Nucleotide excision repair: From E. coli to man

Biochimie 81 (1999) 15−25 © Société française de biochimie et biologie moléculaire / Elsevier, Paris

Nucleotide excision repair: From E. coli to man Claude Petit*, Aziz Sancar University of North Carolina at Chapel Hill, School of Medicine, Department of Biochemistry and Biophysics, 405 Manning Drive, Chapel Hill, NC 27599-7260, USA (Received 21 June 1998; accepted 30 November 1998) Abstract — Nucleotide excision repair is both a ‘wide spectrum’ DNA repair pathway and the sole system for repairing bulky damages such as UV lesions or benzo[a]pyrene adducts. The mechanisms of nucleotide excision repair are known in considerable detail in Escherichia coli. Similarly, in the past 5 years important advances have been made towards understanding the biochemical mechanisms of excision repair in humans. The overall strategy of the repair is the same in the two species: damage recognition through a multistep mechanism involving a molecular matchmaker and an ATP-dependent unwinding of the damaged duplex; dual incisions at both sides of the lesion by two different nucleases, the 3' incision being followed by the 5'; removal of the damaged oligomer; resynthesis of the repair patch, whose length matches the gap size. Despite these similarities, the two systems are biochemically different and do not even share structural homology. E. coli excinuclease employs three proteins in contrast to 16/17 polypeptides in man; the excised fragment is longer in man; the procaryotic excinuclease is not able by itself to remove the excised oligomer whereas the human enzyme does. Thus, the excinuclease mode of action is well conserved throughout evolution, but not the biochemical tools: this represents a case of evolutionary convergence. © Société française de biochimie et biologie moléculaire / Elsevier, Paris excision nuclease / molecular matchmaker / DNA damage / DNA repair

1. Introduction Nucleotide excision repair (NER), sometimes called more briefly excision repair, is a major DNA repair pathway whose function is to remove a wide range of DNA damage through dual incisions on both sides of the lesion followed by DNA resynthesis, using the complementary strand as a template, and ligation. The concerted enzymatic system responsible for the dual incisions is also called excision nuclease or excinuclease (from the latin word excidere that means to cut out). To date, this very versatile system has been found in all living organisms tested. Although the primary function of nucleotide excision repair is to remove bulky lesions from DNA, for which it is the sole system, it is able to repair a wide range of DNA adducts or UV damages. These two properties, versatility and universality in the biological world, confer to nucleotide excision repair a unique place among DNA repair pathways. Nucleotide excision repair has been first extensively studied in prokaryotes, mainly Escherichia. coli, and in the last decade in eukaryotes. Biological relevance has been attested by genetic diseases observed in man related to mutations in this pathway and its role in the first stage * Correspondence and reprints: Present address: École Nationale vétérinaire, 23, chemin des Capelles, 31076 Toulouse cedex, France

of carcinogenesis is most likely important. In fact, a living species would be unlikely to survive without excision repair [1]. In this review we will compare the biochemical mechanisms in E. coli and the latest data obtained in man. Although the function of excinuclease and the overall sequence of the process are strikingly similar, there is no structural homology between the subunits involved in prokaryotes and in eukaryotes. 2. Nucleotide excision repair in prokaryotes 2.1. Miscellaneous Research leading to the discovery of nucleotide excision repair was first initiated in Escherichia coli in the early 1960s [2–4]. It became clear very quickly that this pathway was crucial for the survival of bacteria under various DNA damage conditions, mainly, but not exclusively, involving bulky lesions such as UV-induced pyrimidine dimers, wich were eventually used as a model for mechanistic studies. It was further discovered that other bacterial species, and most likely all, possess a similar, though not strictly identical, nucleotide excision repair system [5]. Nucleotide excision repair occurs in E. coli through two different modalities: i) preferential, or transcription coupled; and ii) genome-overall repair.

