Download Department of Pathology, Stanford University School of Medicine

1
J. Cell Sci. Suppl. 6, 1-23 (1987)
Printed in Great Britain © The Company of Biologists Limited 1987
T H E M O L E C U L A R BIO LO G Y OF N U C LEO T ID E
E X C I S I O N R E P A IR O F D N A : R E C E N T PROG RESS
E R R O L C. F R IE D B E R G
Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305,
USA
SUMMARY
Recent years have witnessed significant progress towards understanding the molecular mech­
anism of nucleotide excision repair in living cells. Biochemical studies in
and
genetic and molecular studies in lower and higher eukaryotes have revealed an unexpected
complexity suggesting interesting protein-protein and protein-DNA interactions. This review
considers selected aspects of nucleotide excision repair in
and
mammalian cells, with a particular emphasis on new observations and on models that may provide
explanations for the complexity evident from genetic and biochemical studies.
Escherichia coli,
E. coli, Saccharonvyces cerevisiae
INTRO DUCTION
The concept of excision repair of DNA evolved during the mid-1960s (see
Friedberg, 1985, for a review) following the demonstration that when Escherichia
coli cells are ultraviolet (u.v.)-irradiated and then incubated, pyrimidine dimers are
lost from acid-precipitable (high molecular weight) DNA and can be recovered as
acid-soluble material. The additional demonstration of repair synthesis of DNA at
about the same time led to a general model for a multistep process in E. coli consisting
of the following sequential events:
(1) DNA incision 5' to sites of base damage
(2) Concomitant 5'—>3' excision-resvnthesis catalysed by DNA polymerase I
(3) DNA ligation.
Mutants of E. coli defective in the excision of pyrimidine dimers are also
abnormally sensitive to killing by a variety of chemicals known to damage DNA.
This suggested that the model presented above was distinguished by its generality,
i.e. that living cells probably excise all forms of base damage by a single molecular
mechanism. Progress during the past 15 years has proven this concept to be
simplistic. T h e discovery by Tomas Lindahl and his colleagues in the early 1970s of a
repair-specific class of enzymes called DNA glycosylases (see Friedberg, 1985, for a
review), was of fundamental importance, for it provided definitive evidence for
a distinct and novel mechanism for the excision of damaged and inappropriate (e.g.
uracil) bases, involving their excision as free bases. Hence, the phrase base excision
repair was coined to distinguish this mode of excision repair of DNA from so-called
nucleotide excision repair in which damaged bases are excised as part of a nucleotide
structure. Since then, at least one other distinct mode of excision repair has been
discovered. Inis', coli the process of post-replicative mismatch correction involves the
2
E. C. Friedberg
recognition of hemimethylated G A TC sequences and of mismatched bases in newly
replicated DNA (see Friedberg, 1985, for a review). A cell-free system for mismatch
correction has been established (Lu et al. 1983) and although the detailed mech­
anism of this process has not been elucidated, it is evident that it requires a number of
gene products not utilized in either base excision or nucleotide excision repair.
Finally, in at least two biological systems, elements of both base and nucleotide
excision repair are represented in a ‘hybrid’ biochemical mechanism specific for the
removal of pyrimidine dimers from DNA. Thus, in phage T4-infected E. coli and in
Microccus luteus, pyrimidine dimers are excised as part of an oligonucleotide, as is
true of nucleotide excision repair (see Friedberg, 1985, for a review). However, the
mechanism of DNA incision involves a specific DNA glycosylase and in this respect
resembles other examples of base excision repair. It is of ironic historical interest that
for many years the enzyme in T4-infected E. coli and M. luteus served as a general
model for the study of nucleotide excision repair before it was appreciated that the
mechanism of excision repair involving this enzyme is specific for pyrimidine dimers
and is apparently limited to these two organisms.
It is now apparent that the general term excision repair o f DNA is an over­
simplification that embraces multiple diverse biochemical mechanisms. Living cells
utilize discrete processes of base excision repair, nucleotide excision repair or
mismatch correction, depending on the type of damage present in DNA, and
manifest other cellular responses to DNA damage depending on the proximity of
damaged bases to sites of active DNA replication. Both excision repair and other
cellular responses to DNA damage have been subjects of a number of recent reviews
(see Friedberg, 1985, and references therein; Walker, 1985; Walker et al. 1985;
Cleaver, 1986; Sedgwick, 1986; Weiss & Grossman, 1986). This review deals
exclusively with the process of nucleotide excision repair, with particular emphasis
on recent advances in E. coli, the yeast Saccharomyces cerevisiae, and mammalian
cells.
E S C H E R I C H I A C O L I : A GENERAL MODEL FOR NU CLEOTIDE EXCISION
REPAIR?
The demonstration that mutants in the uvrA, uvrB or uvrC genes of E. coli are
defective in the excision of pyrimidine dimers during post-ultraviolet incubation led
to a search for the proteins encoded by these genes. These were first partially purified
by Seeberg and his colleagues using an assay in which ATP-dependent incision of
ultraviolet (u.v.)-irradiated DNA was complemented in extracts of a particular uvr
mutant by fractions from uvr+ cells or from different uvr mutants (Seeberg, 1978,
1981; Seeberg et al. 1978). This was an arduous task since constitutive un­
transformed E. coli cells contain very small amounts of UvrA, UvrB and UvrC
proteins (Sancar et al. 1981a, b; Yoakum & Grossman, 1981). Subsequent isolation
of the relevant uvr genes by molecular cloning facilitated studies on the genes
themselves, as well as their overexpression to yield large amounts of proteins for
biochemical studies.
Nucleotide excision repair of DNA
3
The uvr genes o f E. coli
Regulation. While the isolation and overexpression of the uvrA, uvrB, uvrC and
uvrD genes from recombinant plasmids have greatly facilitated inroads into the
biochemical mechanism of nucleotide excision repair in E. coli (see below), much
remains to be learned about the expression of the chromosomal genes, particularly
those that are controlled by other regulatory loci. The expression of the uvrA and
uvrB genes of E. coli is enhanced fivefold following exposure of cells to DNAdamaging agents, and there is good evidence that the induced expression of these
genes is part of the general SOS phenomenon regulated by the recA/lexA system (see
Friedberg, 1985, for a review). The uvrA gene contains a single typical prokaryotic
promoter with a Pribnow box and a ‘-35 sequence’ appropriately located with respect
to a single transcriptional start-site. This promoter is overlapped at its 5' end by a
consensus SO S box and binding of LexA protein to this operator region has been
demonstrated in vitro (Sancar et al. 1982b).
