(Received for publication, October 27, 1995)
From the
Nucleotide excision repair by mammalian enzymes removes DNA
damage as part of 30-mer oligonucleotides by incising
phosphodiester bonds on either side of a lesion. We analyzed this dual
incision reaction at a single 1,3-intrastrand d(GpTpG)-cisplatin
cross-link in a closed circular duplex DNA substrate. Incisions were
formed in the DNA with human cell extracts in which DNA repair
synthesis was inhibited. The nicks were mapped by restriction fragment
end labeling and primer extension analysis. Principal sites of cleavage
were identified at the 9th phosphodiester bond 3` to the lesion and at
the 16th phosphodiester bond 5` to the lesion. The predominant product
was found to be a 26-mer platinated oligonucleotide by hybridization to
a
P-labeled complementary DNA probe. Oligonucleotides were
formed at the same rate as the 3` cleavage, suggesting that both
incisions are made in a near-synchronous manner. There was, however, a
low frequency of 5` incisions in the absence of 3` cleavage. The dual
incision reaction was reconstituted using the purified mammalian
proteins XPA, RPA, XPC, TFIIH, XPG, and a fraction containing ERCC1-XPF
and IF7. All of these components were required in order to observe any
cleavage.
The dual incisions produced during nucleotide excision repair
(NER) ()in vertebrates have been studied for several DNA
lesions, including UV photoproducts, thymine-psoralen monoadducts,
cisplatin cross-links, and acetylaminofluorene adducts (1, 2, 3, 4) . For these lesions, an
asymmetric pattern of incisions has been found. Oligonucleotides
27-29 nucleotides long are released after cleavage at the 22nd to
24th phosphodiester bonds 5` and at the 5th phosphodiester bond 3` to a
thymine dimer(1) . A few other DNA lesions have been reported
to be removed in this way(2, 3, 4) , leading
to the view that this is the general incision pattern during NER in
eukaryotes.
The incision reaction requires XPA, RPA, XPG, XPC, TFIIH, ERCC1-XPF complex, and IF7(5) , although it is not clear that all of these components directly participate in the formation of both the 3` and 5` incisions. XPA preferentially binds damaged DNA(6) , suggesting a role in damage recognition. The single-stranded DNA-binding protein RPA is also required for the incision stage (7, 8, 9) and acts synergistically with XPA to bind damaged DNA(10) . Two ``structure-specific'' nucleases are involved that can recognize bubble or splayed arm structures, with opposite polarity. The XPG nuclease is responsible for incisions 3` to a DNA lesion(11) , while the human ERCC1-XPF complex is believed to mediate the 5` incision, by analogy with the structure-specific nuclease activity of the homologous RAD1-RAD10 complex of Saccharomyces cerevisiae(12, 13) . The roles of IF7 and XPC are not yet known, although the latter is a single-stranded DNA-binding protein that may stabilize an incision reaction intermediate(14, 15) . The TFIIH complex contains 3` to 5` and 5` to 3` ATP-dependent helicase activities, in the XPB and XPD subunits, respectively(16, 17) . These may allow localized unwinding of DNA and assist opening of the DNA helix so that the structure-specific nucleases can act. It is not known whether the dual incisions are made sequentially or simultaneously, but it is likely that the nucleases achieve selectivity in cleavage of the damaged DNA strand through interactions with other repair proteins. For example, the XPA protein has been found to interact with ERCC1, RPA, and TFIIH(10, 18, 19, 20, 21, 22) .
To investigate the roles of these proteins further we have examined the incision reaction using a closed circular DNA substrate containing a single 1,3-intrastrand d(GpTpG)-cisplatin cross-link at a specific site. Cisplatin is an important anti-tumor drug (23) that reacts with DNA to form intrastrand and interstrand cross-links (24) that are removed by nucleotide excision repair with varying efficiencies. Several new methods are reported here for characterization of the incisions made by human cell extracts during NER and for mapping the structure of the excised oligonucleotide. Although the oligonucleotides released during repair of the 1,3-intrastrand d(GpTpG)-cisplatin cross-link have sizes (4) and repair patches (25) consistent with those observed for several other DNA lesions, we find that the platinum lesion has a novel incision pattern that may be dictated by its unusual structure. This dual incision reaction has been reconstituted using purified repair proteins and this has provided information on the degree to which the incisions are coupled and on the proteins required for cleavage 3` and 5` to a DNA lesion.