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Table I. The three proteins involved in the excinuclease activity in Escherichia coli. TRCF, transcription repair coupling factor (adapted from [17]). Protein

Mr

Sequence motifs

Activity

Role in repair

UvrA

(104)2

Walker ATPase Zinc finger (2) Leucine zipper UvrA superfamily

ATPase Damage-specific DNA binding UvrB binding TCRF binding

Proximal DNA damage recognition Molecular matchmaker Transcription-repair coupling

UvrB

78

Helicase TRCF homology

ATPase (latent) Helicase (latent) Damage-specific ss DNA binding UvrA binding Uvrc binding

Ultimate DNA damage recognition DNA unwinding 3' incision

UvrC

69

UvrB (limited homology)

Non-specific DNA binding UvrB binding

Induction of 3' incision 5' incision

We will mainly focus here onto the general biochemical basis of nucleotide excision repair. This repair can be summarized in three steps whatever the species: damage recognition, dual incisions and repair synthesis followed by a ligation. 2.2. The Escherichia coli excinuclease Excision repair in E. coli requires six proteins, among which three (table I) are involved in the excinuclease activity. The so-called (A)BC excinuclease complex is formed by the three proteins UvrA, UvrB and UvrC. In fact, a complex between the three subunits at the same time does not exist, but rather a sequential interaction as exposed below. 2.3. Damage recognition The goal for damage recognition in nucleotide excision repair is to be both very specific of true DNA damage to avoid futile, and possibly mutagenic activity, and to be versatile enough to function as a ‘multipurpose system’, in contrast with much more specialized repair features such as mismatch repair, photolyases or glycosylases. At first, these two requirements seem to be rather incompatible because of the high diversity of DNA adducts: an isolated enzymatic activity would have to ‘choose’ between a highly stereospecific active site, fitted for one single type of adduct, and thus leading to a narrow repair spectrum, and an active site of multi-substrate specificity, running the risk of artifactual repair. To deal with those requirements, nucleotide excision repair uses a concerted multistep mechanism to ensure the specificity, and non-specific sites of binding to the damaged DNA to accept a wide substrate range. Adducted DNA (or internal lesions resulting from UV damage) is first recognized by UvrA, which also works as a molecular matchmaker. A molecular matchmaker is a

protein which, in an ATP-dependent reaction, brings target DNA and effector protein(s) together, promotes a specific and stable complex formation and then dissociates from the complex to enable the effector protein to carry out its function. ATP hydrolysis may be by the matchmaker itself or the effector molecule [6]. In an ATP-dependent manner, UvrA is able both to perform the first step of recognition and to promote the tight association between the damaged DNA and the UvrB protein, which acts eventually both as an helicase and a 3' nuclease [7]. The system works as follows (figures 1, 2): an A2B1 complex UvrA-UvrB, tracking along the DNA backbone, recognizes a lesion at first through (UvrA)2 which is specific for damage recognition on double-stranded DNA [8]. The complex halts at the damaged site and binds onto. This binding activates the UvrB-dependent helicase function and leads to a local unwinding/kinking that allows the further recognition of the damaged strand by UvrB. The unwinding is about 5 bp around the damage [9] and the DNA kinking is by 130° into the major groove, at phosphodiester bond 11 on the 5' side of the lesion [10]. The unwinding is obviously facilitated by the helix unstability caused by bulky adducts, which can explain why non-helix distorting lesions, although repaired by nucleotide excision repair, are less efficiently processed. However, 8-oxoguanine is an exception, at least in higher mammals, as discussed later. In the second step of recognition, UvrB, whose binding is single-stranded damaged DNA specific [11], and hence would not be possible before the unwinding step, is enabled to form a tighter complex at the lesion site through a hydrophobic, salt-insensitive binding [12]. Since there is no involvement of stereospecific hydrogen or ionic binding, this binding mode allows the processing of a large combination of chemical groups: this characteristic is likely crucial for the versatility of the recognition.