Regulation of the uvrB gene is less straightforward. Sequence analysis has
identified three distinct promoter regions and three transcriptional start-sites have
been mapped by studies in vitro (Sancar et al. 1982a). Two of these promoters (PI
and P2) are located relatively close to each other and to the presumed translation
start-site; the third (P3) is located about 300base-pairs (bp) upstream from them. A
typical SO S box overlaps P2 (the more distal of the two closely spaced promoters),
and in the presence of LexA protein in vitro transcription from this promoter is
blocked while that from the more proximal promoter PI is unaffected (Sancar et al.
1982a). This suggests that constitutive expression of uvrB is from P I, and that
induced expression is from P2 and is regulated by the recA/lexA system. On the
other hand, studies in living cells show that both promoters are active in the
constitutive and induced states, but PI is the stronger of the two (van den Berg et al.
1983). Thus, it is possible that all transcription of uvrB is controlled by PI and that
LexA protein regulates expression from PI by binding to P2. The role, if any, of the
most upstream promoter P3 is unknown; however, there are indications that this
promoter may be regulated by is. coli DnaA protein (see below).
The situation with respect to the uvrC gene is even more complicated. This gene
was not among the many is. coli din (damage mducible) genes isolated by the operon
fusion strategy that used the Mud (ApR lac) system (Kenyon & Walker, 1980).
Using uvrC-galK fusion plasmids, recA//ex4-dependent induction of galactokinase
has been demonstrated (van Sluis et al. 1983). However, in contrast to other SOS
genes, induction of uvrC under these experimental conditions is very slow and is not
effected by treatment of cells with nalidixic acid, characteristically a strong inducer
of SO S genes. The uvrC gene has been cloned and studied in a number of
laboratories; however, the promoter(s) for constitutive expression has not been
unambiguously identified. Current evidence suggests the presence of multiple
promoters of different strength, and of transcriptional regulation by attenuation
(Forster & Strike, 1985). In a recent study two different uvrC fusions to the E. coli
cat gene (one of which contains as much as 1200 bp of uvrC upstream sequence) were
4
E. C. Friedberg
tested for inducible expression of chloramphenicol acetyl-transferase (C A T). No
enhanced expression of CAT was observed in response to treatment with either u.v.
radiation or mitomycin C, and the levels of CAT expression were unaffected by
mutations in recA or lexA known to prevent induction or to result in constitutive
induction of SOS genes (Forster & Strike, 1985). Thus, it would appear that the
uvrC gene is not induced by DNA-damaging agents.
There is compelling evidence from cellular-biological studies for involvement of
the uvrD gene in nucleotide excision repair in E. coli. Unlike uvrA, uvrB and uvrC
strains in uvrD mutants, excision of pyrimidine dimers is not completely abolished.
However, such mutants show a reduced rate and extent of loss of thymine-containing
pyrimidine dimers during post-irradiation incubation (see Friedberg, 1985, for a
review). In addition, the extent (but not the initial rate) of ATP-dependent DNA
incision in semipermeabilized u.v.-irradiated cells is reduced in uvrD mutants
relative to uvr+ cells (Ben-Ishai & Sharon, 1981). There is also good evidence that
the uvrD gene is inducible and under SOS regulation. uvrD: :Mud (ApR lac)
insertion mutants have been identified and yield enhanced /3-galactosidase activity
following treatments known to induce SOS genes. Furthermore, the genetics of this
response is consistent with recA/lexA regulation (Maples & Kushner, 1982; Siegel,
1983; Kumura & Sekiguchi, 1984). In addition, sequence analysis of the uvrD gene
revealed a typical LexA binding box, which functions as an operator for purified
LexA protein in vitro and is situated downstream from a single promoter (Easton &
Kushner, 1983). In the absence of LexA protein a significant fraction of the mRNA
transcribed from this promoter in vitro terminates after about 60 nucleotides at a
sequence that resembles a rho-independent terminator (Easton & Kushner, 1983).
Hence, like the uvrC gene, expression of uvrD may be subject to regulation by
transcriptional attenuation.
Structural features. The uvr and polA genes have been sequenced (Joyce et al.
1982; Finch & Emmerson, 1984; Sancar et al. 1984; Arikan et al. 1986; Backendorf
et al. 1986; Husain et al. 1986) and examination of the predicted amino acid
sequences of the proteins has revealed a number of interesting homologies. For
example, a number of nucleotide binding proteins have regions of amino acid
sequence homology that include the tripeptide G K S (T ) (single-letter code for amino
acids) (Walker et al. 1982). (The third position of the tripeptide is sometimes G,
which has a hydropathic index very similar to that of S and T (Kyte & Doolittle,
1982).) Some of the proteins that contain the G K S (T or G) sequence are also purine
nucleoside triphosphatases and include the ¿s. coli RecA, UvrD (Finch & Emmerson,
1984) and UvrA (Husain et al. 1986) proteins. A related sequence is also present in
UvrB protein (Arikan et al. 1986; Backendorf et al. 1986) as well as in UvrC
and DNA polymerase I (Fig. 1). Since the latter protein binds deoxynucleoside
triphosphates it can be considered a nucleotide binding protein. Neither triphos­
phatase nor nucleotide binding activities have been associated with UvrB or UvrC
protein; however, a mixture of UvrA and UvrB protein has more ATPase activity
than UvrA protein alone (see below), suggesting the possible activation of such an
activity in UvrB protein. The sequence of UvrA protein shows two G K S (T or G)
Nucleotide excision repair of DNA
IM A
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Fig. 1. Sequence homology between the predicted amino acid sequences of regions of
and yeast nucleotide excision repair genes. T he single-letter amino acid code
is used throughout. Boxes denote identical amino acids or amino acids with similar
hydropathic values.
E. coli
regions and examination of their predicted secondary structure shows a striking
similarity between them and to other known ATPases, suggesting the presence of
two nucleotide binding sites in the protein (Husain et al. 1986).
A comparison of the amino acid sequences of the predicted UvrB and UvrC
polypeptides shows two other regions of homology (Backendorf et al. 1986; Husain
et al. 1986) and it is perhaps relevant in this regard that antisera to UvrB and to UvrC
proteins crossreact with both proteins (Backendorf et al. 1986). A region near the
amino-terminal end of UvrB protein also shows homology with a region of the
predicted amino acid sequence of the alkA gene product, a DN A glycosylase called
3-methyladenine-DNA glycosylase II.
