AbstractThe eukaryotic nucleotide excision–repair pathway has been reconstituted in vitro, an achievement that should hasten the full enzymological characterization of this highly complex DNA-repair pathway

Hoeijmakers, J.H.J.

Jaspers, N.G.J.

The eukaryotic nucleotide excision-repair pathway has been reconstituted in vitro, an achievement that should hasten the full enzymological characterization of this highly complex DNA-repair pathway

Jaspers, N.G.J. (Nicolaas)

Hoeijmakers, J.H.J. (Jan)

Erasmus University Digital Repository

N.G.J. JASPERS AND J.H.J. HOEIJMAKERS DNA REPAIR Nucleotide excision-repair in the test tube The eu karyotic nucleotide excision-repair pathway has been reconstituted in vitro, an achievement that should hasten the full enzymological characterization of this highly complex DNA-repair pathway. All living organisms have evolved mechanisms to cope with the problem of genomic instabhty. Cells have a number of different mechanisms for repairing the various types of damage - resulting from errors of internal meta- bolism or the action of external agents - that their DNA is prone to. Many of these mechanisms are relatively sim- ple and speciahzed for the repair of strictly defined types of DNA lesions: those dealing with strand mismatches or small base adducts, for example. The nucleotide exci- sion-repair (NER) pathway, however, can recognize a broad spectrum of heh-distorting lesions, mostly gener- ated by environmental DNA-damaging agents, such as ultraviolet (UV) light. In eukaryotes, this versatility is accomplished by intricate interactions between many pro- teins, which mediate the excision and replacement of a single-strand patch substantially larger than the structurally affected DNA region itself. The basic features of NER have remained unchanged throughout eukaryotic evolu- tion, h m  simple yeasts up to hlgher mammals, and many parallels extend even to prokaryotes such as Escherichia coli. The complexity of eukaryotic NER is reminiscent of the other fundamental DNA metabolic processes - transcrip- tion, replication and recombination - and mutual inter- actions between these processes are now being uncovered. The classical biochemical approach to dissecting such processes involves the separation of the individual compo- nents and their reconstitution to a fully operative system. While simplified in vitro systems for basic transcription and replication have been available for some time, the recon- stitution of NER has just recently been reported by Aboussekra et al. [I]. This delay is remarkable as, unlike replication and transcription, NER is only essential in the presence of environmental stress, so that mutants afTected in this system can readily be identified and genetic analysis is possible. The well-known human inherited disorders xeroderma pigmentosum (XP), Cockayne's syndrome (CS) and trichothiodystrophy are caused by mutations, defining at least ten complementation groups, affecting NER; many other, less well-known, NER mutants have been identified in yeast and cultured mammalian cells. The first attempts to set up a mammalian cell-free NER system date back to 1976 [2]. However, this and other early systems lacked stability, reproducibhty or specifi- city. Twelve years later, and twenty years after Cleaver's discovery that NER is defective in cells from XP patients, Wood et al. [3], followed independently by Sibghat- Ullah et al. [4], described how carefully isolated mam- mahan cell extracts can repair damage on simple plasmid DNAs. The conditions appeared to mimic the NER sys- tem rather closely, as most of the factors affected in the known mammahan NER mutants were required. Extracts from different mutants that complement each other in vivo - that is, after cell fusion - were generally also able to do so in the cell-free system. The in vitro assay was soon successfully exploited for a number of enzymological advances. These included the isolation of the XPA, XPC and XPG proteins [5-71, the separation of the incision and excisiodgap-filling steps, the discovery of the involvement of the replication fac- tors PCNA (proliferating cell nuclear antigen) and RPA [8-111, the identification of the ERCCl/XPF enzyme complex [12,13] and the sizing of the excised patch to 