Nucleotide excision repair

Figure 1. Model for transcription-independent nucleotide excision repair of DNA in Escherichia coli. 1. The A2B1 complex locates the damage by tracking along DNA and forms an unstable A2B1-DNA complex in an ATP-independent step. DNA is kinked and unwound by UvrB through an ATP-dependent reaction, leading to a stable complex of UvrB-damaged strand. 2. The molecular matchmaker UvrA dissociates. UvrC binds to UvrB-DNA complex, activating UvrB that makes the 3' incision. 3. UvrC is activated and makes the 5' incision a few seconds after the 3'. 4. UvrD (helicase II) releases the excised oligomer and UvrC. 5. DNA PolI fills the gap and releases UvrB at the same time. The repair patch is then ligated.

The tight binding of UvrB produces a conformational change that weakens the complex UvrA-UvrB, leading to dissociation of (UvrA)2, leaving on the site of the damage

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Figure 2. Model for transcription-independent nucleotide excision repair of DNA in humans. 1. The damage is first recognized in an ATP-independent step by the short-lived XPA⋅RPA complex. In a second, ATP-dependent step, the damaged DNAbound XPA⋅RPA complex recruits XPC and TFIIH, to form the preincision complex 1 (PIC1). TFIIH possesses both 3'-5' and 5'-3' helicase activities, respectively through its XPB and XPD subunits and unwinds DNA by about 20 base pairs around the damage. 2. XPG binds the PIC1 complex while the molecular matchmaker XPC dissociates, leading to the more stable PIC2 excinuclease complex. 3. PIC2 recruits XPF⋅ERCC1 (F-1) to form PIC3. XPG makes the 3' incision and F-1 makes the 5' incision a fraction of second later, in a concerted but asynchronous mechanism. 4. The excised damaged fragment is released by the excinuclease complex, leaving in place a post-incision complex whose exact composition is still unclear. PCNA is loaded on DNA as Pole/δ clamp, replacing then the post-incision complex with repair synthesis proteins. 5. The gap is filled and the repair patch is ligated.

18 a stable UvrB-DNA preincision complex [13, 14]. The release of (UvrA)2 also leads to an isomerisation of the preincision complex, to form [UvrB-DNA]' less stable but incision-competent [15]. This [UvrB-DNA]' complex enables the binding of UvrC, that will initiate the dual incisions. 2.4. Dual incisions The removal of the lesion is performed by incisions of the DNA strand bearing the adduct at the 8th phosphodiester bond 5' to the lesion and at the 4th or 5th bond 3' [7], leading to the formation of a 12–13 nt long oligomer. The 3' incision is performed first, immediately after the recognition and binding or UvrC to the complex UvrBDNA [16], activating UvrB that makes the incision. The 3' incision requires ATP binding but not hydrolysis [17]. A few seconds later, the 5' incision is made by UvrC [16]. At this step, the complex UvrB-damaged excised oligomer is stable and tends to remain in place, while less tightly bound UvrC slowly dissociates spontaneously. The subsequent binding of the protein UvrD (helicase II) to the nicks dissociates the oligomer and facilitates also the dissociation of the remaining UvrC, while the UvrBgapped DNA complex is still stable. 2.5. Repair synthesis and ligation The dissociation of UvrC frees the hydroxyl residue at the 5' incision site, that becomes accessible to the repair specific DNA polymerase I (PolI) which synthetizes the repair patch. At least 90% of the repair patches perfectly match the excision gap [18] indicating that no gap enlargement occurs prior to or during repair synthesis. The DNA ligase then completes the repair. To summarize, the high specificity of nucleotide excision repair for DNA adducts is provided by the multistep recognition mechanism (double strand specific binding, ‘checked’ after unwinding by further single strand recognition that triggers the first incision). One may suppose that if UvrA would bind by error a damage-free region of DNA, it would be very unlikely that a second misrecognition by UvrB occurs, and the system would be then uncoupled and would stop. On the contrary, the wide specificity for a large range of adducts is due to a hydrophobic binding in the active site of at least UvrB, hence avoiding stereospecific interactions. This system is similar in its principle to that observed in the active sites of cytochrome P-450 dependent monooxygenases, whose specificity in xenobiotic metabolism is very broad as well [19]. The fidelity of the repair is in most cases not different from that of classical semi-conservative DNA replication: nucleotide excision repair is thus grossly considered ‘error free’, by opposition to the ‘error prone’ system of tolerance of the lesions using Umu DC proteins in the SOS repair [20].