Are uvr genes involved in other DNA transactions?
T h e study of the E. coli uvr genes has perhaps led to the implicit (if not explicit)
assumption that the process of nucleotide excision repair defines their exclusive role
in D N A metabolism. T h is section examines evidence that some of these genes may
be involved in other DN A transactions. I particularly wish to address the possibility
that in E. coli DN A repair may be linked to processes such as DN A replication,
through one or more gene products common to both events. If such linked functions
do indeed exist for particular uvr genes, they could have interesting and important
regulatory potential. T hu s, for example, the sequestration of Uvr proteins by an
excision repair complex could limit the amount of these proteins available for a
replication complex, hence providing a mechanism for regulating the initiation
and/or progression of semi-conservative DN A synthesis during nucleotide excision
repair.
Several provocative examples of the possible involvement of uvr genes in DNA
replication have been documented. Early studies demonstrated that uvrD mutants
that are also defective in th epolA gene are inviable (Horiuchi & Nagata, 1973; Siegel,
1973; Sm irnov et al. 1973), implicating the uvrD gene in D N A replication. More
6
E. C. Friedberg
recently, it has been shown that the uvrD gene encodes an ATP-dependent DNA
helicase called DNA helicase II (Arthur et al. 1982; Maples & Kushner, 1982;
Kumura & Sekiguchi, 1984). Antiserum raised against purified DNA helicase II
inhibits the replication of phage A or ColEl DNA in subcellular systems (Klinkert
et al. 1980); and the addition of DNA helicase II to a cell-free system stimulates the
replication of artificially forked X DNA (Kuhn & Abdel-Monem, 1982). Mutants
defective in just the uvrD gene are viable; hence DNA helicase II is apparently not
absolutely required for DNA replication. Nonetheless, the results just summarized
are consistent with an involvement of UvrD protein in normal DNA replication in
E. coli.
The double mutant uvrBpolA is also inviable; an observation that dates back to the
early 1970s (Shizuya & Dykhuizen, 1972; Morimyo & Shimazu, 1976). More
recently, evidence has emerged for the possible regulation of the uvrB gene by E. coli
DnaA protein. DnaA protein is a polypeptide of Mr ~ 4 8 x l 0 3 required for the
initiation of bidirectional replication at oriC, the E. coli origin of replication (Korn­
berg, 1980). Studies on the specific cooperative binding of DnaA protein to oriC
have defined a minimal binding sequence with the consensus 5' T T A T ^ C A ^ A 3'
(Fuller et al. 1984). This sequence is highly conserved in the chromosomal rep­
lication origin of other prokaryotes, in the replication origin of a number of plasmids,
and in the upstream regulatory region of a number of E. coli genes, including the
dnaA gene itself, p y r B l, argF, polA, dam and uvrB (Fuller et al. 1984). In the case
of the dnaA gene, DnaA protein appears to act as a negative auto-regulator of
expression. Thus, in cells transformed with dnaA-lacZ fusion plasmids, the ex­
pression of /3-galactosidase correlates inversely with levels of DnaA protein (Fuller
et al. 1984).
In the uvrB gene two putative DnaA protein binding sites are located in inverted
orientation in a region with potential stem-loop structure that includes part of P3,
the most 5' of the three functional promoters identified in this gene (see above) (van
den Berg et al. 1985). The possible interaction of DnaA protein with the uvrB gene
represents another potential mechanism for coupling uvrB expression to DNA
replication. Fuller et al. (1984) have suggested that the replication of oriC during
initiation of DNA synthesis could generate sequences that may act as a sink for highaffinity binding of DnaA protein, resulting in the derepression of genes negatively
regulated by this protein. The uvrB gene (and perhaps other genes that are regulated
by dnaA) might encode products that play a role in replication initiation. Thus, in
the case of UvrB protein, we have a possible example of a gene product involved in
both nucleotide excision repair and DNA replication, the enhanced expression of
which is independently regulated for both functions by using distinct operator/
promoter sites. In cells containing damaged DNA the amount of available UvrB
protein may limit replication initiation during excision repair or vice versa, depen­
ding on the relative affinity of UvrB protein for the excision repair and replication
complexes.
Nucleotide excision repaii' of DNA
7
Finally, it is interesting to consider another gene involved in DN A replication in
E. coli, mutations in which can result in an abnormal excision-repair phenotype. T h e
d n aB gene of E . coli encodes a protein of molecular weight « 5 0 X 1 0 3 essential for
replication of the E. coli chromosome (Kornberg, 1980). A strain designated
CM 1031 carries a temperature-sensitive mutation in the dn aB gene (Bridges, 1975).
At permissive temperatures, this strain has normal resistance to u.v. radiation but is
deficient in host-cell reactivation of bacteriophage A. Bridges et al. (1976) observed
that this mutant loses sites sensitive to a dimer-specific enzyme probe at a reduced
rate relative to the parental strain, suggesting a partial defect in excision repair. T h e
possible involvement of DnaB protein in nucleotide excision repair represents a third
potential component of the E. coli replication complex, which may be limited in cells
exposed to D N A damage (Fig. 2).
The JJvrA, UvrB an d UvrC proteins
Isolation of the uvrA, uvrB and uvrC genes and their overexpression in i?, coli has
greatly facilitated the purification of the proteins they encode and their use in
biochemical studies. T h e measured molecular weights of the UvrA, UvrB and UvrC
proteins are ~ 1 1 4 X 103, —84 X 103 and ~ 6 7 x 103, respectively (Thom as et al. 1985),
and agree well with values calculated from the coding regions of the cloned genes
(1 0 37 4 9 , 76 118 and 660 3 8 ) (Sancar et al. 1984; A. Sancar, personal communi­
cation) .