27-29 nucleotides [14]. From 1984 on [15], most of the NER genes were gradually cloned by painstaking gene- transfer experiments carried out using the mutant cell cultures (reviewed in [16]). In adhtion, the human multi- protein TFIIH basal transcription factor complex was isolated and unexpectedly found to harbour at least three known repair proteins [17,18]. It is mainly the combina- tion of these developments that has made the full in vitro reconstitution of NER possible. The approach used by Aboussekra et al. [I] and, indepen- dently but worked out in less detail, by Mu et al. [19] was as classical as it was effective. First, a repair-competent mamrnahan extract was fiactionated into at least eight parts using standard chromatographic procedures. Then, these fractions were tested one by one for the presence of known repair enzymes. Finally, the fractions were replaced by purified enzyme preparations, either derived from individual repair genes overexpressed in bacterial or insect cells, or from detailed classical purification protocols starting with HeLa nuclear extracts. The initial stages of NER, comprising the damage recog- nition step and a dual incision around the lesion, were found to require the single proteins XPA and XPG, and the multi-protein complexes TFIIH, RPA (hSSB), ERCC1 /XPF and XPC/HHR23B. The XPE-related protein UV-DDB acted as a dispensable stimulator. A new essential factor IF7 (incision factor 7), as yet only partially characterized, was also identified. Completion of the NER reaction, which involves patch-displacement, gap-filling and religation, could be accomplished by DNA polymerase E and ligase I, in combination with the accessory replication factors PCNA, RPA and RF-C. Interestingly, this final stage could be performed just as O Current Biology 1995, Vol 5 No 7 DISPATCH 701well by a combination of the E. coli DNA polymerase IKlenow fragment and ligase.A total of between 25 and 30 proteins (summarized inTable 1) are minimally required to reconstitute the fullNER reaction; this is, however, just an estimate, as someuncertainties remain. For instance, the TFIIH prepara-tion used in the experiments contained at least six poly-peptides, known as the 'core complex', which are alsorequired to support in vitro transcription. It is, however,very likely that (minor) accessory factors present in thispreparation, such as cyclin-dependent kinase CDK7 [20],are also involved in NER. As a second example, thecomposition of the ERCC1/XPF complex is still uncer-tain, as it could not be purified to homogeneity usingclassical chromatography, a failure also experienced bytwo other groups [19,21]. Besides ERCC1 and anunknown 110 kD protein, identified by South-westernblotting [1] and immunoprecipitation [21], the complexcarries the complementing activities of the XPF and702 Current Biology 1995, Vol 5 No 7ERCC11 mutants [12,13]. Finally, the newly identifiedfactor IF7 also remains uncharacterized.The in vitro reconstitution of NER clearly opens thedoor to further dissection of its individual steps, theirorder and the protein-protein interactions involved. Inthe years to come, our knowledge of mammalian NERcan be expected to become at least as detailed as that ofthe much simpler uvrABCD excinuclease system ofE. coli. Despite all these promises for the future, adaptingthe basic NER reaction to simplified in vitro conditionshas its price. Cells repair the transcribed strand of theiractive genes much faster than the remainder of thegenome, by an NER subpathway called transcription-coupled repair (TCR). In patients with CS, TCR isimpaired, but the global NER facility is unaffected [22].The gene products CSA and CSB are not required forthe reconstituted in vitro NER reaction, and extracts ofCS mutant cells (both human and hamster) have normalNER activity. This implies that the test-tube conditionsselectively reflect the global repair subpathway, which isnot entirely unexpected as the damaged plasmid used asthe substrate is not transcribed in the experimental con-ditions. It also fits with the need for the XPC/HHR23Bcomplex, which is known to be essential for the globalNER subpathway only [22].Recent attempts to set up a repair assay with a substratecarrying an active transcription unit and ribonucleotidesadded