From the
There are two types of structural anomalies that lead to
mutation, a permanent change in DNA sequence. The first class involves
normal bases in abnormal sequence context (mismatch, bulge, loop). The
second class, which is referred to as DNA damage or DNA lesion,
involves abnormal nucleotides (modified, fragmented, cross-linked) in
normal sequence context. DNA lesions, in addition to causing mutations,
also constitute replication and transcription blocks.
Both types of
structural anomalies are rectified by a series of enzymatic reactions
referred to by the general term DNA
repair(1, 2, 3, 4, 5) . The
repair reactions employed for correcting mismatches and lesions are
similar in principle. The incorrect or damaged base is removed either
as a base (base excision) or as an (oligo)nucleotide (nucleotide
excision), the single-stranded gap resulting from the excision reaction
is filled in by a polymerase (repair synthesis), and the newly
synthesized DNA is ligated. Hence, there are two basic assays for
measuring repair(6) : the ``incision/excision assay''
and the ``repair synthesis assay.''
In base excision repair the mismatched or damaged base is
cleaved off the deoxyribose by a DNA glycosylase, and the resulting
apurinic/apyrimidinic (AP)
In excision
repair, both procaryotes and eucaryotes hydrolyze the 3rd to 5th
phosphodiester bond 3` to the lesion; on the 5` side the procaryotes
hydrolyze the 8th (4) and the eucaryotes hydrolyze the 21st to
25th phosphodiester bond(7, 8) . Thus procaryotes excise
damage in 12-13-nt-long oligomers and eucaryotes excise
27-29-nt-long fragments. This dual incision activity is referred
to as excision nuclease (excinuclease). The single-stranded gap
generated by either type of excision is filled in by DNA polymerases
and sealed by ligase.
The excision repair genes (uvrA, uvrB, and uvrC) of E. coli show no homology to the human
excision repair genes(1) . In contrast, the sequences of
excision repair genes in mammalian cells and yeast are highly
homologous, and the enzymology of excision repair in these two systems
is very similar (1, 3). Only mammalian excision repair will be covered
in this review. Three human diseases are caused by a defect in excision
repair(9) : xeroderma pigmentosum, Cockayne's syndrome,
and trichothiodystrophy.
Xeroderma pigmentosum patients suffer from
photosensitivity, photodermatoses including skin cancers, and in some
cases from neurological abnormalities. XP patients are defective in
excision repair. Mutations in 7 genes, XPA through XPG, cause XP. In addition, there is a group of patients with
classic symptoms of XP but with normal excision repair. These are
called XP variants (XP-V). Cells from XP-V patients are moderately
sensitive to UV light but excise UV photoproducts at a normal rate and
are defective in a biochemically ill defined phenomenon called
postreplication repair (10).
Cockayne's syndrome patients
suffer from growth failure, mental and neurological abnormalities,
cataracts, dental caries, and photosensitivity and related dermatoses.
Mutations in two groups of genes appear to cause Cockayne's
syndrome. The CS-A and CS-B (ERCC-6) mutants exhibit classical CS
symptoms without an increased rate of skin cancer. Cells from these
patients have near normal UV sensitivity. A second group of patients
manifest XP symptoms in addition to CS symptoms. Patients in this group
have mutations in the XPB, XPD, or XPG genes.
Trichothiodystrophy (TTD) patients have ichthyosis and
brittle hair and suffer from photosensitivity, skeletal abnormalities,
and mental retardation. The patients may or may not have an increased
rate of skin cancer. Mutations in three genes are associated with TTD.
In the XP/TTD overlapping syndrome, the mutation is in either XPB or XPD. In classical TTD (TTD-A), the mutation is
presumably in one of the other subunits of TFIIH(1) .
In
addition to the 9 genes identified by human diseases to be involved in
excision repair, many rodent excision repair mutants have been isolated
and characterized in order to define the entire set of excision repair
genes(11, 12) . The rodent mutants fall into 11
complementation groups, and the majority of these correspond to human
XP and CS complementation groups as indicated. In fact, some of the
human XP genes were cloned by virtue of complementing rodent mutant
cell lines and hence are also referred to as excision repair cross
complementing (ERCC) genes. Of these genes, XPE and ERCC6 through ERCC11 are not required for the basal excision
reaction(13) .
summarizes some of the properties of excision
repair proteins. Most of these proteins are in complexes in
vivo, and hence the activity associated with a solitary protein in vitro may or may not be relevant to its function in
excision repair. Human excision nuclease has been reconstituted in a
defined system by mixing six highly purified polypeptides or
polypeptide complexes(13) .