Purified U vrA protein is a DNA-independent A TPase (Seeberg & Steinum,
1982). Neither of the other two Uvr proteins has demonstrable catalytic activity of
any kind. However, as indicated above, a mixture of UvrA and UvrB proteins shows
more A TPase activity than UvrA protein alone (Thom as et al. 1985). It is not known
Fig. 2. T h e UvrD and UvrB proteins are known to be involved in nucleotide excision
repair inis.
and may also be involved in DNA replication. DnaB protein is required
for DNA replication, and may also be involved in nucleotide excision repair.
coli,
8
E. C. Friedberg
whether this reflects stimulation of the activity associated with UvrA protein or the
unmasking of an activity in the UvrB protein that is dependent on binding to UvrA
protein. None of the Uvr proteins in isolation or in any pairwise combinations
catalyses the incision of damaged DNA. But when u.v.-irradiated DNA is incubated
with a mixture of all three Uvr proteins incision is observed and is strictly dependent
on the presence of Mg2+ and A TP (Seeberg, 1978; Sancar & Rupp, 1983; Yeung
et al. 1983). On the basis of the long-standing preconception of the molecular
mechanism of nucleotide excision repair in E. coli discussed in the Introduction, it
was anticipated that this reaction would generate incisions exclusively 5' to sites of
base damage, thus setting the stage in vivo for coordinated excision/resynthesis
mediated by DNA polymerase I. Surprisingly, incision occurs on both sides of each
lesion of a given DNA strand (Sancar & Rupp, 1983; Yeung et al. 1983). With
respect to cyclobutyl pyrimidine dimers as well as so-called (6-4) or Py-C products
(the two major photoproducts in DNA) the UvrABC catalytic activity hydrolyses the
eighth phosphodiester bond 5' and the fourth or fifth phosphodiester bond 3' to the
lesions (Sancar & Rupp, 1983). A fundamentally similar mode of incision in vitro has
been observed with DNA containing monoadduct base damage caused by photo­
activated psoralen or A^-acetoxy-acetylaminofluorene (Sancar et al. 1985). The only
difference is that with these substrates incisions 3' to sites of base damage are
exclusively at the fifth phosphodiester bond. The pattern of DNA incision is
insensitive to the ratio of psoralen monoadducts and psoralen crosslinks and hence it
has been suggested that DNA containing interstrand crosslinks is cut in the same way
as DNA containing monoadduct or dipyrimidine base damage (Sancar et al. 1985).
Incision of DNA by the purified UvrABC activity has been observed with a variety of
other chemicals that produce bulky base adducts in DNA (Fuchs & Seeberg, 1984;
Kacinski & Rupp, 1984; Husain et al. 1985) and, while not specifically
demonstrated, it is a reasonable assumption that in all cases a bilateral incision
mechanism operates.
The demonstration in vitro of bilateral DNA incision that is strictly dependent on
all three Uvr proteins raises a number of interesting questions concerning its
mechanism. How do the Uvr proteins interact with DNA and with one another?
How is a catalytically active endonuclease constituted? How is incision apparently
confined to the DNA strand containing sites of base damage? How are the bilateral
incisions placed at precise locations with respect to sites of base damage? How does
bilateral incision facilitate excision of base damage? Answers to these questions are
beginning to emerge. There are no indications that the Uvr proteins constitute a
preformed complex prior to incision of damaged DNA. On the contrary, the
available evidence suggests that individual proteins bind to damaged DNA in a
sequential fashion and that a catalytically active UvrABC complex is generated on
the substrate (Weiss & Grossman, 1986; Yeung et al. 1986). UvrA protein binds
preferentially to double-stranded u.v.-irradiated DNA relative to unirradiated
DNA, and this binding is stimulated in the presence of ATP and inhibited by ADP
(Weiss & Grossman, 1986; Yeung et al. 1986). Binding of A TP to UvrA protein
results in conformational alterations of the protein that may be important for the
Nucleotide excision repair of DNA
9
constitution of a catalytically active UvrABC complex (Weiss & Grossman, 1986).
Thus, in the presence of A TP or non-hydrolysable ATP analogues such as A TP[yS]
UvrA protein is more readily iodinated at tyrosine residues. Furthermore, in the
absence of A TP UvrA protein forms dimers that exist in an equilibrium state with
monomers, whereas in the presence of A TPfyS] the protein exists almost exclusively
in the dimerized form. The combination of UvrA protein with UvrC protein does
not increase its binding affinity for u.v.-irradiated DNA, and UvrB protein alone
does not bind to u.v.-irradiated DNA. However, when UvrA and UvrB proteins are
mixed both proteins bind to u.v.-irradiated DNA to form a DNA-protein complex
with a half-life significantly longer than that of the UvrA-DNA complex (Weiss &
Grossman, 1986; Yeung et al. 1986). Once UvrA and UvrB proteins bind to u.v.irradiated DNA the addition of UvrC protein results in incision. Evidence suggests
that UvrC protein binds directly to the U vrA B-DN A complex without first binding
to DNA and that the formation of a stable U vrAB-DN A complex is the rate-limiting
step in the incision reaction catalysed by the UvrABC endonuclease (Weiss &
Grossman, 1986; Yeung et al. 1986).
Following incision of u.v.-irradiated DNA by the UvrABC endonuclease in vitro
the DNA-protein complex persists, thus limiting the turnover of Uvr proteins.
However, the addition of UvrD protein and DNA polymerase I alters the stability of
this complex and also has a significant effect on the incision reaction itself (Caron
et al. 1985; Husain et al. 1985; Kumura et al. 1985). When the amount of UvrC
protein is rate-limiting for the UvrABC endonuclease-catalysed incision reaction the
addition of UvrD protein stimulates the incision of u.v.-irradiated DNA, suggesting
that this protein causes UvrC protein in the complex to turn over (Caron et al. 1985).
However, the complete turnover of UvrA, UvrB and UvrC proteins requires the
addition of both UvrD protein and DNA polymerase I (Husain et al. 1985). The role
of DNA polymerase I is at least partially dependent on concurrent DNA polym­
erization (i.e. repair synthesis), since less stimulation of nicking of u.v.-irradiated
DNA is observed in the absence of deoxyribonucleoside triphosphates (Husain et al.
1985). T 4 DNA polymerase or the Klenow fragment of DNA polymerase I can
replace intact E. coli DNA polymerase I; however, E. coli Rep protein cannot
substitute for UvrD protein (Husain et al. 1985). The complete reaction involving
the four Uvr proteins and DNA polymerase I is not associated with an increase in the
initial rate of DNA incision (Husain et al. 1985). Thus, UvrD protein and DNA
polymerase I apparently do not influence the kinetics of formation of the UvrABC
complex on damaged DNA, or its intrinsic endonucleolytic activity.
The precise mechanism of the release (excision) of the oligonucleotide created by
bilateral incision of u.v.-irradiated DNA is not yet established. Studies by Caron
et al. (1985) have shown that under non-denaturing conditions the oligonucleotide
fragment is not released in vitro and is dependent on the addition of both DNA
helicase II and DNA polymerase I. On the other hand, Husain et al. (1985) observed
some removal of cisplatin adducts from DNA in the absence of the latter two
proteins, but, as is true for the incision reaction, the extent of damage excision was
stimulated by their addition.