Petit and Sancar 3. Excision repair in humans 3.1. Miscellaneous Nucleotide excision repair in eukaryotes is both very similar to prokaryotes regarding the biochemical strategy used (i.e., recognition, unwinding, 3' and 5' sequential dual incisions, patch repair synthesis and ligation) and very different in the nature and number of proteins involved. Significant studies have been done in UV-sensitive mutant yeasts, leading to the discovery of the RAD (radiation sensitive) loci. Over 30 RAD loci, organized in three epistasis groups, have been identified to date [21]. Characterization of yeast repair genes has been useful in understanding nucleotide excision repair in man. Although human nucleotide excision repair is not quite as well understood as in E. coli, considerable understanding of the phenomenon has been achieved in the past 5 years. The main differences between E. coli and man nucleotide excision repair are the following [17]: a) 16 to 17 polypeptides, associated in six factors, are required for the recognition/excinuclease complex; at least 25 are used in the whole nucleotide excision repair pathway; b) the length of the repair patch is 24–32 mers, hence both longer and more variable in length than in prokaryotes; and c) there is no sequence conservation of the uvr loci throughout species evolution. Rather, eukaryotes, dealing with much more complicated DNA/chromatin structures (see [22]), have developed an original, and closely genetically related, nucleotide excision repair pathway conserving the strategy but not the tools. 3.2. Genetics Three rare human genetic diseases have provided powerful models for the researchers in the field of nucleotide excision repair in man: xeroderma pigmentosum, Cockayne syndrom and trichothiodystrophy. 3.2.1. Xeroderma pigmentosum

Xeroderma pigmentosum (XP) was named by Hebra and Kaposi in 1874 [23]. Xeroderma means in Greek ‘dry skin’, suggesting the most evident abnormality observed in that disease which is caused by the absence (or sometimes the severe reduction) of excision repair [24]. In addition, XP is associated with a huge photosensitivity and sometimes ocular and/or neurological abnormalities. The photosensitivity is correlated with a very high risk of several forms of skin cancer: basal cell or squamous cells carcinomas, malignant melanomas, most of them occurring in the teens [25]. The overall rate is 2000-fold higher in XP individuals under the age of 20 as compared to the rest of the population, which may be the highest increase in rate documented for any recessive human hereditary disease [25]. Internal cancers also occur at 10–20-fold higher rate.

Nucleotide excision repair

19

Table II. The six factors involved in the excinuclease activity in humans. GTF, general transcription factor; CAK, cdk-activating kinase (adapted from [17]). Fraction

Proteins

Sequence motif

Activity of the fraction

Role in repair

XPA

XPA

Zinc finger

DNA binding

Damage recognition

RPA

p70 p34 p11

Zinc finger

XPA binding DNA binding

Damage recognition

TFIIH

XPB XPD p62 (TFB1) p52 p44 (SSL1) Cdk7 CycH p34

3'-5' helicase 5'-3' helicase

DNA-dependent ATPase Helicase GTF

Formation of preincision complexes PIC 1-2-3 Transcription-repair coupling

Zinc finger S/T kinase Cyclin Zinc finger

CAK

XPC HHR23B

Ubiquitin

XPC

DNA binding

Molecular matchmaker Stabilization of PIC1

XPG

XPG

Nuclease

3' incision

XPF

XPF ERCC1

Nuclease

5' incision

Ocular abnormalities occur in approximately 40% of the cases. Neurological abnormalities, including mental retardation, deafness and progressive ataxia, are observed less frequently, in 30–40% of the patients [25]. UV-sensitive XP cells in culture obtained from XP patients, or their equivalents in rodents, have provided a system leading to the discovery of seven complementation groups in man named XPA through XPG. The XP genes have now been cloned (except for XPE), many of them first as ERCC (for excision repair cross complementing), by complementation of the UV-sensitive phenotypes in rodent cell lines [26].

