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UV-induced alterations in the spatial distribution of the basal transcription factor TFIIH: an early event in nucleotide excision repair
Fundamental and Molecular
Mechanisms of Mutagenesis
ELSEVIER
Mutation Research404 (1998) 129-131
UV-induced alterations in the spatial distribution of the basal transcription factor TFIIH: an early event in nucleotide excision repair P. Karmarkar a, A. Leer-van Hoffen a, A.T. Natarajan L.H.F. Mullenders a,b,*
a,b, A.A.
van Zeeland a,b,
a MGC-Department of Radiation Genetics and Chemical Mutagenesis, Leiden University Medical Centre, P.O. Box 9503, 2300 RA Leiden, Netherlands b J.A. Cohen Institute, Interuniversity Research Institute f o r Radiopathology and Radiation Protection, Leiden, Netherlands
Received 20 March 1998; accepted30 March 1998
Abstract
New findings concerning the molecular mechanisms of nucleotide excision repair (NER) are discussed. 9 1998 Elsevier Science B.V. All rights reserved. Keywords:
Transcription-coupledrepair; Global gcnomerepair; Bulky DNA lesion; Alkylationdamage;Mutagenesis
1. Introduction
DNA is a highly reactive molecule, sensitive to attack by numerous agents. Evidently, integrity of the genome is of crucial importance to the survival and proper functioning of any organism. To cope with this fundamental problem, a complex set of repair machineries has evolved to undo virtually any type of DNA damage. In recent years, many genes and proteins involved in DNA repair have been isolated. This holds particularly for one of the most versatile and important repair pathways: nucleotide excision repair (NER). The occurrence of three distinct genetic disorders in man: Cockayne's syndrome (CS), Xeroderma pigmentosum (XP) and trichoth* Corresponding author.
iodystrophy due to defects in NER, have been of pivotal importance and clinical relevance. Detailed analysis of these disorders have led to an impressive increment of insight into the molecular mechanisms of NER, and have uncovered three unexpected links with transcription [1-3]: (i) Active genes are repaired much faster than non-transcribed parts of the genome; (ii) the basal transcription factor THFIIH, that is required for transcription initiation by RNA polymerase II, is also involved in NER, and (iii) upon induction of DNA damage, transcription is transiently repressed, suggesting a tight coordination between repair and transcription regulation. The underlying mechanisms of these inter-connections are obscure. Furthermore, DNA repair systems face the unique requirement to reach DNA lesions in any location of the genome, independent of chromatin
0027-5107/98/$19.00 9 1998 Elsevier Science B.V. All rights reserved. PII: S0027-5107(98)00105-5
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P. Karmarkar et al. / Mutation Research 404 (1998) 129-131
conformation or stage in the cell cycle. The above implies dynamic interactions and a highly sophisticated organization of NER in the nucleus. As mentioned above, DNA damage induced by a broad range of agents including UV light threatens proper functioning of transcfipton. Experiments with cell free systems employing substrates with specific lesions and purified enzymes or cellular extracts have indicated that the interference of DNA lesions with transcription and replication is mediated by direct blockage of the elongation step in the synthesis. Also in intact cells, UV-induced phototesions are capable of inactivating transcription by elongation blockage [4,5]. Only some evidence exists that in cells, inhibition of transcription could also occur by trans-acting factors. Takeda et al. [6] observed that irradiation of a small area of the cell by a UV-microbeam could produce a uniform inhibition of RNA synthesis. Inactivation of transcription by trans-acting factors might be accomplished by temporary recruitment of key factors from processes such as replication and transcription into the NER machinery [1]. Most strikingly, multi-subunit transcription factor TFIIH essential for transcription initiation of RNA polymerase II promoters appears to play a role in NER. The XPB and XPD gene products have subsequently been identified as helicase subunits of TFIIH presumably required for local opening of the DNA helix within the context of transcription initiation and excision repair. The involvement of TFIIH in NER is not limited to the repair of transcribed genes but also extends to the total genome as certain mutations in TFIIH lead to complete abolishment of NER. The requirement of TFIIH for repair of DNA lesions all over the genome might provide a mechanistic link to transcription inhibition at the initiation level. Recently, evidence has been obtained in yeast that different forms of TFIIH for transcription (holo-TFIIH) and DNA repair (repairosome) may exist [7]. An interchange between holo-TFIIH and the repairosome could underlie the transcription-repair coupling. When associated with RNA polymerase II at a promoter, the core might bind transcription factors more readily, whereas in the presence of DNA damage the core might undergo conformational changes resulting in a release of transcription factors and association with NER proteins.
