide excision repair (NER), including the two subpathways of transcription-coupled repair (rCR) and global genome repair (GOR), functions primarily to remove potentially lethal and/or mutagenic DNA lesions from living organisms. The functions of most of the prote ins involved in NER have recently been established from measurements of excision and repair synthesis using reconstituted repair systems in vitro. The XPC prote in, deficient in patients suffering from xeroderma pigmentoswn, complemetation group C (XPe), is required for GGR and for repair of purified ONA substrates in vitro, but not for TCR . Thus , the precise role of the XPC prote in remains unclear. We have used the S-bromouracil buoyant denSity shift method to measure excision repair patch lengths in both UV-irrad iated repair-proficient human cells and XPe primary fibroblasts. The patch size was found to be about 30 nucleotidcs for both cell types, correlating With tbe size of the DNA fragments excised in vitro by the dual incisions of the structure-specific nucleases XPG and ERCCI -XPF. These results suggest that the XPC protein is not required to target the excision nucleases to sites of DNA cleavage in transcribed strands of expressed genes or to protect the newly incised DNA from funher processing by exonucleases. Models for the possible roles of the XPC protein in GGR will be discussed. Keyword(s) : XPC; repair patch length