Covalently
closed circular DNA containing a single 1,3-intrastrand
d(GpTpG)-cisplatin cross-link (Pt-GTG) was produced by priming 30
µg of plus strand M13mp18GTGx (150 ng/µl) with a 5-fold molar
excess of 5`-phosphorylated platinated oligonucleotide in a 200-µl
reaction mixture containing 10 mM Tris-HCl (pH 7.9), 50 mM NaCl, 10 mM MgCl, 1 mM DTT, 600
µM each of dATP, dCTP, dGTP, and TTP, 40 units of T4 DNA
polymerase gp43 subunit (HT Biotechnology Ltd.) and 36 Weiss units of
T4 DNA ligase (New England Biolabs) for 4 h at 37 °C. Closed
circular DNA was isolated by CsCl/EtBr density gradient centrifugation
and purified by consecutive butanol extraction, centrifugation in a
Centricon-10 microconcentrator (Amicon) and a Sephadex G-50 (Pharmacia)
column. DNA substrates were stored at -80 °C in 10 mM Tris-HCl, 1 mM EDTA, pH 8.0. Control DNA substrate
(Con-GTG) was produced by the same method except that a nonplatinated
oligonucleotide was used to prime M13mp18GTGx. The vector M13mp18GTGx
was constructed by replacing the 230-bp BsaHI-EcoRI
fragment of M13mp18GTG (11) with a synthetic DNA duplex
containing a unique XhoI site, formed by annealing the
oligonucleotides 5`-AATTCGGTCATAGCTGTTTCCTGCTCGAGGG-3` and
5`-CGCCCTCGAGCAGGAAACAGCTATGACCG-3`. M13mp18GTGx was transformed into
the F
Escherichia coli strain TG-1 for the
preparation of replicative form and single-stranded DNA(26) .
Figure 5:
Analysis of incision at the cisplatin
cross-link by UvrABC endonuclease. A, autoradiograph of a
denaturing 10% polyacrylamide gel demonstrating incisions made 5` to
the cisplatin cross-link. DNA was incubated with UvrABC endonuclease,
purified, digested with HindIII, and 5`-labeled with P prior to electrophoresis. Lane 1, 50 nM UvrA, 500 nM UvrB, and 270 nM UvrC. Lanes G,
A, T, and C are dideoxy sequencing reactions of the
single-stranded M13mp18GTGx DNA using a
P-labeled 15-mer
primer 5`-AGCTTGCATGCCTGC-3` which has a 5` end that corresponds to the
5` terminus produced by HindIII digestion of the damaged DNA
strand. The expanded region shows the DNA sequence of the damaged DNA
strand around the cisplatin cross-link. The position of the cisplatin
cross-link is shown alongside the autoradiograph. An arrow indicates the predominant 5` incision at the 8th phosphodiester
bond from the lesion. B, autoradiograph of a denaturing 10%
polyacrylamide gels demonstrating incisions made 3` to the cisplatin
cross-link. DNA was incubated with purified UvrABC endonuclease and
primer extension analysis performed as described except that digestion
with PvuI was omitted. Lanes 1 and 2,
incubation with buffer only. Lanes 3 and 4,
incubation with UvrA (14 nM), UvrB (105 nM), and UvrC
(40 nM). The bracket delineates the size range of
extension products blocked at incisions. The position of the cisplatin
cross-link is shown alongside the autoradiograph. An arrow indicates the predominant 3` incision at the 4th phosphodiester
bond from the lesion.