As a consequence, RNA polymerase II promoters become deprived from essential initiation factors leading to inhibition of transcription initiation. Conceivably, factors that interact with TFIIH might be modified by DNA damage induction and in this way render the core available for either transcription or repair. In this study, we investigated the dynamic aspects and three dimensional organization of NER in the nucleus and its inter-connection with the transcription apparatus by employment of specific antibodies raised against components of TFIIH and labelling of repair sites and transcription. 2. Results and discussion
To achieve this goal, we have analysed the distribution of repair sites and factors, i.e., components of TFIIH and repair proteins, in interphase nuclei of primary human fibroblasts after exposure to UV light. Employing a permeabilized cell system [8,9] sites of repair and transcription were labelled with biotin modified nucleotides and BrUTP, respectively. Briefly, in order to obtain intact chromatin, primary human fibroblasts were encapsulated in agarose microbeads and permeabilized in isotonic buffers containing streptolysine O (SLO). Prior to permeabilization, the cells are UV-irradiated and allow to start repair. Subsequently, repair synthesis is performed in vitro in a run-on type of reaction and repair sites are monitored by immunostaining and confocal microscopy. In a series of experiments, confluent human fibroblasts were exposed to UVC light and analysed at different time intervals for repair synthesis. Repair patches manifested as small loci, are detected at low UV dose within a few minutes after UV radiation. No gross changes occur in time with respect to distribution of repair patches, but intensity reached a maximal level after 30 min and went down with time. Two components of TFIIH were analysed with specific antibodies. In non-treated cells p62 and p89 (XPB) resided in a limited number of distinct foci which co-localized with the sites of transcription. Upon UV radiation and subsequent incubation, the fluorescent signals of p62 and p89 became very enhanced and dispersed throughout the nucleus without any detectable increase of proteins, as measured by Western blotting. When dual labelling was per-
P. Karmarkar et al. / Mutation Research 404 (1998) 129-131
formed, p62 c o - l o c a l i z e d with repair patches during the initial period after U V (i.e., the first 30 min), but less so f o l l o w i n g extended p o s t - U V time. P r o l o n g e d incubation o f normal ceils resulted in quick restoration o f the distribution o f p62 and p89 back to that seen in non-treated cells. This restoration was absent or v e r y m u c h delayed in X P A cells which are deficient in N E R . The situation with regards to CS cells deficient in transcription repair, is currently under investigation. To explain the e n h a n c e d fluorescent signals and the different distributions of p62 and p89, we propose that upon infliction o f D N A damage, T F I I H is recruited for repair either by transport f r o m factories to sites o f damage, or by m o d i f i c a t i o n o f its c o m p o n e n t s and thus, m i g h t actually represent a very early stage of the process o f N E R . This process occurs independently o f the expression o f X P A , X P C or CS gene-products. Interestingly, the restoration o f T F I I H back to its original distribution, does not require c o m p l e t e r e m o v a l o f U V photolesions f r o m the g e n o m e overall.
References [1] D. Bootsma, J.H. Hoeijmakers, DNA repair. Engagement with transcription, Nature 363 (1993) 114-115, [news; comment].
131
[2] C. Chalut, V. Moncollin, J.M. Egly, Transcription by RNA polymerase 1I: a process linked to DNA repair, Bioessays 16 (1994) 651-655. [3] I. Mellon, G. Spivak, P.C. Hanawalt, Selective removal of transcription-blocking DNA damage from the transcribed strand of the mammalian DHFR gene, Cell 51 (1987) 241-249. [4] W. Sauerbier, Gene and transcription mapping by radiation effects, Ann. Rev. Genet. 12 (1978) 329-363. [5] M. Protic-Sabljic, K.H. Kraemer, One pyrimidine dimer inactivates expression of a transfected gene in Xeroderma pigmentosurn cells, Proc. Natl. Acad. Sci. U.S.A. 82 (1985) 66226626. [6] S. Takeda, S. Naruse, R. Yatani, Effects of ultraviolet microbeam irradiation of various sites in HeLa cells on the synthesis of RNA, DNA and protein, Nature 18 (1967) 696697. [7] J.W. Svejstrup, Z. Wang, W.J. Feaver, X. Wu, D.A. Bushnell, T.F. Donahue, E.C. Friedberg, R.D. Kornberg, Different forms of TF~H for transcription and DNA repair: holo-TFIIH and nucleotide excision repairosome, Cell 80 (1995) 21-28. [8] K. Bouayadi, A. van der Leer-van Hoffen, A.S. Balajee, A.T. Natarajan, A.A. van Zeeland, L.H.F. Mullenders, Enzymatic activities involved in the DNA resynthesis step of nucleotide excision repair are firmly attached to chromatin, Nucl. Acids Res. 25 (1997) 1056-1063. [9] D. Jackson, A.S. Balajee, L.H.F. Mullenders, P.R. Cook, Sites in human nuclei where DNA damage by ultraviolet light is repaired: visualization and localization relative to the nucle oskeleton, J. Cell Sci. 107 (1994) 1745-1752.
