Mahmud K. K. Shivji,* Mark K. Kenny,t and Richard D. Wood'. *Imperial Cancer Research Fund. Clare Hall Laboratories. South Mimms, Herts EN6 3LD. England.
Cell, Vol. 69, 367-374,
April 17, 1992, Copyright
0 1992 by Cell Press
Proliferating Cell Nuclear Antigen Is Required for DNA Excision Repair Mahmud K. K. Shivji,* Mark K. Kenny,t and Richard D. Wood’ *Imperial Cancer Research Fund Clare Hall Laboratories South Mimms, Herts EN6 3LD England fDepartment of Biochemistry University of Dundee Dundee DDI 4HN Scotland
Summary Fractionation of extracts from human cell lines allows nucleotide excision repair of damaged DNA to be resolved into discrete incision and polymerization stages. Generation of incised intermediates depends on the XP-A protein, a polypeptide that recognizes sites of damaged DNA, and on the human single-stranded DNA-binding protein HSSB. The proliferating cell nuclear antigen (PCNA) is required for the DNA synthesis that converts the nicked intermediates to completed repair events. This need for PCNA implies that repair synthesis is carried out by DNA polymerase 6 or E. The ability to visualize repair intermediates in the absence of PCNA facilitates dissection of the multiprotein reaction that leads to incision of damaged DNA in a major pathway of cellular defense against mutagens. Introduction Nucleotide excision repair of DNA is the main pathway by which mammalian cells remove the carcinogenic lesions caused by sunlight and other common mutagens. The process can be broadly divided into two stages: first, incision at sites of DNA damage and second, synthesis of new DNA to replace the damaged nucleotides. In the first stage, a multiprotein endonuclease assembles at a site of a lesion and catalyzes enzymatic cleavage of the damaged strand. After the damaged oligonucleotide and the incision proteins are displaced, DNA synthesis proceeds to form a short patch using the nondamaged strand as a template, and repair is completed by a DNA ligase. Most of the known proteins involved in nucleotide excision repair of human DNA have been identified by studying mutant cells that exhibit sensitivity to ultraviolet (UV) light. The best known cells of this type are those derived from individuals with the inherited human syndrome xeroderma pigmentosum (XP). There are eight different genetic complementation groups in this disease. Cells from most or all of the XP groups display incision defects by various assays (Hoeijmakers, 1991), and so XP products are generally thought to be involved in the first stage of repair. Since the second stage of DNA repair involves DNA synthesis, there is scope for an overlap between the proteins used for mammalian DNA replication and those used
for DNA repair. Recently, it was found that the HSSB protein (also known as RP-A and RF-A) is required for nucleotide excision repair synthesis in vitro (Coverley et al., 1991). HSSB had originally been isolated as one of the cellular factors essential for replication of SV40 viral DNA (Tsurimoto et al., 1990; Weinberg et al., 1990; Hurwitz et al., 1990). Another important participant in DNA replication is the proliferating cell nuclear antigen (PCNA). PCNA is a 36 kd protein that is required for the in vitro replication of SV40 and is strongly indicated to function in replication of cellular DNA (Prelich et al., 1987a; Jaskulski et al., 1988; Zuber et al., 1989). The protein is known to interact with DNA polymerase S (Prelich et al., 1987b; Bravo et al., 1987). PCNA also acts in conjunction with the RF-C protein to bind at model DNA primer-template junctions (Tsurimoto and Stillman, 1991; Leeet al., 199la). During DNA replication, a form of PCNA is localized at sites of DNA synthesis and this can be detected by immunostaining of S phase nuclei with anti-PCNA antibodies after appropriate fixation of cells (Celis et al., 1987; Wilcock and Lane, 1991). Interestingly, following exposure of cells to UV irradiation, a tightly bound form of PCNA can be detected in nuclei in all phases of the cell cycle (Celis and Madsen, 1986; Toschi and Bravo, 1988). These observations suggest that PCNA might be involved in DNA repair synthesis as well as in DNA replication. To investigate a possible role for PCNA in DNA nucleotide excision repair, we have undertaken studies with fractionated cell extracts. The data show that