vivo and in vitro. Due to this, the human excinuclease, which exhibits striking similarities with the yeast excinuclease [32], indicating a highly useful system for the eucaryotic life correlated with a high degree of conservation, at least of the function, throughout evolution of species, is now understood in detail. 3.3. The human excinuclease The human excinuclease is much more complex than in E. coli since it uses 16/17 proteins that are in six repair factors (table II). This is in contrast with the three polypeptides in the prokaryotic excinuclease.

3.2.2. Cockayne’s syndrome and trichothiodystrophy

Cockayne syndrom (CS) and trichothiodystrophy (TTD) are less clearly DNA repair deficiency syndromes and even considered more of transcription defect diseases [17, 27, 28]. Mutations in the genes CSA(ERCC8) and CSB(ERCC6) seem to be primarily responsible for CS [29, 30] and TTD-A for TTD. In addition, some XPB, XPD and XPG mutations give rise to an XP/CS overlap syndrome [31]. Another less closely related disease is Fanconi’s anemia, which is linked to a defect in interstrand cross-link repair, hence not involving nucleotide excision repair alone [5]. To recapitulate, the most useful model for the elucidation of the genetic pattern of human nucleotide excision repair were xeroderma cell lines, and their rodent homologs in CHO, which provided powerful tools both in

3.4. Damage recognition Damage recognition by human excision nuclease is a multi-step ATP-dependent process, which like the E. coli excinuclease system, employs a molecular matchmaker [33]. In the following, three interrelated issues regarding damage recognition will be addressed: the repair factors involved in DNA recognition, the structure of the DNA within the recognition complex, and the protein composition of the preincision complexes. 3.4.1. Binding of repair factors to damaged DNA

It has been shown that RPA [34, 35], XPA [36, 37] and XPC [33, 38] have higher affinities for damaged DNA than for undamaged DNA. However, the discrimination between undamaged and damaged DNA by all these

20 proteins is modest and cannot account for the high specificity of human excinuclease. It has been shown that the combination of XPA and RPA increases specificity [39, 40]. However, even this synergistic effect of two damage recognition factors does not provide a high enough selectivity comparable to those of other repair proteins such as photolyase or various DNA glycosylases. Similarly, the combination of the three repair factors XPA, RPA and XPC implicated in damage recognition does not improve specificity as tested by pull-down [41] or gel retardation [33] assays. These experiments have shown that the first high specificity complex forms with the combination of four repair factors, XPA, RPA, XPC and TFIIH. This complex, which is referred to as preincision complex 1 (PIC 1) is formed in an ATP-dependent manner, is rather unstable, and easily disassembles during electrophoretic separation. Binding of XPG to PIC1 greatly increases the stability of the DNA-protein complex and leads to the formation of preincision complex 2 which is of very high affinity and specificity, in complete agreement with human excinuclease. It is noteworthy that the XPF-ERCC1 complex cannot associate with PIC1 [33] and that the nuclease function of XPG is not required for the formation of PIC2 [42]. Finally, binding of XPF-ERCC1 to PIC2 leads to formation of preincision complex 3 which does not have much greater specificity than PIC2 but is an essential intermediate on the pathway to dual incision [41]. Thus, it is apparent that XPG, in addition to its 3' nuclease function, is involved in recruiting the XPF-ERCC1 5' nuclease to the excinuclease complex [42]. To recapitulate our current understanding of the damage recognition by human excinuclease, although three repair factors (XPA, RPA, XPC) have some specific affinity for damaged DNA none of these factors qualify as ‘the damage recognition factor’. Rather, a preexcinuclease (pre-repairosome) complex consisting of XPA, RPA, XPC, and TFIIH is the ATP-dependent highaffinity damage recognition factor and XPG contributes to damage recognition by increasing the stability of the complex. 3.4.2. Structure of DNA within the recognition complex