10
E. C. Friedberg
Collectively the data summarized above suggest the following model for the
molecular mechanism of nucleotide excision repair in E. coli (Husain et al. 1985;
Weiss & Grossman, 1986; Yeung et al. 1986). UvrA protein binds A TP and
undergoes a conformational change that favours the formation of protein dimers. In
this state UvrA protein has a high affinity for sites in duplex DNA with confor­
mational alterations in secondary structure. UvrB protein binds specifically to
UvrA—DNA complexes and these complexes are recognized by UvrC protein, the
binding of which generates a catalytically active endonuclease that nicks the DNA at
precise locations relative to DNA adducts. The events that determine strand
specificity for DNA incision and the locations of these incisions relative to each lesion
are uncertain at present. However, it is presumably not pure coincidence that the
distance separating the two incisions flanking each lesion approximates one helical
turn of fi-DN A . It is also likely that the conformational distortion generated by bulky
base adducts is asymmetrical with respect to the helical axis of DNA and this may
facilitate preferential binding of UvrA protein to the damaged strand. The structure
of the UvrABC complex subsequently assembled on the DNA may provide for
contact of catalytic sites separated by a distance that is close to a single helical turn.
Alternatively, the binding of the UvrABC complex to DNA may unwind the DNA
helix, generating regions of local denaturation precisely 12 (and in the case of
pyrimidine dimers sometimes 13) nucleotides long. The enzyme may then cleave the
DNA strand to which it is preferentially bound at the boundaries of the denaturation
bubble. Following DNA incision UvrD protein alone (or in combination with DNA
polymerase I) promotes turnover of UvrC protein, which is freed to interact with
other U vrA B-D N A complexes. A complex consisting of UvrA and UvrB proteins
bound to a potential oligonucleotide 12 (or 13) nucleotides long remains relatively
stable until both UvrD protein and DNA polymerase I (or DNA polymerase II or
I I I ) effect its displacement from the DNA duplex with concurrent repair synthesis
and DNA ligation.
This model is consistent with the phenotype of mutants defective in the uvr or
polA genes, uvr A, uvrB and uvrC strains are completely defective in DNA incision.
Mutants defective in the uvrD or polA genes should not manifest a defect in the initial
rate of DNA incision. However, because these genes are required for turnover of
UvrA, UvrB and UvrC proteins, one would expect mutations in these genes to
reduce the extent of DNA incision relative to wild-type strains. As indicated above, a
reduced efficiency of DNA incision in a uvrD mutant was demonstrated some years
ago by Ben-Ishai & Sharon (1981), and both uvrD and polA mutants have been
shown to be deficient in the loss of thymine-containing pyrimidine dimers (excision)
from DNA during post-irradiation incubation in vivo (see Friedberg, 1985).
The model presented above predicts that DNA incision, damage excision, repair
synthesis (and presumably DNA ligation) are highly coordinated events possibly
effected by a multiprotein complex (repairosome ) that acts in a strictly processive
fashion (Weiss & Grossman, 1986). Assuming stoichiometric interactions of Uvr
proteins and DNA polymerase, and in the absence of competing reactions such as
DNA replication, the rate of nucleotide excision repair in E. coli is presumably
Nucleotide excision repair of DNA
11
limited by the amount of UvrA, UvrB and UvrC protein, all of which are present at
levels of < 100 molecules per cell (Husain et al. 1985). DNA polymerase I and UvrD
protein are present in larger amounts and are not expected to be rate-limiting for
nucleotide excision repair in the presence of semiconservative DNA synthesis.
However, if, as suggested earlier, UvrB is also involved in DNA replication and
DnaB protein is involved in excision repair, these proteins could limit the rate of
replication during nucleotide excision repair.
N U C L E O T ID E EXCISION REPAIR IN EUKARYOTES
At the present time, E. coli is the only organism for which any detailed information
exists on the biochemistry of nucleotide excision repair. However, recent years have
witnessed interesting developments in at least two eukaryotic systems that offer the
potential for exploring the generality of this process as we know it in E. coli.
The yeast S. cerevisiae
Close to 100 yeast mutants (some of which may represent alleles of the same gene)
have been isolated with the phenotype of abnormal sensitivity to killing by DNAdamaging agents (see Haynes & Kunz, 1981, for a review). Using u.v. radiation as a
source of DNA damage the sensitivity of some mutants known to be defective in
different single genes was compared with that of mutants defective in more than one
of these genes. If mutations in both genes impart no more u.v. sensitivity than
mutations in either gene alone the genes are said to be epistatic, and the most obvious
interpretation of an epistatic interaction is that the two genes function in the same
biochemical pathway (see Haynes & Kunz, 1981, for a review). Such experiments,
when combined with the results of studies that directly measure parameters of
nucleotide excision repair in u.v.-irradiated yeast cells (e.g. loss of thyminecontaining pyrimidine dimers or introduction of strand breaks during post-u.v.incubation) have identified 10 loci unique to the nucleotide excision repair (so-called
RAD3) epistasis group. Two other mutant loci also behave epistatically with mutants
in this group, but are also epistatic with mutant loci in other groups, suggesting an
involvement in more than one type of cellular response to DNA damage. One of
these was originally designated r), but has been renamed RAD24 (F . EckhardtSchump, W. Siede & J. C. Game, personal communication). The rad24- 1 mutant is
epistatic to other mutants in the RAD3 epistasis group with respect to u.v. radiation
sensitivity and is epistatic to mutants in the RAD52 group with respect to sensitivity
to X - or y-radiation (F. Eckhardt-Schump, W. Siede & J. C. Game, personal
communication). Similarly, a strain mutated in the CDC8 gene that encodes thymidylate kinase is u.v.-sensitive and epistatic to a ra d l mutant (belonging to the RAD3
epistasis group) (Prakash et al. 1979). In addition, this mutant shows reduced u.v.
mutability, and in this regard is epistatic to rad6 from the RAD6 epistasis group
(Prakash et al. 1979). Thus, at least 12 genetic loci may be involved in nucleotide
excision repair in yeast (Fig. 3).
12
E. C. Friedberg
G E N E T I C C O M P L E X I T Y O F EXCI S I O N R E P A I R IN Y E A S T
R A D 3 Epistasis Group
RADI
RAD2
RAD3
RAD»
RAD7
RADIO
RADI»
RAD16
RAD23
(R A D 2 4 )
MdS19
(C D C 8 )
RAD3
Fig. 3. Genes in the
epistasis group are thought to be involved in nucleotide
excision repair in yeast. The involvement of the two genes marked in parenthesis has not
been firmly established.
