The Cockayne syndrome B protein, involved in transcription‐coupled DNA repair, resides in an RNA polymerase II‐containing complex

Transcription‐coupled repair (TCR), a subpathway of nucleotide excision repair (NER) defective in Cockayne syndrome A and B (CSA and CSB), is responsible for the preferential removal of DNA lesions from the transcribed strand of active genes, permitting rapid resumption of blocked transcription. Here we demonstrate by microinjection of antibodies against CSB and CSA gene products into living primary fibroblasts, that both proteins are required for TCR and for recovery of RNA synthesis after UV damage in vivo but not for basal transcription itself. Furthermore, immunodepletion showed that CSB is not required for in vitro NER or transcription. Its central role in TCR suggests that CSB interacts with other repair and transcription proteins. Gel filtration of repair‐ and transcription‐competent whole cell extracts provided evidence that CSB and CSA are part of large complexes of different sizes. Unexpectedly, there was no detectable association of CSB with several candidate NER and transcription proteins. However, a minor but significant portion (10–15%) of RNA polymerase II was found to be tightly associated with CSB. We conclude that within cell‐free extracts, CSB is not stably associated with the majority of core NER or transcription components, but is part of a distinct complex involving RNA polymerase II. These findings suggest that CSB is implicated in, but not essential for, transcription, and support the idea that Cockayne syndrome is due to a combined repair and transcription deficiency.

[1]  D. Lilley,et al.  DNA Repair , 1998, Nucleic Acids and Molecular Biology.

[2]  J. Hoeijmakers,et al.  Cockayne syndrome: defective repair of transcription? , 1997, The EMBO journal.

[3]  F. Gruijl,et al.  Defective Transcription-Coupled Repair in Cockayne Syndrome B Mice Is Associated with Skin Cancer Predisposition , 1997, Cell.

[4]  James T Kadonaga,et al.  SWI2/SNF2 and Related Proteins: ATP-Driven Motors That Disrupt-Protein–DNA Interactions? , 1997, Cell.

[5]  J. Hoeijmakers,et al.  Cloning and characterization of p52, the fifth subunit of the core of the transcription/DNA repair factor TFIIH , 1997, The EMBO journal.

[6]  C. Chang,et al.  Cdc73p and Paf1p are found in a novel RNA polymerase II-containing complex distinct from the Srbp-containing holoenzyme , 1997, Molecular and cellular biology.

[7]  A. Sancar,et al.  Human Transcription-Repair Coupling Factor CSB/ERCC6 Is a DNA-stimulated ATPase but Is Not a Helicase and Does Not Disrupt the Ternary Transcription Complex of Stalled RNA Polymerase II* , 1997, The Journal of Biological Chemistry.

[8]  R. Halaban,et al.  UV-induced ubiquitination of RNA polymerase II: a novel modification deficient in Cockayne syndrome cells. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[9]  R. Wood,et al.  Xeroderma Pigmentosum Group F Caused by a Defect in a Structure-Specific DNA Repair Endonuclease , 1996, Cell.

[10]  P. J. van der Spek,et al.  Mutational analysis of the human nucleotide excision repair gene ERCC1. , 1996, Nucleic acids research.

[11]  P. Sung,et al.  RAD26, the Yeast Homolog of Human Cockayne's Syndrome Group B Gene, Encodes a DNA-dependent ATPase* , 1996, The Journal of Biological Chemistry.

[12]  L. Mullenders,et al.  The sensitivity of Cockayne's syndrome cells to DNA-damaging agents is not due to defective transcription-coupled repair of active genes , 1996, Molecular and cellular biology.

[13]  Chikahide Masutani,et al.  XPC and human homologs of RAD23: intracellular localization and relationship to other nucleotide excision repair complexes , 1996, Nucleic Acids Res..

[14]  Danny Reinberg,et al.  A human RNA polymerase II complex associated with SRB and DNA-repair proteins , 1996, Nature.

[15]  J. Hoeijmakers,et al.  TFIIH: a key component in multiple DNA transactions. , 1996, Current opinion in genetics & development.

[16]  N. Iyer,et al.  Interactions involving the human RNA polymerase II transcription/nucleotide excision repair complex TFIIH, the nucleotide excision repair protein XPG, and Cockayne syndrome group B (CSB) protein. , 1996, Biochemistry.

[17]  R. Wood.  DNA repair in eukaryotes. , 1996, Annual review of biochemistry.

[18]  D. Reinberg,et al.  A human RNA polymerase II complex associated with SRB and DNA-repair proteins , 1996, Nature.

[19]  R. Conaway,et al.  The RNA polymerase II elongation complex , 1995, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[20]  U. Schibler,et al.  A mammalian RNA polymerase II holoenzyme containing all components required for promoter-specific transcription initiation , 1995, Cell.

[21]  E. Friedberg,et al.  The Cockayne syndrome group A gene encodes a WD repeat protein that interacts with CSB protein and a subunit of RNA polymerase II TFIIH , 1995, Cell.

[22]  J. Hoeijmakers,et al.  Partial characterization of the DNA repair protein complex, containing the ERCC1, ERCC4, ERCC11 and XPF correcting activities. , 1995, Mutation research.

[23]  A. Hyman.  Microtubule Dynamics: Kinetochores get a grip , 1995, Current Biology.

[24]  R. Wood,et al.  Detection and measurement of nucleotide excision repair synthesis by mammalian cell extracts in vitro , 1995 .