Experiments with cell extracts [43] and purified repair factors [41] have revealed that DNA within the repair complex is unwound around the damage by 15–20 bp. The unwinding is executed by the 3' to 5' and 5' to 3' helicase activities of TFIIH. The study with the purified repair factors has shown that in PIC1 the DNA is unwound by ≈ 10 bp 5' and 5 bp 3' to the damage. There is no evidence of kinking, in contrast to E. coli. In PIC2, the extent of unwinding is the same, however, the equilibrium has shifted to open complex formation, as probed by KMnO4 sensitivity of thymines. The extent of unwinding is the same in PIC3 formed upon binding of XPF-ERCC1 to PIC2 containing catalytically active or inactive XPG

Petit and Sancar protein. In PIC3, which forms with catalytically active XPG, dual incisions release the excised oligomer and expose 20–30 nucleotides in the undamaged strand to the KMnO4 probe [41]. Thus, in the immediate post-incision complex most of the bases in the excision gap are relatively unprotected. 3.4.3. XPC is a molecular matchmaker

When the compositions of the PIC1, -2 and -3 of human excinuclease were analyzed by a variety of methods including ‘antibody supershift’ and protein tagging, it was found that neither PIC2 nor PIC3 contained XPC protein [33]. Since the formation of these complexes requires ATP hydrolysis, XPC satisfies all of the requirements of a molecular matchmaker. Thus, XPC helps in damage recognition and in unwinding of DNA to form PIC1, which contains XPA, RPA, TFIIH and XPC, and then recruits XPG to stabilize the complex to form PIC2, but it must dissociate from the preincision complex upon entry of XPG. Hence, PIC2 contains XPA, RPA, TFIIH, and XPG; and PIC3 contains XPA, RPA, TFIIH, and XPFERCC1 [33]. This explains why XPC is not needed in transcription-coupled repair [44] and for repair of lesions adjacent to or within a mismatch bubble [45, 46]. To summarize, human excinuclease employs a multistep recognition mechanism in which the complementary protein and DNA surfaces are created by using the energy released from ATP hydrolysis aided by the XPC molecular matchmaker. It is perhaps more productive to analyze the question of damage recognition in existentialist terms, that is, by describing the way it happens rather than what makes it happen and why it happens. 3.5. Dual incisions Initiation of nucleotide excision repair is universal to all three forms of life, bacteria [47], eukarya [48] and archaea [49]. In man, dual incisions occur in a concerted but asynchronous manner, releasing fragments 24–32 nucleotides in length with those in the 26–29 nt range predominating [48, 50]. The 3' incision is at the 6th ± 3 phosphodiester bond 3' to the damage [48, 50, 51] with the incisions at the 4–6th phosphodiester bond predominating [48, 49, 52]. The 5' incision occurs at the 20 ± 5th phosphodiester bond with those at 22 ± 2 being the most prevalent [48, 50, 51, 53]. The precise sites of incision are influenced by the type of lesion and the sequence context [17, 54]. The two subunits with the nuclease activity are XPG and XPF⋅ERCC1. Both have junction cutting nuclease activities. XPG incises at the vicinity of the 3' junction of a bubble structure [55, 56] and XPF⋅ERCC1 incises in the vicinity of the 5' junction [57]. Both activities are stimulated by the RPA protein [52, 57]. It has been shown that in the excinuclease complex these nucleases perform the predicted reactions: XPG makes the 3' incision [51, 53]