The three formal steps of excision repair are damage
recognition, dual incision (excision), and repair synthesis and
ligation.
First, damaged bases are not the
sole substrate for the enzyme. Human excinuclease excises mismatched
bases and 1-3-nt loops as well(38) . However, in contrast
to the true mismatch repair system, the excinuclease apparently has no
way of discriminating the correct and incorrect strands and as a
consequence excises the mismatch from either strand.
Regarding the
problem of identifying the damaged strand from the undamaged one, the
example of mismatch repair by excinuclease shows that the enzyme may
not always be able to discriminate the damaged and undamaged strands.
This point has not been investigated in detail. However, with thymine
cyclobutane dimer there was no excision of the undamaged strand at a
rate of 5% of the damaged strand (the detection limit of the assay).
Thus, clearly with damage as opposed to mismatch, the enzyme has a
mechanism of discriminating the right and wrong strands.
This fact leads to the third question that was
raised with regard to substrate recognition: what is the molecular
basis of damage recognition? The simple answer at present is: we
don't know. The following facts are of relevance in searching for
an answer for this question. (i) Although lesions that cause gross
helical deformity are repaired, those that do not are also repaired,
and there is no linear relationship between the specificity coefficient (k
The XPA-RPA complex binds to the
damage site; then XPA recruits TFIIH, which makes a preincision complex
in an ATP hydrolysis-dependent manner. XPC helps stabilize the
precincision complex. The ATP-dependent unwinding of the DNA by TFIIH
primes it for nuclease attack by the two XP proteins known to have
nuclease activity. XPG is recruited by TFIIH and incises on the 3`
side(13, 44) , and ERCC1-XPF, which is recruited by XPA,
incises on the 5` (44) side of the damage. The dual incision is
absolutely dependent on ATP hydrolysis(8) .
The major sites
of incision are relatively precise and are at the 5th phosphodiester
bond 3` and the 24th phosphodiester bond 5` to the
lesion(7, 8) . However, the incision sites show some
variability. The site of 3` incision extends from the 3rd through the
8th phosphodiester bond(7, 8) , and the site of 5`
incision extends from the 20th through the 26th phosphodiester
bond(8) . The combination of these incision patterns usually
results in excision of fragments 24-32 nt in length; however,
27-29-nt fragments are the dominant species. The sites of
incision are influenced by several factors, including the type of
lesion (38) and the sequence context(37, 39) .
The same incision pattern has been observed in vivo in Xenopus eggs (8) and in cell-free extracts from Schizosaccharomyces pombe
Transcribed sequences and in particular the template strand
within a transcribed sequence are repaired at a higher rate than
non-transcribed sequences(50) . Cells from CS patients are
defective in strand-specific repair(51) . In E. coli, a
transcription-repair coupling factor encoded by the mfd gene
displaces stalled RNA polymerase and releases the stalled complex while
recruiting the damage recognition complex of excinuclease(52) .
However, at present there is no in vitro system for
transcription-repair coupling in mammalian cells. The CSB gene
encodes a protein of 160 kDa, which contains the so-called helicase
motifs and is likely to function in a manner analogous to the E.
coli Mfd protein(50) . Thus, a simple model for
strand-specific repair based on behavior of CS-A and CS-B mutants and
of the proteins is as follows.
RNA polymerase II stalled at a lesion
is recognized by the CSA-CSB complex, which causes the polymerase to
back off the lesion without disrupting the ternary complex. The CSA-CSB
complex also recruits XPA and TFIIH to the lesion site and thus helps
in the assembly of the excinuclease. The lesion is excised and the
excision gap is filled in. The backed off RNA polymerase elongates the
truncated transcript(53) .
It appears that mammalian cells do not possess an SOS
response like that in E. coli where DNA damage by bulky agents
increases the transcription of excision repair genes(54) .
Similarly, damage-induced post-translational modification of repair
proteins (55, 56) does not affect the activity of human
excinuclease(57) .