in the reaction mixture have not yet resulted ina clear-cut CSB-dependence or CSA-dependence. Itappears that much more is needed to reproduce theTCR subpathway in vitro; presumably the chromatinstructure of the repair substrate has to be taken intoaccount as well. SV40 minichromosomes with a partiallyintact chromatin structure can be repaired in vitro [6],but this repair activity also appears largely to reflect theglobal subpathway, as it is strongly dependent on XPC/HHR23B. As a matter of fact, the human homologs ofthe yeast global repair proteins RAD7 and RAD16could not be traced in the in vitro system yet.Most of the NER proteins exist as stable complexes, butrecent work has suggested that a minor fraction maycome in the form of a multi-protein supercomplex, ten-tatively called the 'repairosome' [23]. The reconstitutionstudies however, indicate that such a structure, if presentin the in vitro conditions, can be readily assembled fromand separated into its individual components. In the nearfuture, detailed work with the reconstituted system willundoubtedly reveal the significance of the appealingrepairosome concept.References1. Aboussekra A, Biggerstaff M, Shivji MKK, Vilpo JA, Moncollin V,Podust VN, Protic M, HObscher U, Egly J-M, Wood RD: MammalianDNA nucleotide excision repair reconstituted with purified compo-nents. Cell 1995, 80:859-868.2. Mortelmans K, Friedberg EC, Slor H, Thomas G, Cleaver JE: Defec-tive thymine dimer excision by cell-free extracts of xeroderma pig-mentosum cells. Proc Natl Acad Sci USA 1976, 73:2757-2761.3. Wood RD, Robins P, Lindahi T: Complementation of the xerodermapigmentosum repair defect in cell-free extracts. Cell 1988, 53:97-106.4. Sibghat-Ullah, Husain I, Carlton W, Sancar A: Human nucleotideexcision repair in vitro: repair of pyrimidine dimers, psoralen andcisplatin adducts by HeLa cell-free extract. Nucleic Acids Res 1989,17:4471-4484.5. Robins P, Jones C, Biggerstaff M, Lindahi T, Wood RD: Comple-mentation of DNA repair in xeroderma pigmentosum group Aextracts by a protein with affinity for damaged DNA. EMBO 1 1991,10:3913-3921.6. Masutani C, Sugasawa K, Yanagisawa J, Sonoyama T, Ui M,Enomoto T, Takio K, Tanaka K, van der Spek PJ, Bootsma D, et al.:Purification and cloning of a nucleotide excision repair complexinvolving the xeroderma pigmentosum group C protein and ahuman homolog of yeast RAD23. EMBOJ 1994, 13:1831-1843.7. O'Donovan A, Scherly D, Clarkson SG, Wood RD: Isolation ofan active recombinant XPG protein, a human DNA repair endo-nuclease. Biol Chem 1994, 269:15965-15968.8. Nichols AF, Sancar A: Purification of PCNA as a nucleotide excisionrepair protein. Nucleic Acids Res 1992, 20:2441-2446.9. Coverly D, Kenny MK, Lane DP, Wood RD: A role for human singlestranded DNA binding protein hSSB/RPA in an early stage ofnucleotide excision repair. Nucleic Acids Res 1992, 20:3873-3880.10. Shivji MKK, Kenny MK, Wood RD: Proliferating cell nuclear antigenis required for DNA excision repair. Cell 1992, 69:367-374.11. Calsou P, Salles B: Measurement of damage-specific DNA incisionby nucleotide excision repair in vitro. Biochem Biophys Res Comm1994, 202:788-795.12. Biggerstaff M, Szymkowsky DE, Wood RD: Co-correction of theERCC1, ERCC4 and xeroderma pigmentosum group F repair defectsin vitro. EMBO J 1993, 12:3685-3692.13. Van Vuuren AJ, Appeldoorn E, Odijk H, Yasui A, Jaspers NGJ, Hoeij-makers JHJ: Evidence for an repair enzyme complex involvingERCC1 and the correcting activities of ERCC4, ERCC11 and xero-derma pigmentosum group F. EMBOJ 1993, 12:3693-3701.14. Huang JC, Svoboda DL, Reardon T, Sancar A: Human nucleotideexcision nuclease removes thymine dimers from DNA by incisingthe 22nd phosphodiester bond 5' and the 6th phosphodiesterbond 3' to the photodimer. Proc Natl Acad Sci USA 1992, 89:3664-3668.15. Westerveld A, Hoeijmakers JHJ, VanDuin M, De Wit J, Odijk H,Pastink A, Wood RD, Bootsma D: Molecular cloning of a humanDNA repair gene. Nature 1984, 310:425-429.16. JHJ Hoeijmakers: Human nucleotide excision repair syndromes-molecular clues to unexpected intricacies. Eur Cancer 1994,30:1912-1921.17. Schaeffer L, Roy R, Humbert S, Moncollin V, Vermeulen W, Hoeij-makers JHJ, Chambon P, Egly J-M: DNA repair helicase: a compo-nent of the BTF-2 (TFIIH) basic transcription