Mutations in five of these loci (RADI, RAD2, RAD3, RAD4, RADIO) confer
marked sensitivity to killing by u.v. radiation and a complete defect in phenotypes
associated with nucleotide excision repair. Mutations in five others (RAD7 , RAD14,
RAD16, RAD23, MMS19) result in less sensitivity to u.v. radiation and retain a
significant residual capacity for repair, though at levels less than those in wild-type
cells (see Friedberg, 1986; Friedberg et al. 1986). (The nucleotide excision repair
capacity of the rad.24 and cdc8 mutants has not been reported.) This segregation of
gene functions into those absolutely required for nucleotide excision repair and those
that apparently are not resembles that which distinguishes the uvrA, uvrB and uvrC
genes from the uvrD and polA genes of E. coli, and suggests the possibility that the
R adi, Rad2, Rad3, Rad4 and RadlO proteins are involved in the incision of DNA
while the second group of gene products are involved in post-incision events. This
comparison is not intended to suggest that the Radi, Rad2, Rad3, Rad4 and RadlO
proteins function as a specific homologue of the UvrABC complex of E. coli or that
the Rad7, R adl4, Radl6, Rad23 and M msl9 proteins are strictly analogous to E. coli
UvrD protein and DNA polymerase I.
The RAD genes and R ad proteins. The RADI, RAD2, RAD3 and RADIO genes
have been cloned by screening yeast genomic libraries for multicopy plasmids
containing sequences that complement the u.v. sensitivity of appropriate mutants
(see Friedberg, 1986; Friedberg et al. 1986). Such screening failed to reveal
recombinant plasmids containing the RAD4 gene. Recent studies in my laboratory
(R. Fleer, C. Nicolet, G . Pure & E. C. Friedberg, unpublished) have shown that
RAD4 is located very close to a gene called SPT2, and that a recombinant plasmid
previously isolated by Roeder et al. (1985) contains the entire RAD4 gene. However,
Nucleotide excision repair of DNA
13
in this plasmid, as well as in other plasmids propagated in E. coli, the gene is
mutationally inactivated. Deletion of defined regions of the mutant plasmid-borne
gene and repair of the resultant gaps by gene transfer from yeast genomic sequences
reconstitutes circular plasmids with a functional RAD4 gene that complements the
u.v. sensitivity of all rad4 mutants tested (R. Fleer, C. Nicolet, G. Pure & E. C.
Friedberg, unpublished data). However, propagating such plasmids ini?, coli again
results in inactivation of the gene. These results suggest that Rad4 protein is toxic to
E. coli and explain the failure to isolate the functional gene on a plasmid from a yeast
library cloned in this bacterium. The confines of the RAD4 gene have been
established by insertional mutagenesis at defined sites and by mapping the
transcriptional start-sites, and indicate that the gene is —2-3 X103 bp in size, thus
yielding a calculated molecular weight of —9 0 X103 for Rad4 protein.
The nucleotide sequences and predicted amino acid sequences of the RADI,
RAD2, RAD3 and RADIO genes and the proteins they encode have been determined
(Yang & Friedberg, 1984; Naumovski et al. 1985; Reynolds et al. 1985a,b ; Nicolet
et al. 1985; Weiss & Friedberg, 1985; Madura & Prakash, 1986) and have revealed
several points of interest. (1) The four genes can encode proteins of calculated
molecular weight —110X103, —118X 103, —8 9 X 1 0 3 and = 2 4 x l0 3, respectively.
Hence, if the products of all 10 genes known to be involved in nucleotide excision
repair assemble as a single multiprotein complex at sites of base damage in yeast
chromatin, the size of such a complex is at least 500X 103M r and could be as large as
10(>MI. (2) Intervening sequences have not been detected in any of these genes and
all are present in the yeast genome in single copy. (3) Hydropathicity profiles of the
predicted polypeptides show a balanced distribution of hydrophobic and hydrophilic
domains in the Radi, Rad3 and RadlO polypeptides. Rad2 protein appears to be
unusually hydrophilic (Fig. 4). (4) We have not detected extensive nucleotide
sequence homology between any of these genes, or with other genes evaluated by
computer search. Furthermore, using the cloned RAD genes as probes we have not
observed hybridization to total human, rodent, Drosophila or E. coli DNA. Several
limited regions of amino acid sequence homology have been noted between the Rad
proteins themselves and between one or fnore Rad proteins and other proteins. The
most interesting of these are the following: (a) the tripeptide G K S (T or G) observed
in the amino acid sequence of the E. coli Uvr proteins and DNA polymerase I (see
Structural features, p. 4) is also common to the Rad3 (Reynolds et al. 1985a;
Naumovski & Friedberg, 1986), Radi and RadlO proteins of 5. cerevisiae (Fig. 1),
suggesting a convergent evolutionary relationship between these three yeast proteins
and the excision repair proteins of E. coli. The homology between the yeast Rad3
protein and the E. coli UvrA, UvrB and UvrD proteins is particularly impressive.
Over a stretch of 22 amino acids, 19 are common to two or more of these four proteins
and the pentapeptide G S (T )G K T (S ) is common to all of them. This has focused
attention on the possibility that Rad3 protein may possess ATPase, GTPase and/or
nucleotide binding activity (see below), (b) There is significant homology between
the carboxy-terminal half of the RadlO polypeptide and a region of a protein
designated E rccl, which is believed to be involved in nucleotide excision repair of
E. C. Friedberg
RAD3
RAD2
HYDROPATH I C I T Y
HYDROPATH I C I T Y
Nucleotide excision repair of DNA
RADIO
IS
HYDROPATH I C I T Y
Fig. 4. Hydropathicity profiles of the R ad i, Rad2, Rad3 and RadlO proteins. The light
areas highlight peaks with values greater than +2-5 or -2 - 5 .
D N A in human cells (van Duin et al. 1986) (see below). Over a stretch of —100
consecutive amino acids, 39 are common to both proteins and a further 37 are closely
related (van Duin et al. 1986). (c) A number of prokaryotic specific DN A binding
proteins share a consensus amino acid sequence consistent with the <*-helix-turn-<*helix secondary structure that is characteristic of their DN A binding domain. Among
the lower eukaryotes many of the proteins with so-called homeo boxes match this
consensus rather well, as do regions in the yeast DN A binding proteins Mated and
Matcv2. Comparison of a number of these proteins with RadlO and Rad3 suggests
that these two proteins may interact with DN A through a or-helix—turn—ar-helix
motif (van D uin et al. 1986; Naumovski & Friedberg, 1986).