Nucleotide excision repair

21

and XPF⋅ERCC1 makes the 5' incision [42, 51]. In contrast to E. coli, formation of the 3' incision does not depend on the presence of the 5' incision nuclease. Thus in the absence of XPF⋅ERCC1 the PIC2 assembles normally and the 3' incision is made at a normal rate without 5' incision [53]. However, under physiologic conditions where both XPG and XPF⋅ERCC1 are present in the reaction mixture such ‘uncoupled’ 3' incisions are rare and uncoupled 5' incisions are not observed. Under these conditions the 3' incision occurs first and is followed by 5' incision within a fraction of a second [53]. Hence the term ‘concerted but asynchronous’ incisions is used to describe the dual incisions reaction. Dual incisions generate an oligomer of 24–32 nt carrying the damage. This oligomer, in contrast to the E. coli excinuclease, is released by the human excinuclease without the aid of additional factors. Hence the human enzyme better fits the description of excinuclease, an enzyme that excises a single-stranded oligomer from a damaged duplex.

tant mutagenic oxidative damage, though non-helix distorting is repaired more efficiently in vitro by human purified repair factors than the classical nucleotide excision repair substrate, the cyclobutane pyrimidine dimer [63]. The biological significance of this finding will be discussed later. In contrast to human excinuclease, Uvr(A)BC, although able to incise thymine glycols [64], does not significantly process 8-oxoguanine [65]. Interestingly, human excision repair recognizes and processes mismatches as well [46]. Although the functional relevance of this activity remains to be demonstrated it may explain some cases of mutagenicity of nucleotide excision repair, once regarded as ‘error free’ in E. coli. In fact, there are indications of a mutagenic excision repair (type II UV mutagenesis) in this organism [66]. In addition, it has been suggested that excision repair could contribute in fixing frameshift mutations, since Uvr(A)BC binds and excises substrates that mimic slipped mutagenic intermediates [67].

3.6. Repair synthesis and ligation

4. Concluding comments

Following the release of the excised oligomer some, as yet not known, components of the excinuclease remain bound to the post-incision gap [58] and protects the single stranded region from non-specific degradation. The gap is then filled in by DNA polymerases and ligated. The repair synthesis requires PCNA [59, 60] and since Pol δ and Pol e are PCNA-dependent polymerases, one or most likely both of these enzymes carry out the repair synthesis. Indeed, a yeast pol e mutant is defective in excision repair, consistent with the role of this enzyme in repair synthesis [59]. Whether or not RFC, which in replication loads PCNA onto the template, plays any role in repair synthesis is not clear at present. Similarly, which of the three human DNA ligases completes the repair reaction is not known. Most likely, any of the ligases can seal the repair patch to the parental duplex. The size of the repair patch precisely matches the excision gap indicating that excision/repair synthesis/ligation occur in a rather concerted manner without an opportunity for the enlargement of the gap by the exonuclease activities of XPG and XPF⋅ERCC1 nucleases [62].

4.1. Differences and similarities between E. coli and man

3.7. Repair spectrum The repair spectrum of nucleotide excision repair, in E. coli as well as in man, is very large and basically the same in both species. Table III summarizes some of the main DNA lesions repaired by excision repair and the interactions with other DNA repair systems. Nucleotide excision repair have been long considered to be a repair system for bulky lesions with the capability of repairing other damages such as methylated bases albeit with lower efficiency. An exciting new finding is that 8-oxoguanine, an impor-

There are major biochemical differences between the two systems: the human excision nuclease has more components which show no homology to the prokaryotic system; the replication protein RPA and the transcription factor TFIIH are essential subunits of the human excinuclease; the substrate hierarchies of the two systems and the effect of sequence context on excision repair are different. However, the similarities between the two are even more striking. Despite the lack of sequence similarities between the subunits of the prokaryotic and eukaryotic excision nucleases the overall strategy of repair is the same: two-step damage recognition, localized helix unwinding, and 3' incision followed by 5' incision. Thus, excision repair represents evolutionary convergence in a biochemical pathway. 4.2. Inducibility of nucleotide excision repair In E. coli, nucleotide excision repair is inducible through the SOS system. The uvrA and uvrB genes possess SOS boxes that allow the binding of and repression by the LexA repressor [68–70]. Strongly DNA damage-inducible, RecA-dependent proteins also likely interact with nucleotide excision repair independently from LexA [71]. Until recently, with the exception of reports suggesting Weigle reactivation-like phenomena in eukaryotic cells [72, 73], there was no true evidence of an SOSresponse like phenomena in eukaryotes [17]. However, recently it was shown that in yeast the repair by nucleotide excision repair of non-dimer photoproducts was inducible,