factor. Science 1993,260:58-63.18. Vermeulen W, Van Vuuren AJ, Chipoulet M, Schaeffer L, Appel-doom E, Weeda G, Jaspers NGJ, Priestly A, Arlett CF, Lehman AR, etal.: Three unusual repair deficiencies associated with transcriptionfactor TFIIH - evidence for the existence of a transcription syn-drome. Cold Spring Harb Symp Quant Biol 1994, 59: in press.19. Mu D, Park CH, Matsunaga T, Hsu DS, Reardon JT, Sancar A:Reconstitution of human DNA excision repair endonuclease in ahighly defined system. J Biol Chem 1995, 270:2415-2418.20. Roy R, Adamczewsky JP, Seroz T, Vermeulen W, Tassan JP, Schaef-fer L, Nigg EA, Hoeijmakers JHJ, Egly J-M: The MO15 cell cyclekinase is associated with the TFIIH transcription DNA repair factor.Cell 1995, 79:1093-1101.21. Van Vuuren A, Appeldoorn E, Odijk H, Humbert S, Eker APM,Jaspers NGJ, Egly J-M, Hoeijmakers JHJ: Partial characterization ofthe DNA repair protein complex containing ERCC1, ERCC4,ERCC11 and XP-F correcting activities. Mutation Res 1995, in press.22. Venema J, Mullenders LHF, Natarajan AT, Van Zeeland AA, MayneLV: The genetic defect in Cockayne syndrome is associated with adefect in DNA repair of UV-induced DNA damage in transcription-ally active DNA. Proc Natl Acad Sci USA 1990, 87:4707-4711.23. Svejstrup JQ, Wang ZG, Feaver WJ, Wu XH, Bushnell DA, DonahueTF, Friedberg EC, Kornberg RD: Different forms of TFIIH for tran-scription and DNA repair: holo-TFIIH and a nucleotide excisionrepairosome. Cell 1995, 80:21-28.Nicolaas GJ. Jaspers and Jan H.J. Hoeijmakers, Depart-ment of Cell Biology and Genetics, Erasmus University,Rotterdam, The Netherlands.

Nucleotide excision repair in the test tube.

Elsevier - Publisher Connector 

N.G.J. JASPERS AND J.H.J. HOEIJMAKERS DNA REPAIR 
Nucleotide excision-repair in the test tube 
The eu karyotic nucleotide excision-repair pathway has been reconstituted 
in vitro, an achievement that should hasten the full enzymological 
characterization of this highly complex DNA-repair pathway. 
All living organisms have evolved mechanisms to cope 
with the problem of genomic instabhty. Cells have a 
number of different mechanisms for repairing the various 
types of damage - resulting from errors of internal meta- 
bolism or the action of external agents - that their DNA 
is prone to. Many of these mechanisms are relatively sim- 
ple and speciahzed for the repair of strictly defined types 
of DNA lesions: those dealing with strand mismatches 
or small base adducts, for example. The nucleotide exci- 
sion-repair (NER) pathway, however, can recognize a 
broad spectrum of heh-distorting lesions, mostly gener- 
ated by environmental DNA-damaging agents, such as 
ultraviolet (UV) light. In eukaryotes, this versatility is 
accomplished by intricate interactions between many pro- 
teins, which mediate the excision and replacement of a 
single-strand patch substantially larger than the structurally 
affected DNA region itself. The basic features of NER 
have remained unchanged throughout eukaryotic evolu- 
tion, h m  simple yeasts up to hlgher mammals, and many 
parallels extend even to prokaryotes such as Escherichia coli. 
The complexity of eukaryotic NER is reminiscent of the 
other fundamental DNA metabolic processes - transcrip- 
tion, replication and recombination - and mutual inter- 
actions between these processes are now being uncovered. 
The classical biochemical approach to dissecting such 
processes involves the separation of the individual compo- 
nents and their reconstitution to a fully operative system. 
While simplified in vitro systems for basic transcription and 
replication have been available for some time, the recon- 
stitution of NER has just recently been reported by 
Aboussekra et al. [I]. This delay is remarkable as, unlike 
replication and transcription, NER is only essential in the 
presence of environmental stress, so that mutants afTected 
in this system can readily be identified and genetic analysis 
is possible. The well-known human inherited disorders 
xeroderma pigmentosum (XP), Cockayne's syndrome 
(CS) and trichothiodystrophy are caused by mutations, 
defining at least ten complementation groups, affecting 
NER; many other, less well-known, NER mutants have 
been identified in yeast and cultured mammalian cells. 