The connection between the p53 tumor
suppressor protein and DNA repair has been the source of much
speculation and debate because the p53 protein is stabilized by DNA
damage and it is a transcriptional regulator. It has been reported that
p53 protein binds to XPB (58) and RPA(59) , both of which
are essential for basal excision repair. However, p53(-/-)
cells excise the two major UV photoproducts, pyrimidine dimers, and
6-4 photoproducts at the same rate as wild type cells and are
equally resistant to UV(60) . Similarly, p53 protein at nearly
micromolar concentrations has no effect on excision repair in a defined
system.
In contrast, the recent discovery that Cdk7 and cyclin
H, which make up the Cdk-activating kinase, are constituents of TFIIH (62, 63) raises interesting possibilities regarding cell
cycle regulation and DNA repair. Excision repair capability of the cell
does not change during the cell cycle(64) . However, replication
and repair may be coordinated by differential effects of p21(Cip/WAF)
on replicative and repair DNA synthesis(65) . Future research is
likely to uncover interesting interconnections between DNA repair,
replication, cell cycle, and apoptosis.
INTRODUCTION
Excision Repair
Genetics of Excision Repair
Structure and Function of Excision Repair Proteins
Mechanism of Excision Repair
Transcription-Repair Coupling
Regulation of Excision Repair
FOOTNOTES
REFERENCES
(
)deoxyribose is
released by sequential actions of an AP lyase which cleaves 3` and an
AP endonuclease which cleaves 5` to the AP site. The one-nucleotide gap
is filled in and ligated (Fig. 1).
Figure 1:
Mismatch and damage excision repair.
For simplicity a deaminated C is taken as an example of both mismatch
and damage. The repair of mismatch and lesions by base excision follows
the same pathway. Removal of damage by nucleotide excision occurs by
dual incision, whereas removal of mismatches by nucleotide excision
occurs by endonuclease/exonuclease action. The size of repair patches
are 1-4 nt for base excision (66, 67), 27-29 nt for damage
repair by nucleotide excision (7), and 300-500 nt for mismatch
repair by the nucleotide (general) mismatch repair system
(2).
Nucleotide excision
repair, conceptually, can be accomplished by two basic mechanisms. In
one, a phosphodiester bond is hydrolyzed 5` or 3` to the mismatch
(lesion), and then the incorrect base is removed by a 5` to 3` (or 3`
to 5`) exonuclease, which hydrolyzes DNA one nucleotide at a time
starting at the nick and digesting past the lesion. This is the repair
mode employed by both Escherichia coli and human general
mismatch correction (repair) systems(1, 2) . This
endonuclease/exonuclease reaction pathway is not utilized for removing
damaged bases from DNA. A possible explanation for this is that most
base adducts eliminated from DNA by excision repair inhibit
exonucleases. One way to circumvent this problem is to have an enzyme
system that nicks the damaged strand on both sides of the lesion at
some distance removed from the lesion. This second mechanism, indeed,
is the excision repair mechanism found in all species investigated. As
a matter of common practice, ``excision repair'' without
further qualification means nucleotide excision repair of DNA damage,
and hence it will be used as such in this review.
XPA
This protein of 31 kDa has a zinc finger and is
involved in damage recognition(14) . It also interacts with
several other components of excision repair and hence may function as a
nucleation factor for excinuclease. XPA interacts through its
N-terminal domain with the ERCC1-XPF heterodimer (6) to form a
relatively stable complex (15, 16); it also binds to TFIIH through its
C-terminal domain(17) . Finally, RPA (HSSB) binds to XPA and
increases its specificity for damaged DNA(18) . In addition to
XPA, other proteins that specifically bind to damaged DNA have been
identified. One of these, the DDB protein, is absent in some XPE
patients(19, 20, 21, 22) . However, this
protein is not required for excision repair(13) , and its
relation to XPE gene is unclear at present. Another class of
proteins that bind to certain types of damaged DNA have the HMG (high
mobility group) domain(23, 24) ; however, these proteins
inhibit excision repair (25).
RPA(HSSB)
This trimer of
(p70)(p34)
(p11)
is essential for
DNA replication (26, 27) and for repair
synthesis(28) . It is also absolutely required for the dual
incision step of excision repair(13) . It binds to damaged DNA
with moderate affinity and makes a complex with XPA that binds to
lesions with higher affinity than either component alone(18) .