Mechanisms of Mutagenesis
ELSEVIER
Mutation Research404 (1998) 129-131
UV-induced alterations in the spatial distribution of the basal transcription factor TFIIH: an early event in nucleotide excision repair P. Karmarkar a, A. Leer-van Hoffen a, A.T. Natarajan L.H.F. Mullenders a,b,*
a,b, A.A.
van Zeeland a,b,
a MGC-Department of Radiation Genetics and Chemical Mutagenesis, Leiden University Medical Centre, P.O. Box 9503, 2300 RA Leiden, Netherlands b J.A. Cohen Institute, Interuniversity Research Institute f o r Radiopathology and Radiation Protection, Leiden, Netherlands
Received 20 March 1998; accepted30 March 1998
Abstract
New findings concerning the molecular mechanisms of nucleotide excision repair (NER) are discussed. 9 1998 Elsevier Science B.V. All rights reserved. Keywords:
Transcription-coupledrepair; Global gcnomerepair; Bulky DNA lesion; Alkylationdamage;Mutagenesis
1. Introduction
DNA is a highly reactive molecule, sensitive to attack by numerous agents. Evidently, integrity of the genome is of crucial importance to the survival and proper functioning of any organism. To cope with this fundamental problem, a complex set of repair machineries has evolved to undo virtually any type of DNA damage. In recent years, many genes and proteins involved in DNA repair have been isolated. This holds particularly for one of the most versatile and important repair pathways: nucleotide excision repair (NER). The occurrence of three distinct genetic disorders in man: Cockayne's syndrome (CS), Xeroderma pigmentosum (XP) and trichoth* Corresponding author.
iodystrophy due to defects in NER, have been of pivotal importance and clinical relevance. Detailed analysis of these disorders have led to an impressive increment of insight into the molecular mechanisms of NER, and have uncovered three unexpected links with transcription [1-3]: (i) Active genes are repaired much faster than non-transcribed parts of the genome; (ii) the basal transcription factor THFIIH, that is required for transcription initiation by RNA polymerase II, is also involved in NER, and (iii) upon induction of DNA damage, transcription is transiently repressed, suggesting a tight coordination between repair and transcription regulation. The underlying mechanisms of these inter-connections are obscure. Furthermore, DNA repair systems face the unique requirement to reach DNA lesions in any location of the genome, independent of chromatin
0027-5107/98/$19.00 9 1998 Elsevier Science B.V. All rights reserved. PII: S0027-5107(98)00105-5
130
P. Karmarkar et al. / Mutation Research 404 (1998) 129-131
conformation or stage in the cell cycle. The above implies dynamic interactions and a highly sophisticated organization of NER in the nucleus. As mentioned above, DNA damage induced by a broad range of agents including UV light threatens proper functioning of transcfipton. Experiments with cell free systems employing substrates with specific lesions and purified enzymes or cellular extracts have indicated that the interference of DNA lesions with transcription and replication is mediated by direct blockage of the elongation step in the synthesis. Also in intact cells, UV-induced phototesions are capable of inactivating transcription by elongation blockage [4,5]. Only some evidence exists that in cells, inhibition of transcription could also occur by trans-acting factors. Takeda et al. [6] observed that irradiation of a small area of the cell by a UV-microbeam could produce a uniform inhibition of RNA synthesis. Inactivation of transcription by trans-acting factors might be accomplished by temporary recruitment of key factors from processes such as replication and transcription into the NER machinery [1]. Most strikingly, multi-subunit transcription factor TFIIH essential for transcription initiation of RNA polymerase II promoters appears to play a role in NER. The XPB and XPD gene products have subsequently been identified as helicase subunits of TFIIH presumably required for local opening of the DNA helix within the context of transcription initiation and excision repair. The involvement of TFIIH in NER is not limited to the repair of transcribed genes but also extends to the total genome as certain mutations in TFIIH lead to complete abolishment of NER. The requirement of TFIIH for repair of DNA lesions all over the genome might provide a mechanistic link to transcription inhibition at the initiation level. Recently, evidence has been obtained in yeast that different forms of TFIIH for transcription (holo-TFIIH) and DNA repair (repairosome) may exist [7]. An interchange between holo-TFIIH and the repairosome could underlie the transcription-repair coupling. When associated with RNA polymerase II at a promoter, the core might bind transcription factors more readily, whereas in the presence of DNA damage the core might undergo conformational changes resulting in a release of transcription factors and association with NER proteins.