Figure 1: Closed circular duplex DNA containing a single 1,3-intrastrand d(GpTpG)-cisplatin cross-link. A, structure of Pt-GTG DNA, a modified M13mp18 molecule containing a specifically located 1,3-intrastrand d(GpTpG)-cisplatin cross-link in the(-)-DNA strand. The expanded region illustrates the DNA sequence flanking the cisplatin cross-link. The eight BstNI restriction fragments and the target sites of other restriction enzymes are indicated. Nondamaged Con-GTG DNA contains a sequence identical to Pt-GTG DNA, without a Pt lesion. B, 0.8% agarose gel demonstrating the presence of the cisplatin cross-link. Lane 1, uncut Pt-GTG; lanes 2-7, digestion of Pt-GTG with Bsu36I (Bsu), HindIII (H), ApaLI (A), EcoRI (E), XhoI (X), and BsaHI (Bsa). Lane 8, uncut Con-GTG. Lanes 9-14, digestion of Con-GTG with the same enzymes. The mobilities of covalently closed circular (ccc), linear (lin), and nicked circular (nc) Pt-GTG and Con-GTG DNA are indicated alongside the gel.
Figure 4:
Analysis of incised DNA intermediates. A, primer extension. Autoradiograph of a denaturing 12%
polyacrylamide gel demonstrating damage-dependent incisions 3` to the
lesion and blocking of Sequenase by the cisplatin cross-link. DNA was
incubated with HeLa whole cell extract (wce) for 30 min as
described. Purified DNA was digested with PvuI, a P-labeled 17-mer primer was annealed to denatured DNA and
extended by Sequenase prior to electrophoresis. Lanes 1 and 2, no incubation of DNA prior to primer extension analysis (N/A, not applicable). Lanes 3 and 4,
incubation with buffer only. Lanes 5 and 6, HeLa cell
extract. Lanes 7 and 8, HeLa cell extract in the
presence of aphidicolin. The lanes G, A, T, and C show dideoxy sequencing reactions of Con-GTG using the same
P-labeled 17-mer primer. The DNA sequence of the
complementary strand surrounding the cisplatin cross-link is written at
the side. The positions of blocks to extension at the cisplatin
cross-link, the PvuI restriction site and those resulting from
cleavage of the damaged DNA strand are indicated at the left
side. An arrow indicates a predominant 3` incision at the
9th phosphodiester bond from the lesion. B, restriction
fragment end labeling analysis. Autoradiograph of a denaturing 12%
polyacrylamide gel demonstrating damage-specific incisions around the
cisplatin cross-link. DNA was incubated for the times incubated (min)
with HeLa cell extract, purified, digested with XhoI, and
labeled with [
-
P] dNTPs using DNA
polymerase I (Klenow fragment) prior to electrophoresis. Lanes
1-10, +aphidicolin; lanes 11-16,
-aphidicolin. The expanded region illustrates the DNA sequence
surrounding the cisplatin cross-link. An arrow indicates a
predominant 3` incision at the 9th phosphodiester bond from the lesion.
The positions of strand cleavage were determined by comparison with
dideoxy sequencing ladders and by comparison with the mobility of
primer extension reaction products. Phosphodiester bonds are indicated
by bars at the left side; the bold bars show
the bonds within the cross-link.
Figure 2: Inhibition of DNA repair synthesis at the site of the cisplatin cross-link. Autoradiographs of 12% polyacrylamide gels demonstrating the occurrence of DNA repair synthesis in the region of the cisplatin cross-link. DNA was incubated with cell extract, purified, and digested with BstNI before electrophoresis. Lanes 1-9, HeLa cell extract. Lanes 10-18, HeLa cell extract in the presence of aphidicolin. The size of the eight BstNI restriction fragments is shown alongside each autoradiograph. In order to make the 68-bp band visible, lanes 10-18 are shown at a darker exposure than lanes 1-9.
Several strategies were used to detect incisions produced during repair. These are summarized schematically in Fig. 3and discussed in turn below.