The first attempts to set up a mammalian cell-free NER 
system date back to 1976 [2]. However, this and other 
early systems lacked stability, reproducibhty or specifi- 
city. Twelve years later, and twenty years after Cleaver's 
discovery that NER is defective in cells from XP patients, 
Wood et al. [3], followed independently by Sibghat- 
Ullah et al. [4], described how carefully isolated mam- 
mahan cell extracts can repair damage on simple plasmid 
DNAs. The conditions appeared to mimic the NER sys- 
tem rather closely, as most of the factors affected in the 
known mammahan NER mutants were required. Extracts 
from different mutants that complement each other in 
vivo - that is, after cell fusion - were generally also able 
to do so in the cell-free system. 
The in vitro assay was soon successfully exploited for a 
number of enzymological advances. These included the 
isolation of the XPA, XPC and XPG proteins [5-71, the 
separation of the incision and excisiodgap-filling steps, 
the discovery of the involvement of the replication fac- 
tors PCNA (proliferating cell nuclear antigen) and RPA 
[8-111, the identification of the ERCCl/XPF enzyme 
complex [12,13] and the sizing of the excised patch to 
27-29 nucleotides [14]. From 1984 on [15], most of the 
NER genes were gradually cloned by painstaking gene- 
transfer experiments carried out using the mutant cell 
cultures (reviewed in [16]). In adhtion, the human multi- 
protein TFIIH basal transcription factor complex was 
isolated and unexpectedly found to harbour at least three 
known repair proteins [17,18]. It is mainly the combina- 
tion of these developments that has made the full in vitro 
reconstitution of NER possible. 
The approach used by Aboussekra et al. [I] and, indepen- 
dently but worked out in less detail, by Mu et al. [19] was 
as classical as it was effective. First, a repair-competent 
mamrnahan extract was fiactionated into at least eight 
parts using standard chromatographic procedures. Then, 
these fractions were tested one by one for the presence of 
known repair enzymes. Finally, the fractions were replaced 
by purified enzyme preparations, either derived from 
individual repair genes overexpressed in bacterial or insect 
cells, or from detailed classical purification protocols 
starting with HeLa nuclear extracts. 
The initial stages of NER, comprising the damage recog- 
nition step and a dual incision around the lesion, were 
found to require the single proteins XPA and XPG, 
and the multi-protein complexes TFIIH, RPA (hSSB), 
ERCC1 /XPF and XPC/HHR23B. The XPE-related 
protein UV-DDB acted as a dispensable stimulator. A 
new essential factor IF7 (incision factor 7), as yet only 
partially characterized, was also identified. Completion of 
the NER reaction, which involves patch-displacement, 
gap-filling and religation, could be accomplished by 
DNA polymerase E and ligase I, in combination with the 
accessory replication factors PCNA, RPA and RF-C. 
Interestingly, this final stage could be performed just as 
O Current Biology 1995, Vol 5 No 7 
DISPATCH 701
well by a combination of the E. coli DNA polymerase I
Klenow fragment and ligase.
A total of between 25 and 30 proteins (summarized in
Table 1) are minimally required to reconstitute the full
NER reaction; this is, however, just an estimate, as some
uncertainties remain. For instance, the TFIIH prepara-
tion used in the experiments contained at least six poly-
peptides, known as the 'core complex', which are also
required to support in vitro transcription. It is, however,
very likely that (minor) accessory factors present in this
preparation, such as cyclin-dependent kinase CDK7 [20],
are also involved in NER. As a second example, the
composition of the ERCC1/XPF complex is still uncer-
tain, as it could not be purified to homogeneity using
classical chromatography, a failure also experienced by
two other groups [19,21]. Besides ERCC1 and an
unknown 110 kD protein, identified by South-western
blotting [1] and immunoprecipitation [21], the complex
carries the complementing activities of the XPF and
702 Current Biology 1995, Vol 5 No 7
ERCC11 mutants [12,13]. Finally, the newly identified
factor IF7 also remains uncharacterized.