[25]  R. Wood,et al.  Mammalian DNA nucleotide excision repair reconstituted with purified protein components , 1995, Cell.

[26]  A. Sancar,et al.  Structure and Function of Transcription-Repair Coupling Factor , 1995, The Journal of Biological Chemistry.

[27]  J D Tucker,et al.  Characterization of the XRCC1-DNA ligase III complex in vitro and its absence from mutant hamster cells. , 1995, Nucleic acids research.

[28]  Jaap,et al.  RAD26, the functional S. cerevisiae homolog of the Cockayne syndrome B gene ERCC6. , 1994, The EMBO journal.

[29]  A. Sancar,et al.  Mechanisms of transcription-repair coupling and mutation frequency decline. , 1994, Microbiological reviews.

[30]  P. Hanawalt,et al.  Repair and Transcription: Collision or collusion? , 1994, Current Biology.

[31]  Yang Li,et al.  A multiprotein mediator of transcriptional activation and its interaction with the C-terminal repeat domain of RNA polymerase II , 1994, Cell.

[32]  D. Reinberg,et al.  Where transcription meets repair , 1994, Cell.

[33]  K. Tanaka,et al.  Purification and cloning of a nucleotide excision repair complex involving the xeroderma pigmentosum group C protein and a human homologue of yeast RAD23. , 1994, The EMBO journal.

[34]  S. Humbert,et al.  Correction of xeroderma pigmentosum repair defect by basal transcription factor BTF2 (TFIIH). , 1994, The EMBO journal.

[35]  Richard A. Young,et al.  An RNA polymerase II holoenzyme responsive to activators , 1994, Nature.

[36]  W. Vermeulen,et al.  Three unusual repair deficiencies associated with transcription factor BTF2(TFIIH): evidence for the existence of a transcription syndrome. , 1994, Cold Spring Harbor symposia on quantitative biology.

[37]  J. Hoeijmakers.  Human nucleotide excision repair syndromes: molecular clues to unexpected intricacies. , 1994, European journal of cancer.

[38]  L. Mullenders,et al.  Deficient repair of the transcribed strand of active genes in Cockayne's syndrome cells. , 1993, Nucleic acids research.

[39]  A. Yasui,et al.  Evidence for a repair enzyme complex involving ERCC1 and complementing activities of ERCC4, ERCC11 and xeroderma pigmentosum group F. , 1993, The EMBO journal.

[40]  R. Wood,et al.  Identical defects in DNA repair in xeroderma pigmentosum group G and rodent ERCC group 5 , 1993, Nature.

[41]  P. Chambon,et al.  DNA repair helicase: a component of BTF2 (TFIIH) basic transcription factor. , 1993, Science.

[42]  J. Hoeijmakers,et al.  Structure and expression of the excision repair gene ERCC6, involved in the human disorder Cockayne's syndrome group B. , 1993, Nucleic acids research.

[43]  J. Hoeijmakers,et al.  Xeroderma pigmentosum complementation group G associated with Cockayne syndrome. , 1993, American journal of human genetics.

[44]  J. Hoeijmakers,et al.  ERCC6, a member of a subfamily of putative helicases, is involved in Cockayne's syndrome and preferential repair of active genes , 1992, Cell.

[45]  W. Herr,et al.  Ethidium bromide provides a simple tool for identifying genuine DNA-independent protein associations. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[46]  D. Hunting,et al.  Transcription-dependent and independent DNA excision repair pathways in human cells. , 1992, Mutation research.

[47]  R. Wood,et al.  Proliferating cell nuclear antigen is required for DNA excision repair , 1992, Cell.

[48]  M. Nance,et al.  Cockayne syndrome: review of 140 cases. , 1992, American journal of medical genetics.

[49]  P. Chambon,et al.  Purification and interaction properties of the human RNA polymerase B(II) general transcription factor BTF2. , 1991, The Journal of biological chemistry.

[50]  V. Bohr,et al.  Gene specific DNA repair. , 1991, Carcinogenesis.

[51]  L. Mullenders,et al.  The genetic defect in Cockayne syndrome is associated with a defect in repair of UV-induced DNA damage in transcriptionally active DNA. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[52]  M. Smerdon,et al.  Site-specific DNA repair at the nucleosome level in a yeast minichromosome , 1990, Cell.

[53]  L. Mullenders,et al.  The residual repair capacity of xeroderma pigmentosum complementation group C fibroblasts is highly specific for transcriptionally active DNA. , 1990, Nucleic acids research.

[54]  E. Harlow,et al.  Antibodies: A Laboratory Manual , 1988 .

[55]  P. Hanawalt,et al.  Selective removal of transcription-blocking DNA damage from the transcribed strand of the mammalian DHFR gene , 1987, Cell.

[56]  P. Hanawalt,et al.  DNA repair in an active gene: Removal of pyrimidine dimers from the DHFR gene of CHO cells is much more efficient than in the genome overall , 1985, Cell.

[57]  P. Sharp,et al.  In vitro transcription: whole-cell extract. , 1983, Methods in enzymology.

[58]  A. Lehmann,et al.  Failure of RNA synthesis to recover after UV irradiation: an early defect in cells from individuals with Cockayne's syndrome and xeroderma pigmentosum. , 1982, Cancer research.

[59]  W. Mangel,et al.  RNA polymerase , 2020, Nature.

[60]  A. van der Eb,et al.  A new technique for the assay of infectivity of human adenovirus 5 DNA. , 1973, Virology.