Figure 3: Strategies for analysis of incised DNA intermediates and oligonucleotides formed by dual incisions. A, extension from a primer annealed 3` to the lesion on the damaged DNA strand locates the cisplatin cross-link and incisions 3` to the lesion. Linearization with PvuI enables detection of translesion DNA synthesis and also of completed repair events. B, end labeling the damaged DNA strand at XhoI reveals 3` incisions and uncoupled 5` incisions. C, end labeling the damaged DNA strand at HindIII allows analysis of 5` incisions. D, an oligonucleotide complementary to the DNA spanning the site of the cisplatin cross-link was used for Southern hybridization to detect oligonucleotides formed by the dual incision reaction. The distance from the cisplatin cross-link to the 3` termini of oligonucleotides was determined by treatment with 3` to 5` exonuclease. E, the 5` termini of oligonucleotides was determined by primer extension from an oligonucleotide complementary to the DNA spanning the site of the cisplatin cross-link.
After
incubation of Pt-GTG with HeLa cell extract in the presence of
aphidicolin, primer extension revealed a pattern of products
corresponding to distances from 2 to 22 phosphodiester bonds 3` to the
lesion. The principal band at the 9th phosphodiester bond 3` to the
lesion appeared to represent the most frequent primary incision product (Fig. 4A, lane 7). In the absence of aphidicolin,
little 3` nicking was detected (Fig. 4A, lane 5),
consistent with completion of most repair events by repair synthesis
and ligation. In such reactions, there was a concomitant increase in
extension to the PvuI restriction site, representing
restoration of an undamaged DNA strand. Quantification of the
diagnostic bands in Fig. 4A indicates that
10-20% of Pt-GTG DNA molecules are incised.
In addition, a weaker new pattern of bands appeared that corresponded to cleavage at the 16th to 20th phosphodiester bonds 5` to the lesion (Fig. 4B, lanes 2-7). In order to be detectable by 3` end labeling, the fragments representing incision 5` to the lesion must have occurred in the absence of a 3` incision and are henceforth referred to as ``uncoupled 5` incisions.'' These incisions 5` to the lesion would not have been detected by the primer extension assay above, because the polymerase was efficiently blocked by the cisplatin cross-link. Omitting aphidicolin from the reaction buffer led to the loss of the uncoupled 5` incisions and >80% reduction in the level of 3` incisions at the 9th phosphodiester bond from the lesion (compare lanes 4 and 12 in Fig. 4B), consistent with completion of the majority of repair events.
Figure 6: Analysis of the incisions made by PCNA-depleted (CFII) cell extracts and purified mammalian repair proteins. Autoradiographs of denaturing 12% polyacrylamide gels demonstrating damage-specific incisions made around the cisplatin cross-link by primer extension (A) and restriction fragment end labeling analysis (B) as described in the legends to Fig. 3and Fig. 4, except that PvuI digestion prior to primer extension was omitted. The samples in A and B are aliquots from the same reactions after purification of incised DNA intermediates. Lane 1, HeLa CFII extract and 100 ng RPA. Lane 2, XPG415A CFII extract, 100 ng of RPA and 30 ng of purified XPG protein (47) . Lane 3, XPG415A CFII extract and 100 ng of RPA. Lanes 4-9, combinations of purified repair proteins as indicated below each lane. The following amounts of purified repair proteins were used: 30 ng of XPA, 150 ng of RPA, 30 ng of XPC, 50 ng of XPG, 12 µg of ERCC1-XPF complex (step 5 CM-Sepharose fraction containing IF7 activity(5) ), and 150 ng of TFIIH. The expanded region in B illustrates the DNA sequence surrounding the cisplatin cross-link. Brackets delineate the size range of extension products (A) or incision fragments (B) resulting from cleavage of the damaged DNA strand. An arrow indicates a predominant 3` incision at the 9th phosphodiester bond from the lesion. The positions of strand cleavage in B were determined by comparison with dideoxy sequencing ladders and with the mobility of primer extension reaction products.
Oligonucleotides 24-32 nucleotides in length were formed during incubation with HeLa cell extract (Fig. 7A, lanes 2-8). A 26-mer platinated oligonucleotide predominates after 20-min incubation with the next most abundant 29- and 30-mer oligonucleotides reaching only 50% of the level of the 26-mer. XPG-defective cell extract formed no oligonucleotides (Fig. 7A, lane 11), but complementation with purified XPG protein fully restored dual incision activity (Fig. 7A, lanes 12 and 13). An identical (but weaker) pattern of platinated oligonucleotides was also observed in reactions that contained purified XPA, XPG, RPA, XPC, TFIIH, ERCC1-XPF complex, and IF7 (not shown).