repair can be resolved in vitro into incision and polymerization stages. HSSB is involved in generating or stabilizing incisions in damaged DNA, while PCNA is required for the DNA synthesis that converts the nicked intermediates to repaired products. Results Fractionation of Cell Extracts and Reconstitution of Repair Synthesis In these experiments, repair synthesis is detected in plasmid DNA that has been irradiated with UV light and freed from nicked forms. As an internal control, the irradiated DNA is mixed with nonirradiated DNA circles of slightly larger size. A mixture of the two closed-circular plasmids is incubated with whole cell extract in a buffer that includes deoxyribonucleoside triphosphates, [a-32P]dATP, ATP, and an ATP-regenerating system. During this incubation, some of the pyrimidine dimer photoproducts are removed from irradiated DNA by excision repair. DNA repairsynthesis can be measured after recovery of the plasmid DNAs from the reaction mixture by linearization of the DNAs with a restriction enzyme, gel electrophoresis, autoradiography, and scintillation counting of excised bands (Wood et al., 1988). To evaluate a possible role for PCNA in the repair synthesis reaction and to explore further the role of HSSB, a
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flowthrough DEAE-Biogel
Figure quired
flow-through
bound
HSSB
PCNA
1. Fractionation for DNA Repair
of Cell Extract
1 2 34
XP-A + other incision proteins topoisomerases DNA polymerases DNA ligases
into Different
Components
5 6 7891011
Re-
whole cell extract from HeLa cells was fractionated according to the scheme shown in Figure 1. Studies of the proteins required for SV40 DNA replication have demonstrated that HSSB and PCNA flow through phosphocellulose columns at 0.1 M salt while other necessary components, including DNA polymerases, are adsorbed and elute at higher ionic strength (Prelich et al., 1987a; Wold et al., 1989). Chromatography of the phosphocellulose flow-through fraction (CFI) on DEAE-Biogel was used to separate the HSSBfrom PCNA(Prelich et al., 1987a; Wold et al., 1989). lmmunoblotting of fractions CFIA, CFIB, and CFII with anti-HSSB and anti-PCNA antibodies confirmed that HSSB was present only in CFIA, and PCNA only in CFIB (data not shown). The three fractions CFIA, CFIB, and CFII, were mixed in various combinations as shown in Figure 2. Appreciable repair synthesis in damaged DNA was detectable only when all three fractions were combined (Figure 2, lane 7). Generally, >200 fmol of dAMP was incorporated in the course of a 3 hr incubation at 30°C with the reconstituted system, an amount of synthesis comparable with that found with unfractionated whole cell extracts (Robins et al., 1991). Replacement of CFIA and CFIB by Purified Proteins Since previous experiments had indicated that HSSB was involved in nucleotide excision repair, we asked whether CFIA could be omitted and replaced with homogeneous HSSB protein from HeLa cells. It was found that purified HSSB could substitute, apparently fully, for CFIA (Figure 2, lane 11). Increasing the amount of HSSB from 180 ng to 540 ng led to increased repair synthesis (Figure 3, reactions 3-5), consistent with other indications that the protein is one of the limiting components of the in vitro repair reaction (Coverley et al., 1991). Using a similar strategy, we tested the possibility that CFIB could be replaced by PCNA. PCNA was purified to m99% homogeneity from Escherichia coli cells that overexpressed the protein from the cloned human gene. As little as 10 ng of PCNA could substitute for CFIB and restore repair synthesis to the reaction mixture (Figure 3). Accumulation of Incised Intermediates in the Absence of PCNA The above results indicated a remarkably strict requirement for PCNA in repair synthesis. Given the known activi-
Figure 2. Reconstitution Fractions
of Repair
Synthesis
Requires
All Three
A mixture of 250 ng of UV-irradiated plasmid and 250 ng of nonirradiated plasmid was incubated for 3 hr at 30% in reaction buffer with fractions as indicated by the filled boxes. The amounts of protein used were 24 ug of CFIA or 0.5 ug of HSSB. 8 pg of CFIB, and 50 ug of CFII. DNA was extracted, treated with BamHl to convert both plasmids to linear forms, and electrophoresed on an agarose gel (top) before autoradiography (lower panel).