As a consequence, RNA polymerase II promoters become deprived from essential initiation factors leading to inhibition of transcription initiation. Conceivably, factors that interact with TFIIH might be modified by DNA damage induction and in this way render the core available for either transcription or repair. In this study, we investigated the dynamic aspects and three dimensional organization of NER in the nucleus and its inter-connection with the transcription apparatus by employment of specific antibodies raised against components of TFIIH and labelling of repair sites and transcription. 2. Results and discussion
To achieve this goal, we have analysed the distribution of repair sites and factors, i.e., components of TFIIH and repair proteins, in interphase nuclei of primary human fibroblasts after exposure to UV light. Employing a permeabilized cell system [8,9] sites of repair and transcription were labelled with biotin modified nucleotides and BrUTP, respectively. Briefly, in order to obtain intact chromatin, primary human fibroblasts were encapsulated in agarose microbeads and permeabilized in isotonic buffers containing streptolysine O (SLO). Prior to permeabilization, the cells are UV-irradiated and allow to start repair. Subsequently, repair synthesis is performed in vitro in a run-on type of reaction and repair sites are monitored by immunostaining and confocal microscopy. In a series of experiments, confluent human fibroblasts were exposed to UVC light and analysed at different time intervals for repair synthesis. Repair patches manifested as small loci, are detected at low UV dose within a few minutes after UV radiation. No gross changes occur in time with respect to distribution of repair patches, but intensity reached a maximal level after 30 min and went down with time. Two components of TFIIH were analysed with specific antibodies. In non-treated cells p62 and p89 (XPB) resided in a limited number of distinct foci which co-localized with the sites of transcription. Upon UV radiation and subsequent incubation, the fluorescent signals of p62 and p89 became very enhanced and dispersed throughout the nucleus without any detectable increase of proteins, as measured by Western blotting. When dual labelling was per-
P. Karmarkar et al. / Mutation Research 404 (1998) 129-131
formed, p62 c o - l o c a l i z e d with repair patches during the initial period after U V (i.e., the first 30 min), but less so f o l l o w i n g extended p o s t - U V time. P r o l o n g e d incubation o f normal ceils resulted in quick restoration o f the distribution o f p62 and p89 back to that seen in non-treated cells. This restoration was absent or v e r y m u c h delayed in X P A cells which are deficient in N E R . The situation with regards to CS cells deficient in transcription repair, is currently under investigation. To explain the e n h a n c e d fluorescent signals and the different distributions of p62 and p89, we propose that upon infliction o f D N A damage, T F I I H is recruited for repair either by transport f r o m factories to sites o f damage, or by m o d i f i c a t i o n o f its c o m p o n e n t s and thus, m i g h t actually represent a very early stage of the process o f N E R . This process occurs independently o f the expression o f X P A , X P C or CS gene-products. Interestingly, the restoration o f T F I I H back to its original distribution, does not require c o m p l e t e r e m o v a l o f U V photolesions f r o m the g e n o m e overall.
References [1] D. Bootsma, J.H. Hoeijmakers, DNA repair. Engagement with transcription, Nature 363 (1993) 114-115, [news; comment].
131
[2] C. Chalut, V. Moncollin, J.M. Egly, Transcription by RNA polymerase 1I: a process linked to DNA repair, Bioessays 16 (1994) 651-655. [3] I. Mellon, G. Spivak, P.C. Hanawalt, Selective removal of transcription-blocking DNA damage from the transcribed strand of the mammalian DHFR gene, Cell 51 (1987) 241-249. [4] W. Sauerbier, Gene and transcription mapping by radiation effects, Ann. Rev. Genet. 12 (1978) 329-363. [5] M. Protic-Sabljic, K.H. Kraemer, One pyrimidine dimer inactivates expression of a transfected gene in Xeroderma pigmentosurn cells, Proc. Natl. Acad. Sci. U.S.A. 82 (1985) 66226626. [6] S. Takeda, S. Naruse, R. Yatani, Effects of ultraviolet microbeam irradiation of various sites in HeLa cells on the synthesis of RNA, DNA and protein, Nature 18 (1967) 696697. [7] J.W. Svejstrup, Z. Wang, W.J. Feaver, X. Wu, D.A. Bushnell, T.F. Donahue, E.C. Friedberg, R.D. Kornberg, Different forms of TF~H for transcription and DNA repair: holo-TFIIH and nucleotide excision repairosome, Cell 80 (1995) 21-28. [8] K. Bouayadi, A. van der Leer-van Hoffen, A.S. Balajee, A.T. Natarajan, A.A. van Zeeland, L.H.F. Mullenders, Enzymatic activities involved in the DNA resynthesis step of nucleotide excision repair are firmly attached to chromatin, Nucl. Acids Res. 25 (1997) 1056-1063. [9] D. Jackson, A.S. Balajee, L.H.F. Mullenders, P.R. Cook, Sites in human nuclei where DNA damage by ultraviolet light is repaired: visualization and localization relative to the nucle oskeleton, J. Cell Sci. 107 (1994) 1745-1752.