Figure 7:
Analysis of oligonucleotides formed by the
dual incision reaction. A, autoradiographs of Southern blots.
Oligonucleotides formed during repair were transferred onto a nylon
membrane and hybridized with a P-labeled complementary
probe. The sizes (in nucleotides) of oligonucleotides are indicated
alongside the autoradiograph and were determined by comparison with the
mobility of the platinated 24-mer oligonucleotide used to make the
substrate (see part B). A: lanes 1-10,
HeLa cell extract; lanes 11-13, XPG415A cell extract in
the absence or presence of 30 ng of XPG protein or aphidicolin as
indicated. Reaction times are in minutes. B, autoradiograph of
a Southern blot. Lanes 1 and 2, HeLa cell extract. Lanes 3 and 4, HeLa cell extract in the presence of
aphidicolin. Lane M, 250 pg of platinated 24-mer
oligonucleotide 5`-TCTTCTTCTGTGCACTCTTCTTCT-3`. The samples in lanes 2 and 4 were incubated with T4 DNA polymerase
in the absence of dNTPs at 37 °C for 30 min prior to
electrophoresis. C, autoradiograph of 12% denaturing
polyacrylamide gel demonstrating blocks to primer extension at the 5`
termini of oligonucleotides. A
P-labeled 16-mer primer was
annealed to denatured DNA and extended by Sequenase prior to
electrophoresis. Reaction mixtures contained HeLa cell extract ±
aphidicolin as indicated. Reaction times (minutes) are shown. Lanes
1-7, 11-13, 8-10, and 14-16 are
aliquots from the reactions shown in Fig. 4B, lanes
1-7, 8-10, 11-13, and 14-16,
respectively. The 3` terminus of the 16-mer primer aligns with the 6th
phosphodiester bond 5` to the lesion in the damaged DNA strand. The bracket alongside the autoradiograph delineates the range of
primer extension blocks, and arrows indicate the predominant
blocks 16, 19, and 20 phosphodiester bonds 5` to the
lesion.
The position of the major 3` incision at the 9th phosphodiester bond from the lesion and the predominant formation of a 26-mer platinated oligonucleotide suggested that the major 5` incision was made 16 phosphodiester bonds 5` to the lesion. This prediction was tested directly by primer extension analysis using a primer that was complementary to the DNA spanning the lesion. The 3` terminus of this oligonucleotide was opposite the 6th phosphodiester bond 5` to the platinum adduct (Fig. 3E), and the positions of blocks to extension reveal the 5` limit of the excised platinated fragment. After incubation of Pt-GTG with HeLa cell extract, the predominant block to extension corresponded to the 16th phosphodiester bond 5` to the lesion, with lower levels of extension products blocked at the 19th and 20th phosphodiester bonds (Fig. 7C). Minor blocks at a few other sites were also detected between the 13th and 20th phosphodiester bonds 5` to the lesion. No significant difference was seen in reactions with or without aphidicolin (compare lanes 4 and 7 with 9 and 10, Fig. 7C).
The principal incisions made during repair of the 1,3-intrastrand d(GpTpG)-cisplatin cross-link are at the 9th phosphodiester bond 3` and at the 16th phosphodiester bond 5` to the lesion, forming a 26-mer platinated oligonucleotide. Secondary 5` cleavage sites for this lesion were also found at the 19th and 20th phosphodiester bonds, resulting in 29- and 30-mer platinated oligonucleotides, respectively. Identical incision patterns were found when incised DNA intermediates were formed either by cell extracts in the presence of aphidicolin or with PCNA-depleted cell extracts. Consistent with this, aphidicolin had no effect on the size or levels of oligonucleotides formed during the reaction. The E. coli UvrABC endonuclease was found to cleave the strand containing the platinum adduct at the 4th phosphodiester bond 3` and the 8th bond 5` to the lesion, as expected (Fig. 8).