ties of PCNA, it seemed reasonable to postulate an involvement in the DNA synthesis stage of repair, rather than the incision stage. To examine this possibility, a mixture of damaged and nondamaged plasmid DNAs was incubated with various combinations of the fractions CFIA, CFIB, and CFII. To visualize closed-circular and nickedcircular forms, DNA from these reactions was directly elec-
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CFIA CFIB
CFII HSSB PCNA Figure
3. Replacement
~UUClD~~mMU ~~~~UUUuuu ¤~~=~~~~~=~ q q tm3~)54oOUUUUtm q q 0 q Uto 22 M 40 90 30 n
q
of Fraction
CFIB
with Purifed
PCNA
A mixture of 250 ng of UV-irradiated plasmid and 250 ng of nonirradiated plasmid was incubated for 3 hr at 30% in reaction buffer with fractions as shown. DNA was extracted, linearized, and electrophoresed on an agarose gel, and bands were excised by scintillation counting to quantify the data. The amounts of protein used were 24 ug of CFIA or HSSB (ng as indicated), 8 ug of CFIB or PCNA (ng as indicated), and 50 ug of CFII. In reactions 7-l 1, synthesis in nondamaged DNA was somewhat elevated but remained proportionally below the amount of synthesis in damaged DNA. The low level of aspecific synthesis may be due to the activity of a trace endonuclease detected in the PCNA preparation. Alternatively, inhibitors of nonspecific nucleases may normally be present in CFIB.
PCNA-Dependent 369
DNA Repair
Synthesis
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nicked
circles
-_----
Figure 4. Incised Intermediates Absence of the PCNA-Containing
Generated Fraction
in Damaged
DNA in the
A mixture of 250 ng of UV-irradiated plasmid and 250 ng of nonirradiated plasmid was incubated for 3 hr at 30% in reaction buffer with fractions as indicated. The amounts of protein used were 24 ug of CFIA or 0.9 ug of HSSB, 6 pg of CFIB, and 50 trg of CFII. DNA was isolated and electrophoresed (without restriction enzyme treatment) on an agarose gel containing ethidium bromide. In all reactions that included CFII, closed-circular forms were converted to relaxed covalently closed circles by the high topoisomerase activity in this fraction. A smaller amount of topoisomerase activity was also present in CFIA (lane 2).
trophoresed (without linearization) on agarose gels containing ethidium bromide. When all three fractions were present (Figure 4, lane 8) no significant differences were apparent between the fraction of nicked circles found in irradiated and unirradiated DNA. Under these conditions, repair synthesis oc-
curs, and most radioactive material is detected in the closed-circular form of the irradiated plasmid (see Figure 5A below). This suggests that the incision stage of repair occurs slowly in the extracts in comparison with the polymerization and ligation steps, so that the steady-state level of incised forms remains low throughout the incubation. However, when fraction CFIB was omitted so that reaction mixtures included only CFII and either CFIA or HSSB, a significantly higher fraction of nicked-circular forms was present in UV-irradiated DNA than in nonirradiated DNA at the end of the incubation (see Figure 4, lanes 6 and 7). As judged by densitometry, about 20% of the irradiated DNA was in the nicked form after 3 hr. This indicated that stable incised intermediates were generated in damaged DNA in the absence of PCNA. Analysis of the time course of the reaction showed that most of the nicked intermediates were formed during the first hour. In similar experiments, we found that these fractions could also introduce damage-dependent nicks in plasmids containing adducts of N-acetylaminoftuorene, another type of lesion that is repaired by the nucleotide excision system (data not shown). Synthesis at Incised Sites upon Addition of PCNA Although incisions could arise in DNA in the absence of PCNA, significant repair synthesis did not occur under these conditions, even though the DNA polymerases in CFII and deoxynucleotides (including radiolabeled dATP) were present throughout the incubation (see Figure 2 and Figure 3). It was therefore of interest to determine whether the intermediate forms could be completely repaired by the addition of PCNA. CFIA and CFII were incubated together with a mixture
Figure
5. Rapid
Synthesis
at Incised
Sites
(A) For the reactions in lanes 1-9, a mixture of 250 ng each of UV-irradiated and nonirradiated plasmid DNA was incubated at 30% in reaction buffer with CFIA, CFIB, and CFII (24,8, and 50 ug of protein, respectively) for the indicated times. For the reactions in lanes 10-15, the plasmid DNAs were incubated with CFIA and CFII for 60 min at 30°C before adding CFIB. Incubation was continued for the indicated times before stopping the reactions with EDTA and chilling on ice. The reaction shown in lane 10 (“0” time) was stopped ~15 s after addition of CFIB. Top, negative of ethidium bromidestained agarose gel; bottom, autoradiograph of the gel. Minus and plus signs indicate the respective positions of nonirradiated and UVirradiated plasmids in the nicked-circular (nc) and closed-circular (cc) forms. (B) The average number of nicks per molecule in UV-irradiated plasmid DNA. The nicked fraction of molecules was obtained from scanning densitometry of a photographic negative of the ethidium bromide gel shown in (A), correcting for the 1.6-fold greater fluorescence of nicked molecules over closed molecules. The average number of nicks per molecule is obtained from the non-nicked fraction by the Poisson equation. Reactions with CFIA, CFIB, and CFII incubated together (lanes l-9 in [A]) are indicated by the unfilled circles and dotted line. Reactions where CFIA and CFII were incubated together for 60 min before addition of CFIB (lanes lo-15 in [A]) are indicated by the closed circles and solid line. (C) Repair synthesis occurring in closed-circular UV-irradiated plasmids, determined by scintillation counting of the appropriate bands from (A), correcting for small variations in the amount of DNA between lanes. Same symbols as in (B).