TFIIH
This is a multiprotein (p89, p80, p62, p44,
p41, p38, p34) complex that contains XPB (p89) and XPD (p80). TFIIH was
initially identified as one of the seven general transcription factors
required for basal transcription by RNA polymerase II(29) . The
accidental discovery that its p89 subunit is identical to XPB (30) and the unexpected finding of failure of XP-B and XP-D
mutant cell-free extracts to complement in excision assay (6) led to the eventual realization that the entire TFIIH
complex is a repair factor(31, 32) . XPB and XPD
proteins are DNA-dependent ATPases, have the so-called helicase motifs,
and they, as well as TFIIH itself(29) , can dissociate short
fragments annealed to single-stranded DNA(1, 3) . This
modest helix unwinding activity is referred to as helicase by some
investigators.
XPC
The sequence of the gene predicts a protein of
125 kDa. This polypeptide co-purifies with a protein of 58 kDa, which
is the human homolog of the yeast Rad23 protein (HHR23B). Thus, the
functional form of XPC is a (p125)(p58)
heterodimer (33). XPC heterodimer binds to TFIIH loosely (31) and binds very tightly to single-stranded DNA(33) .
ERCC1-XPF Complex
This is a very stable complex
such that a heterodimer formed with a mutant protein does not exchange
subunits in vitro and, as a result, cell-free extracts from
XP-F and ERCC-1 do not complement for excinuclease activity(6) .
XPF (120 kDa) and ERCC1 (33 kDa) make a complex with
(p112)(p33)
stoichiometry (13) and bind
to XPA through the N-terminal half of ERCC1(15) . The ERCC1-XPF
complex is an endonuclease specific for single-stranded DNA.
(
)
XPG
This protein has a single-stranded
specific endonuclease activity(34, 35, 36) . It
also acts as a double-stranded specific exonuclease(34) . It
binds loosely to TFIIH (13) and to RPA (18) and is
apparently recruited by these components to the excision nuclease
complex.
Damage Recognition
Excision repair was first identified
by the failure of UV-sensitive E. coli and human cells to
remove thymine dimers from DNA. However, this repair system is not
specific for UV damage as it excises all covalent DNA lesions tested
(37-39). With regard to substrate recognition and preference,
three interrelated questions must be addressed. Does the enzyme system
recognize only DNA with damaged bases, how does the enzyme
``know'' which strand should be cut, and finally, what is the
molecular basis for recognition?
(
)
/k
) of the
excinuclease and the degree of helical
deformity(39, 40) . (ii) Recognition involves a protein
complex that has a preference for damaged DNA (XPA-RPA) and a complex
(TFIIH) with ATP-dependent local unwinding activity that is recruited
to the damage site and, based on the precedent in E. coli,
unwinds DNA and makes the ultimate damaged DNA-protein preincision
complex. (iii) Recently, it has been found that three repair enzymes
with rather narrow substrate specificity, namely DNA photolyase
(pyrimidine dimers), uracil glycosylase (uracil in DNA), and
exonuclease III (AP site), flip out the lesion from the duplex into a
``hole'' within the enzyme to bring the active site cofactor
or residues in close contact with the target
bonds(40, 41, 42) . Whether the excinuclease
system flips out the damaged nucleotide(s) or the entire excised
fragment remains to be seen(43) .
Dual Incision/Excision
The molecular details of
mammalian excision repair are now known in considerable detail. Sixteen
polypeptides are necessary and sufficient for excinuclease activity
(13) as shown in Fig. 2.
(
)and is
considered to be the universal incision pattern for eucaryotes (4).
Repair Synthesis
In contrast to the excision
reaction we know less about the details of the repair synthesis step.
It is known that repair synthesis is PCNA-dependent and hence must be
carried out by Pol and Pol
(45, 46) , and,
since PCNA is the polymerase clamp loaded onto template-primer by RFC
replication factor, RFC may also be required. In a study with cell-free
extract Pol
antibodies specifically inhibited repair
synthesis(47) , and in a highly purified in vitro system for repair synthesis it was found that Pol
and even
Klenow fragment of PolI performed repair synthesis pointing to the
difficulty of assigning repair polymerase from in vitro reconstitution systems(48) . Most likely, both Pol
and
Pol
participate in the repair synthesis step of excision
repair(49) .
Finally, the report that the p53-induced Gadd45
protein stimulates excision repair (61) has not been
confirmed.
Thus, existing data are consistent with the
notion that p53 does not modulate excision repair either positively or
negatively.
Table: The
16 polypeptides required for excision repair in mammalian cells
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.