The in vitro reconstitution of NER clearly opens the
door to further dissection of its individual steps, their
order and the protein-protein interactions involved. In
the years to come, our knowledge of mammalian NER
can be expected to become at least as detailed as that of
the much simpler uvrABCD excinuclease system of
E. coli. Despite all these promises for the future, adapting
the basic NER reaction to simplified in vitro conditions
has its price. Cells repair the transcribed strand of their
active genes much faster than the remainder of the
genome, by an NER subpathway called transcription-
coupled repair (TCR). In patients with CS, TCR is
impaired, but the global NER facility is unaffected [22].
The gene products CSA and CSB are not required for
the reconstituted in vitro NER reaction, and extracts of
CS mutant cells (both human and hamster) have normal
NER activity. This implies that the test-tube conditions
selectively reflect the global repair subpathway, which is
not entirely unexpected as the damaged plasmid used as
the substrate is not transcribed in the experimental con-
ditions. It also fits with the need for the XPC/HHR23B
complex, which is known to be essential for the global
NER subpathway only [22].
Recent attempts to set up a repair assay with a substrate
carrying an active transcription unit and ribonucleotides
added in the reaction mixture have not yet resulted in
a clear-cut CSB-dependence or CSA-dependence. It
appears that much more is needed to reproduce the
TCR subpathway in vitro; presumably the chromatin
structure of the repair substrate has to be taken into
account as well. SV40 minichromosomes with a partially
intact chromatin structure can be repaired in vitro [6],
but this repair activity also appears largely to reflect the
global subpathway, as it is strongly dependent on XPC/
HHR23B. As a matter of fact, the human homologs of
the yeast global repair proteins RAD7 and RAD16
could not be traced in the in vitro system yet.
Most of the NER proteins exist as stable complexes, but
recent work has suggested that a minor fraction may
come in the form of a multi-protein supercomplex, ten-
tatively called the 'repairosome' [23]. The reconstitution
studies however, indicate that such a structure, if present
in the in vitro conditions, can be readily assembled from
and separated into its individual components. In the near
future, detailed work with the reconstituted system will
undoubtedly reveal the significance of the appealing
repairosome concept.
References
1. Aboussekra A, Biggerstaff M, Shivji MKK, Vilpo JA, Moncollin V,
Podust VN, Protic M, HObscher U, Egly J-M, Wood RD: Mammalian
DNA nucleotide excision repair reconstituted with purified compo-
nents. Cell 1995, 80:859-868.
2. Mortelmans K, Friedberg EC, Slor H, Thomas G, Cleaver JE: Defec-
tive thymine dimer excision by cell-free extracts of xeroderma pig-
mentosum cells. Proc Natl Acad Sci USA 1976, 73:2757-2761.
3. Wood RD, Robins P, Lindahi T: Complementation of the xeroderma
pigmentosum repair defect in cell-free extracts. Cell 1988, 53:
97-106.
4. Sibghat-Ullah, Husain I, Carlton W, Sancar A: Human nucleotide
excision repair in vitro: repair of pyrimidine dimers, psoralen and
cisplatin adducts by HeLa cell-free extract. Nucleic Acids Res 1989,
17:4471-4484.
5. Robins P, Jones C, Biggerstaff M, Lindahi T, Wood RD: Comple-
mentation of DNA repair in xeroderma pigmentosum group A
extracts by a protein with affinity for damaged DNA. EMBO 1 1991,
10:3913-3921.
6. Masutani C, Sugasawa K, Yanagisawa J, Sonoyama T, Ui M,
Enomoto T, Takio K, Tanaka K, van der Spek PJ, Bootsma D, et al.:
Purification and cloning of a nucleotide excision repair complex
involving the xeroderma pigmentosum group C protein and a
human homolog of yeast RAD23. EMBOJ 1994, 13:1831-1843.
7. O'Donovan A, Scherly D, Clarkson SG, Wood RD: Isolation of
an active recombinant XPG protein, a human DNA repair endo-
nuclease. Biol Chem 1994, 269:15965-15968.
8. Nichols AF, Sancar A: Purification of PCNA as a nucleotide excision
repair protein. Nucleic Acids Res 1992, 20:2441-2446.