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Table III. Substrates for nucleotide excision repair in man and known interactions with other DNA repair systemsa. Adducts

Repair system overlap

Bulky adducts

Mismatch repair (limited efficiency) 2-Acetylaminofluorene Benzo[a]pyrene diol epoxide Cisplatin 1-3d(GpTpG) 6-4 photoproduct Cisplatin 1-2 d(GpG) Thymine dimers Psoralen monoadducts (pyrone and furan side) Psoralen interstrand cross-linksb

Non-bulky adducts

Base excision 8-Oxoguaninec Thymine glycol Urea residue Synthetic AP sites analogs O6-Methylguanine N6-Methyladenine

Mismatched base pairs

Alkyltransferase Alkyltransferase Mismatch repair

A:G G:G a The substrates are listed in approximate order of catalytic efficiency for each group. In E. coli, the spectrum is the same except for N6-methyladenine (which is not excised), and 8-oxoguanine, but the hierarchy is somehow different. Also, in certain species (both prokaryotes and eukaryotes), thymine dimers and 6-4 photoproducts are repaired by the respective photolyases. b Interstrand cross-links are repaired by a concerted NER/recombination mechanism. c 8-Oxoguanine is repaired in vitro by human NER with higher efficiency than thymine dimers. In E. coli, athough thymine glycol is excised, there is no evidence of significant repair of 8-oxoguanine by Uvr(A)BC (adapted from [17, 26]).

in particular in G1 phase of the cell cycle [74]. In man, there are reports of the existence of a p53-dependent inducible nucleotide excision repair which does not exhibit preferential repair of transcribed genes [75–78]. Further studies are needed to understand the molecular basis of these interesting phenomena. 4.3. Role in cancer It is generally accepted that damage induced mutations are the first step of the multistage process of carcinogenesis. The role of excision repair in preventing skin cancer caused by DNA damage is well known as evidence for the high rate of skin cancer in xeroderma pigmentosum or other cancer prone patients who also exhibit higher rate of internal cancer caused by other agents [79, 80]. Smoking is the major cause of cancer deaths in man, accounting for about 30% of cancer mortality. Bulky adducts produced by benzo[a]pyrene or other polyaromatic hydrocarbons and arylamines found in cigarette smoke [81] are eliminated by nucleotide excision repair. In addition, cigarette smoking induces oxidative DNA damage, both directly and through the local chronic inflammation produced by constant irritation [81, 82]. Although 8-oxoguanine is only one among the many DNA

damages caused by oxidation [83], it has proven to cause G⋅C →T⋅A transversions both in vitro and in vivo [84, 85]. Indeed, a deletion in the OGG1 gene of S. cerevisiae, responsible for base excision repair of 8-oxoguanine, produces a mutator phenotype and increases G⋅C →T⋅A transversions [86] indicating the high importance of repairing this damage which is produced by many endogenous and exogenous factors such as xenobiotics, inflammation, ionizing radiations and internal metabolism. If removal of 8-oxoguanine by nucleotide excision repair [63] proves to be physiologically significant, this repair system may thus play a role helping to maintain genome integrity in the face of constant oxidative aggression, along with the more specific and faster glycosylases [87–89]. Excision repair also plays a role in cancer chemotherapy. Many anticancer drugs are electrophilic compounds that bind DNA and hence produce adducts which are substrates for nucleotide excision repair. Pharmacological approaches which specifically inhibit excision repair in tumor cells may prove useful [90], both to increase the sensitivity to the drugs and to alleviate drug resistance that may be the consequence of increased excision repair activity.

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