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nc
cc
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9
1011121314
I
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.,
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0
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1. HSSB + CFII (60 min) 2. CFIB or PCNA (2 min) 3. cold dATP chase
Figure 6. Pulse-Chase Experiments Showing thesized DNA from Nicked to Closed Forms
Transfer
of Newly Syn-
(A) A mixture of UV-irradiated plasmid (+, 100 ng per lane) and nonirradiated plasmid (-, 400 ng per lane) was incubated for 60 min at 30°C in reaction buffer with (lanes l-7) 24 ug of CFIA and 50 ug of CFII protein per lane, or (lanes 6-14) 0.9 ug of HSSB and 50 ug of CFII protein per lane. CFIB protein (6 ug per lane) was then added, and incubationcontinuedfor2min.Atthis time, unlabeleddATPwasadded to 0.4 mM final concentration, and aliquots were withdrawn at the times indicated in the figure, stopping the reactions by addition of EDTA and chilling. Thus, the samples shown at “0” min had been incubated with CFIB for 2 min. Only the autoradiograph of the agarose gel is shown, for simplicity. (B) Experiment as described in (A), except that PCNA (25 ng of protein per lane) was used instead of CFIB. The positions of nicked-circular (nc) and closed-circular (cc) forms of the plasmids are indicated.
of damaged and nondamaged DNA for 60 min in the presence of all reaction components except the PCNAcontaining fraction, CFIB. After this time, CFIB was added and the repair synthesis in closed-circular and nicked forms was monitored (Figure 5A, lanes 1 O-l 5). Repair synthesis was simultaneously examined in reactions where all of the necessary components were present from the beginning of the incubation (Figure 5A, lanes l-9). Upon addition of CFIB to reaction mixtures, the proportion of nicked UV-irradiated DNA decreased rapidly to the steadystate level seen when all three fractions were present from the start of incubation (Figures 5A, top, and 58). At the same time, radioactive material accumulated in the damaged plasmid (Figure 5C). Within about 10 min, the extent of repair synthesis was equal to that achieved in reactions where all three fractions were present simultaneously. About 15safteradditionof CRIB, asmallamountofsynthe-
sis had already taken place in the nicked-circular form of the irradiated plasmid (Figure 5A, lane lo), and synthesis in this form increased during the next 8 min. At later times, label appeared in the closed-circular form while the proportion of label in the nicked form decreased. After the accumulated incised intermediates had been converted to complete repair patches by addition of CFIB, new repair events occurred during the next hour. These new events appeared to be initiated at a rate similar to that seen in reactions where all components were present from the start of incubation (Figure 5C). To visualize more clearly the completion of repair patches, pulse-chase experiments were performed as follows. CFII and either CFIA or HSSB were first incubated with plasmid DNAs for 60 min in reaction buffer containing the deoxynucleotides and [a-32P]dATP. The PCNA-containing fraction CFIB (Figure 6A) or purified PCNA (Figure 6B) was then added. After 2 min, a 50-fold excess of unlabeled dATP was added to the reaction mixture to prevent detection of synthesis arising from new repair events. At early times after addition of PCNA, radioactive material appeared first in nicked-circular forms, and later in closedcircular forms as ligation of the repair patches occurred to complete repair. Conversion of labeled nicked plasmids to closed forms was slightly slower in reactions that included HSSB in place of CFIA. Dependence of Repair in the Fractionated System on the XP-A Protein To confirm that damage-dependent incisions were a consequence of the multiprotein process of nucleotide excision repair, we studied the generation of incised intermediates in the presence and absence of a known nucleotide excision repair protein, XP-A (Robins et al., 1991). Patients with XP group A have mutations in the XPAC gene that inactivate the XP-A protein (Satokata et al., 1990). Cell extract from an XP-A cell line that lacks XP-A protein was fractionated on phosphocellulose by the procedure outlined in Figure 1. Material that bound to the column at 0.1 M KCI and eluted