9. Coverly D, Kenny MK, Lane DP, Wood RD: A role for human single
stranded DNA binding protein hSSB/RPA in an early stage of
nucleotide excision repair. Nucleic Acids Res 1992, 20:3873-3880.
10. Shivji MKK, Kenny MK, Wood RD: Proliferating cell nuclear antigen
is required for DNA excision repair. Cell 1992, 69:367-374.
11. Calsou P, Salles B: Measurement of damage-specific DNA incision
by nucleotide excision repair in vitro. Biochem Biophys Res Comm
1994, 202:788-795.
12. Biggerstaff M, Szymkowsky DE, Wood RD: Co-correction of the
ERCC1, ERCC4 and xeroderma pigmentosum group F repair defects
in vitro. EMBO J 1993, 12:3685-3692.
13. Van Vuuren AJ, Appeldoorn E, Odijk H, Yasui A, Jaspers NGJ, Hoeij-
makers JHJ: Evidence for an repair enzyme complex involving
ERCC1 and the correcting activities of ERCC4, ERCC11 and xero-
derma pigmentosum group F. EMBOJ 1993, 12:3693-3701.
14. Huang JC, Svoboda DL, Reardon T, Sancar A: Human nucleotide
excision nuclease removes thymine dimers from DNA by incising
the 22nd phosphodiester bond 5' and the 6th phosphodiester
bond 3' to the photodimer. Proc Natl Acad Sci USA 1992, 89:
3664-3668.
15. Westerveld A, Hoeijmakers JHJ, VanDuin M, De Wit J, Odijk H,
Pastink A, Wood RD, Bootsma D: Molecular cloning of a human
DNA repair gene. Nature 1984, 310:425-429.
16. JHJ Hoeijmakers: Human nucleotide excision repair syndromes-
molecular clues to unexpected intricacies. Eur Cancer 1994,
30:1912-1921.
17. Schaeffer L, Roy R, Humbert S, Moncollin V, Vermeulen W, Hoeij-
makers JHJ, Chambon P, Egly J-M: DNA repair helicase: a compo-
nent of the BTF-2 (TFIIH) basic transcription factor. Science 1993,
260:58-63.
18. Vermeulen W, Van Vuuren AJ, Chipoulet M, Schaeffer L, Appel-
doom E, Weeda G, Jaspers NGJ, Priestly A, Arlett CF, Lehman AR, et
al.: Three unusual repair deficiencies associated with transcription
factor TFIIH - evidence for the existence of a transcription syn-
drome. Cold Spring Harb Symp Quant Biol 1994, 59: in press.
19. Mu D, Park CH, Matsunaga T, Hsu DS, Reardon JT, Sancar A:
Reconstitution of human DNA excision repair endonuclease in a
highly defined system. J Biol Chem 1995, 270:2415-2418.
20. Roy R, Adamczewsky JP, Seroz T, Vermeulen W, Tassan JP, Schaef-
fer L, Nigg EA, Hoeijmakers JHJ, Egly J-M: The MO15 cell cycle
kinase is associated with the TFIIH transcription DNA repair factor.
Cell 1995, 79:1093-1101.
21. Van Vuuren A, Appeldoorn E, Odijk H, Humbert S, Eker APM,
Jaspers NGJ, Egly J-M, Hoeijmakers JHJ: Partial characterization of
the DNA repair protein complex containing ERCC1, ERCC4,
ERCC11 and XP-F correcting activities. Mutation Res 1995, in press.
22. Venema J, Mullenders LHF, Natarajan AT, Van Zeeland AA, Mayne
LV: The genetic defect in Cockayne syndrome is associated with a
defect in DNA repair of UV-induced DNA damage in transcription-
ally active DNA. Proc Natl Acad Sci USA 1990, 87:4707-4711.
23. Svejstrup JQ, Wang ZG, Feaver WJ, Wu XH, Bushnell DA, Donahue
TF, Friedberg EC, Kornberg RD: Different forms of TFIIH for tran-
scription and DNA repair: holo-TFIIH and a nucleotide excision
repairosome. Cell 1995, 80:21-28.
Nicolaas GJ. Jaspers and Jan H.J. Hoeijmakers, Depart-
ment of Cell Biology and Genetics, Erasmus University,
Rotterdam, The Netherlands.