In summary, these amino acid sequence comparisons suggest that the E. coli and
some of the yeast nucleotide excision repair proteins share a common domain, which
may be important functionally in nucleotide binding and/or nucleotide triphos­
phatase activity. In addition, Rad3 and RadlO proteins may bind to D N A , perhaps
in a sequence-specific fashion.
T h e functional relationships suggested by these homologies can be explored in a
number of ways. One is to determine whether any of the Rad proteins can comp­
lement phenotypes related to excision repair defects in E. coli and mammalian cells.
T o the extent that such experiments have been completed no interspecies comp­
lementation has been reported. T h e RAD3 and RADIO genes have been expressed in
E. coli and yield proteins of the expected size, yet these proteins do not complement
the u.v. sensitivity of uvrA, uvrB, uvrC or uvrD mutants (L . Naumovski, W. A.
Weiss & E . C. Friedberg, unpublished observations). Similarly, the uvrA and uvrB
genes have been expressed in r a d l , rad2, rad3 and radlO strains without detectable
phenotypic complementation of these mutants (Planque et al. 1984). T h e RAD3 and
RADIO genes have also been tailored into mammalian cell expression vectors (L .
16
E. C. Friedberg
Couto, G. Chu, R. Schultz, P. Berg & E. C. Friedberg, unpublished observations).
At the time of writing, it has not been definitively established that the respective Rad
proteins are actually expressed in mammalian cells transfected with these
recombinant plasmids. Nonetheless, no enhancement of the u.v. resistance of
xeroderma pigmentosum (XP) group A cells (by RAD3) or of a Chinese hamster
ovary (CHO) cell line designated UY20 (see below) (by RADIO) has been observed.
A second way of exploring structural relationships between proteins from different
organisms is by using specific antibodies. We have recently raised rabbit antisera that
react specifically with yeast Rad2 and Rad3 proteins (L. Naumovski, C. Nicolet &
E. C. Friedberg, unpublished observations). The antiserum containing rabbit antiRad3 antibodies does not react with purified UvrA, UvrB or UvrC proteins by
immunoblotting (C. Backendorf, L . Naumovski & E. C. Friedberg, unpublished
observations). Reactions of both antisera against extracts of mammalian cells and of
anti-Rad2 antiserum against Uvr proteins, are in progress.
Finally, it is important to determine whether any of the Rad proteins catalyse
reactions suggested by sequence homology with other proteins. Rad3 protein has
been extensively purified, but no ATPase or GTPase activity has been detected
(L . Naumovski & E. C. Friedberg, unpublished observations). However, we have
not yet excluded the possibility that such activity is dependent on the association of
Rad3 with other Rad protein(s), or on some other specific mechanism of activation.
Studies on nucleotide and DNA binding by Rad3 protein and on DNA helicase
activity in this protein are in progress.
RAD3 is an essential gene in S. cerevisiae. Disruption (as opposed to point
mutagenesis) of the RAD3 gene is lethal to haploid cells, indicating that this gene is
essential for viability. None of the other RAD genes under study has this property.
The nature of the essential function is not known. However, the observation that
rad3 alleles carrying point mutations are viable, but defective in nucleotide excision
repair, suggests that Rad3 protein may have two distinct activities. In an effort to
gain insight into the nature of these activities and their localization at the level of the
primary structure of Rad3 protein, we mutagenized a number of sites in the cloned
RAD3 gene and examined their effect on the phenotypes of u.v. sensitivity and
viability (Naumovski & Friedberg, 1986). Single point mutations in the putative
nucleotide or DNA binding domains had no effect on the essential function of Rad3;
indeed, the essential function of Rad3 protein is very resistant to inactivation by
single point mutations and the only such mutant isolated to date contains a
temperature-sensitive point mutation at codon position 73. A site of tandem point
mutations affecting two adjacent codons in the putative DNA binding domain also
inactivates the essential function, but the site specificity of these mutations is
uncertain, pending study of the effect of tandem mutations in other regions of the
protein. The excision repair function of Rad3 protein is inactivated by single point
mutations at many sites (including both the putative nucleotide binding and DNA
binding domains), an observation that explains the facile isolation of viable, excision
repair-defective mutants. Collectively, these results suggest that whatever the nature
Nucleotide excision repair ofDNA
17
of the nucleotide excision repair function of the Rad3 protein, it is highly sensitive to
conformational changes, whereas the essential function of the protein is not. Hence,
it is likely that the type of protein-protein and/or protein-DNA interactions that
characterize the two Rad3 functions are different.
The essentiality of the RAD3 gene may constitute an example in yeast of the
suggestion made earlier with respect to E. coli, i.e. that some proteins involved in
nucleotide excision repair are also involved in other DNA transactions such as
replication. As indicated above, we have isolated a temperature-sensitive rad3
mutant that is defective for growth at non-permissive temperatures. Studies on DNA
replication in this mutant at permissive and restrictive temperatures may be
illuminating.
The RAD2 gene is inducible by DNA damage. As indicated earlier, the E. coli
UvrA, UvrB and UvrC proteins are constitutively expressed in very small amounts
(«=10—20 copies per cell). In the case of the first two proteins this level is amplified
«fivefold in cells exposed to DNA damage or other treatments that interfere with
normal semiconservative DNA synthesis. At least some of the yeast RAD genes are
also weakly expressed. Accurate quantitative measurements of transcriptional ac­
tivity are difficult to obtain; however, qualitative observations from D N A -R N A
hybridization experiments indicate the presence of very small amounts of RADI,
RAD2, RAD3 and RADIO transcripts (L. Naumovski, C. Nicolet, E. Yang & E. C.
Friedberg, unpublished observations). In addition, fusions of the promoter and S'
coding region of these genes to the E. coli lacZ coding region results in very limited
expression of E. coli /3-galactosidase relative to fusions with highly expressed yeast
genes (Robinson et al. 1986).
When yeast cells containing a single integrated copy of a RAD2-lacZ fusion
plasmid (or an autonomously replicating multicopy fusion plasmid) are exposed to
treatments known to induce E. coli SO S genes, fivefold enhanced expression of
/3-galactosidase is observed (Robinson et al. 1986). Cells transformed with RAD1-,
RAD3- or RADI0-lacZ fusions do not show this result. Consistent with the notion of
enhanced gene expression, untransformed cells exposed to 4-nitroquinoline-l-oxide
(4NQO) show increased levels of RAD2 mRNA. The levels of RADIO messenger are
unaffected by treatment of cells with 4NQO, while RAD3 and RAD4 transcripts
actually decrease in amount (Robinson et al. 1986).