at 1 .O M KCI was designated CFIIXP-A. A mixture of damaged and nondamaged DNA was incubated with CFIIXP-Aand HSSB in reaction buffer including [aJ’P]dATP for 60 min. Then, using the strategy followed for the experiments in Figure 6, CFIB was added for a 2 min pulse, followed by achase with an excess of unlabeled dATP. No damage-dependent labeling of open or closedcircular plasmid forms was detectable after addition of CFIB, showing that repair events did not take place in the absence of XP-A protein (Figure 7, lanes l-7). In a parallel set of reactions, DNA mixtures were incubated in the first stage with 2 ng of homogeneous XP-A protein (Robins et al., 1991) in addition to the CFIIXP-Aand HSSB, before addition of CFIB for a 2 min pulse. In this set of reactions (Figure 7, lanes 8-14) nicked intermediates were generated in damaged DNA, as shown by the repair synthesis that occurred upon addition of the PCNAcontaining fraction. Synthesis occurred first in the nicked intermediates, and these were converted to the closedcircular form during subsequent incubation. As expected, XP-A was required for the incision stage of the reaction
PCNA-Dependent 371
DNA Repair
Synthesis
1 2 3 4 5 6 7 6 9 1011121314 uv nc +
cc + min: 1.
0
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8 12 16 28 58
HSSo”Si,,,ll
XP-A
2. CFIB (2 min) 3. cold dATP chase Figure
7. Dependence
of Excision
0
4
8
1. HSSB + XP-A 2. CFIB 3. cold Repair
12 16 28 58
+ CFIIXP-A protein (60 min) (2 min) dATP chase
on the XP-A Protein
A mixture of W-irradiated plasmid (+, 100 ng per lane) and nonirradiated plasmid (-, 400 ng per lane) was incubated at 30% in reaction buffer with (per lane) 0.9 ng of HSSB protein and 50 ug of CFIIxp-* protein prepared from a lymphoblastoid XP-A cell line. The reaction mixture for lanes 6-14 also contained purified XP-A protein from calf thymus (fraction VII, 2 ng per lane). CFIB protein (6 ug per lane) was added after 60 min, and incubation continued for 2 min. Unlabeled dATP was then added to 0.4 mM, and aliquots were withdrawn at the times indicated, stopping the reactions by addition of EDTA and chilling. Thus the samples shown at “0” min had been incubated with CFIB for 2 min. The autoradiograph of the agarose gel is shown.
rather than the synthesis stage; if XP-A protein was added only during the incubation with CFIB (PCNA), no detectable repair synthesis took place during the 2 min pulse (data not shown). The dependence of damagedependent repair synthesis on XP-A protein confirms that the incisions monitored in this study are generated by nucleotide excision repair. Discussion Requirement for PCNA during Nucleotide Excision Repair in Human Cells The present work provides direct evidence that PCNA is required for nucleotide excision repair. Dissection of the in vitro repair reaction into two distinct stages reveals that PCNA acts after the incision of damaged DNA and before or at the time of polymerization. Thus, in addition to an essential role in semiconservative DNA replication, PCNA is also needed for the formation of the relatively short repair patches created during nucleotide excision repair. PCNA is present in nuclei during all phases of the cell division cycle, but much of the PCNA can be lost upon fixation of cells. After a growing culture is fixed with methanol, most PCNA antibodies stain only S phase nuclei (Celis et al., 1987; Waseem and Lane, 1990). The stained subpopulation of PCNA that remains is tightly bound to foci of replicating DNA. However, following UV irradiation of cells, non-S phase nuclei also exhibit punctate staining with PCNA antibodies (Celis and Madsen, 1988). The pattern of staining resembles the pattern of repair synthesis
as detected by autoradiography of nuclei, and it has been suggested that some PCNA in Gl and G2 phase cells becomes resistant to methanol fixation by migration to sites of DNA repair (Celis and Madsen, 1986). UV irradiation of quiescent (noncycling) human fibroblasts similarly leads to the appearance of PCNA that is retained after methanol fixation (Toschi and Bravo, 1988). A well established activity of PCNA is its ability to affect the processivity of human DNA polymerase 6 (Prelich et al., 1987b; Bravoet al., 1987). Theactivityof DNApolymerase E from HeLa cells can also be stimulated by PCNA (in the presence of RF-C) at moderately high salt concentrations (Lee et