Figure 8: Position of dual incisions around a site-specific 1,3-intrastrand d(GpTpG)-cisplatin cross-link during nucleotide excision repair. The main incisions produced by mammalian cell extracts and purified proteins are on the damaged DNA strand at the 16th phosphodiester bond 5` to the lesion and at the 9th phosphodiester bond 3` to the lesion, resulting in the release of a 26-mer oligonucleotide. UvrABC endonuclease from E. coli mainly incises the damaged DNA strand at the 8th phosphodiester bond 5` to the lesion and at the 4th phosphodiester bond 3` to the lesion resulting in the formation of a 13-mer oligonucleotide.
It has been proposed that after an initial 5` or 3` incision, a 5`-3` exonuclease activity might remove several nucleotides in a controlled manner, before release of the damaged oligonucleotide(37) . However, the major sites of incision that we observed in the duplex DNA by primer extension or end labeling analysis (Fig. 4) were in precise agreement with the structure determined for the excised oligonucleotide (Fig. 7). The same patterns of incision and oligonucleotide formation were observed throughout the time course. The data thus suggest that the principal cleavage sites observed are the ``primary'' ones and have not been significantly altered by an exonuclease. On the other hand, after incubation with cell extracts in which DNA repair synthesis was inhibited, some fragments were observed corresponding to nicks at the 13th, 16th, and 22nd phosphodiester bonds 3` to the lesion ( Fig. 4and Fig. 6). No excised oligonucleotides were found that would correspond to such distant 3` incisions, and therefore it is likely that these particular fragments result from limited exonucleolytic digestion of the plasmid by the cell extract, after the 3` incision has been made. Reactions with purified proteins produced only the major 3` incision products at the 8th and 9th phosphodiester bonds (Fig. 6).
One factor that may determine the incision position is the nature or degree of helical distortion at a particular lesion. The solution structure of a 1,3-intrastrand d(GpTpG)-cisplatin cross-link in a 13-bp DNA duplex has been studied using high-resolution NMR(39) . The model structure showed a helical distortion of the 5`-platinated G and central T residue, with 19° local duplex unwinding and a 20° kink at the platination site. Other studies have found similar distortions caused by 1,3-intrastrand cisplatin cross-links in different DNA sequence contexts(40, 41) . While some aspects of these helical distortions are common to a variety of DNA lesions, including UV photoproducts(42, 43) , the 1,3-intrastrand d(GpTpG)-cisplatin cross-link produces novel base-stacking interactions in the damaged DNA strand(44) . Base-stacking interactions are potentially important in damage recognition (45) and could influence protein-DNA interactions during damage recognition by XPA, RPA, and probably additional repair proteins. Altered protein-DNA contacts may influence subsequent protein-protein interactions, and thus the position on the damaged DNA strand to which structure-specific endonucleases are recruited, leading to variable positions of the dual incisions. It is interesting that although the human repair proteins appear to be susceptible to the unusual structural features of a 1,3-intrastrand d(GpTpG)-cisplatin cross-link, the E. coli UvrABC endonuclease made dual incisions at positions consistent with those observed around other lesions ( (45) and Fig. 5).
Are the two NER incisions normally made in a particular order? The similar time courses of the 3` incision (Fig. 4) and oligonucleotide formation (Fig. 7) suggest that the two incisions are nearly synchronous. However, in this study we observed some uncoupled 5` incisions in the absence of any 3` cleavage, and Matsunaga et al.(38) recently presented evidence for some uncoupled 3` incisions during NER. These data suggest that either incision may occasionally occur without the other. This is compatible with a ``bubble'' model, whereby an opened structure is created during NER, for subsequent processing by structure-specific nucleases such as XPG and the presumed ERCC1-XPF nuclease(11, 12, 13) . Tight coupling between the formation of incisions and subsequent reaction steps would be a preferable strategy, in order to avoid the exposure of a single-stranded gap and DNA ends to degradation by cellular proteins. The kinetics of incision and repair synthesis are nearly the same (Fig. 2, 4B, and 7A), consistent with such tight coupling.