The regulation of this inducible response to DNA damage is currently under
investigation. Investigators have shown that other yeast genes are induced by DNA
damage. These include the CDC9 gene, which encodes DNA ligase (Barker et al.
1985; Peterson et al. 1985) the RAD51 and RAD54 genes (D. Schild & R. K.
Mortimer, personal communication), which are involved in genetic recombination
and which confer sensitivity to ionizing radiation when mutated, and a group of
unidentified genes referred to as damage-inducible (DIN) (Ruby & Szostak, 1985) or
DNA damage-responsive (DDR) genes (McClanahan & McEntee, 1984, 1986).
Whether or not some or all of these genes belong to the RAD2 regulon remains to be
determined.
18
E. C. Friedberg
Nucleotide excision repair in mammalian cells
Information on the molecular biology of nucleotide excision repair in mammalian
cells is very limited. Genetic analyses in human and in rodent cells support the notion
of biochemical complexity evident from studies in yeast. Somatic cell hybridization
using fibroblasts from patients with the human disease xeroderma pigmentosum
(X P) has revealed nine complementation groups, suggesting that at least nine
distinct genes are involved in nucleotide excision repair in humans (see Friedberg,
1985). Similar analyses with u.v.-sensitive and excision repair-defective CHO cells
has demonstrated five complementation groups to date (Thompson et al. 1985a).
Systematic hybridization between human and CHO cells representative of all these
complementation groups has not been completed. However, the hybrids tested to
date show enhanced levels of u.v. resistance (Thompson et al. 1985a), suggesting
that the human and CHO mutations examined are in different loci. It seems
improbable to me that the molecular mechanism of nucleotide excision repair in
these two mammals is completely distinct. Hence, the observation that related
human and hamster genes have not been identified raises the interesting and
awesome possibility that the spectrum of excision repair genes is larger than that
revealed by either of the existing complementation groups alone. Since a case has
been made for the involvement of at least 12 genes in nucleotide excision repair in
yeast, the operational definition of 14 nucleotide excision repair genes in mammalian
cells (9 by analysis of X P cells and 5 by analysis of CHO cells) is perhaps not
unreasonable. In this regard, it is important to bear in mind that the CHO mutants
were selected in the laboratory by screening mutagenized immortalized cells for
enhanced u.v. sensitivity, whereas the X P cell lines are derived from human diploid
strains established from biopsy of human patients. The human cell lines may
therefore reflect a bias against mutations incompatible with normal human
embryogenesis and development, while the CHO lines may (for reasons that are not
clear) represent a bias against mutations in genes that in human cells characterize the
disease X P.
Human excision repair genes and proteins
Attempts to isolate human genes by phenotypic complementation of X P cells in
culture have not been successful (see Schultz et al. 1985). On the other hand,
complementation of u.v.-sensitive CHO mutants by transfection with human DNA
has been observed in several studies (Rubin et al. 1983, 1985; Westerveldei al. 1984)
and has facilitated the isolation of a DNA fragment containing a gene called ERCC-1
(excision repair complementing defective repair in Chinese hamster cells). Trans­
fection of CHO cells from complementation group 2 with cosmids containing this
gene results in enhanced resistance to killing by mitomycin C or u.v. radiation, as
well as enhanced levels of unscheduled DNA synthesis and excision of thyminecontaining pyrimidine dimers (Westerveld et al. 1984; Rubin et al. 1985).
Using a portion of the ERCC-1 gene as a hybridization probe, van Duin and his
colleagues (1986) isolated a series of overlapping cDNAs from which a complete
Nucleotide excision repair of DNA
19
cDNA was reconstructed. This cDNA complements the u.v. sensitivity of appro­
priate CHO mutants, but to date has not been shown to complement human X P cells
(D. Bootsma, personal communication). DNA sequence analysis of the ERCC-1
cDNA revealed an open reading frame consisting of 297 codons, which could encode
a protein of calculated molecular weight 32562. As indicated previously, comparison
of the predicted amino acid sequence of the E rccl polypeptide reveals considerable
homology with the yeast RadlO protein. It is also remarkable that a cDNA missing
302 bp from the 5' end of the gene (including 162 bp of 5' coding sequence) still
complements the phenotype of mutant CHO cells (van Duinei al. 1986). At the time
of writing, it has not been established whether the ERCC-1 gene complements the
phenotype of yeast radlO mutants. Attempts to complement CHO cells from
complementation group 2 with the cloned yeast RADIO gene are in progress in my
laboratory. The human ERCC-1 gene has been mapped to chromosome 19, con­
sistent with a recent report that this chromosome complements the phenotype of a
CHO mutant from complementation group 2 (Thompson et al. 19856).
The use of CHO (and possibly other rodent) cells, in which stable transformation
by DNA-mediated transfection occurs at a reasonable frequency, would appear to be
a useful procedure for screening the human genome for complementing sequences.
One would expect that human genes identified in this way are involved in nucleotide
excision repair in normal human cells, and the amino acid sequence homology
between the human Ercc-1 and yeast RadlO proteins is certainly consistent with this
expectation. However, in view of the failure to demonstrate overlapping mutations in
human X P and CHO cells, it would not be surprising if such human sequences do
not complement X P cells and hence turn out to be uninformative with respect to the
molecular basis of this human disease. Thus, there are compelling imperatives for
the cloning of human genes by direct complementation of the phenotype of X P cells.
In this regard, new strategies for phenotypic complementation by DNA transfer may
be required, since to date X P cells have proven particularly resistant to conventional
approaches to phenotypic complementation.
The author acknowledges the contributions of numerous post-doctoral fellows and students
whose work on nucleotide excision repair in yeast and mammalian cells is described here. I also
thank Aziz Sancar, Claude Backendorf and Lawrence Grossman for unpublished information,
Jane Cooper, Linda Couto, Reinhard Fleer, Charles Nicolet, Gordon Robinson, Roger Shultz and
William Weiss for critical review, Priscilla Cypiot for generating the hydropathicity profiles shown
in Fig. 4, and Jean Oberlindacher and Margaret Beers for preparation of the manuscript. Work
from the author’s laboratory is supported by research grants from the National Cancer Institute,
USPHS, the Council for Tobacco Research and the American Cancer Society, and by contract
with the US Department of Energy.
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