al., 1991 b), although under many conditions PCNA does not affect the activity of this polymerase (Syvaoja et al., 1990). The participation of polymerase 6 or E in DNA excision repair is consistent with other data. For example, nucleotide excision repair in human cells is suppressed by the DNA polymerase a, 6, and E inhibitor aphidicolin (Dresler, 1984; Toschi and Bravo, 1988; Jones et al., 1989) and studies with other chemical inhibitors suggest that DNA polymerase a is not responsible for repair synthesis (Dresler and Frattini, 1986). Significantly, repair synthesis can be restored to permeabilized cell nuclei by addition of purified DNA polymerase E (Nishida et al., 1988; Syvaoja et al., 1990). We have found that DNA repair synthesis mediated by cell extracts is inhibited by aphidicolin, but is unaffected by antibodies that neutralize DNA polymerase a (D. Coverley, M. K. K., and R. W., unpublished data). Thus, it seems very likely that PCNA interacts with DNA polymerase 6 or E in some way during nucleotide excision repair. By regulating the processivity of synthesis, PCNA might serve to control the repair patch size by association and dissociation from the polymerase. A second relevant activity of PCNA is its ability to bind to primer-template junctions in conjunction with the multisubunit protein RF-C. It has been proposed that sequential binding of RF-C, PCNA, and pol 8 to a primer-template junction occurs during the initiation of replicative leadingstrand DNAsynthesis (Tsurimoto and Stillman, 1991). The presence of HSSB bound to the adjacent single-stranded DNA aids in the specificity of binding of these proteins to the junction. The interaction of PCNA with RF-C can also increase the ability of pols 6 and E to utilize low levels of primer ends (Lee et al., 1991a; Lee et al., 1991 b). We do not yet know if RF-C is also involved with PCNA and HSSB in DNA repair in an analogous way, but a similar role seems plausible. Resolution of Nucleotide Excision Repair into Two Stages By using a fractionation scheme that separates PCNA from the other components necessary for repair, we have been able to observe the formation in vitro of incised intermediates in damaged DNA. The ability to examine the incision reaction in the absence of repair synthesis should be of considerable utility for future biochemical efforts to dissect the nucleotide excision repair reaction. It is worth emphasizing that an appropriately purified DNA substrate must be used to attain the sensitivity required for detection of the nicked intermediates created
Cell 372
during repair. The UV-irradiated plasmid molecules used in this study were treated with E. coli Nth protein to remove pyrimidine hydrates from the DNA, and closed-circular forms were reisolated (Wood et al., 1988; Biggerstaff et al., 1991). Without such a purification step, the level of nicked molecules introduced by pyrimidine hydrate-DNA glycosylase in mammalian cell extracts makes it difficult to visualize incisions generated by the nucleotide excision repair enzymes. In the absence of PCNA, repair events remain trapped in an incised and stable intermediate state. From the number of incisions introduced and the subsequent synthesis upon addition of PCNA, the repair patch size can be estimated. After a 1 hr incubation with CFIA and CFII from HeLa cells, the UV-irradiated plasmids contained an average of ~0.4 nicks per molecule (see Figure 58). About 0.1 nicks per molecule were damage-dependent incisions that were rejoined within 15 min of adding the PCNAcontaining fraction. The reaction mixture contained 130 fmol of UV-irradiated plasmid DNA circles, and so ~13 fmol of damage-dependent incisions were introduced during the incubation. During the 15 min after addition of CFIB, ~100 fmol of dAMP (400 fmol nt) was incorporated as synthesis took place to complete these repair events (Figure 5C). Thus, the average patch size per repair event was (400/13) = 31 nt. This size is consistent with the estimate of approximately 30 nt determined for repair of acetylaminofluorene lesions by unfractionated whole cell extracts (Hansson et al., 1989; Coverley et al., 1991). The number is also similar to the in vivo repair patch size of ~20 nt measured in HeLa cells after UV irradiation (Th’ng and Walker, 1986). Synthesis and ligation of patches is completed rapidly, with most sites repaired within 10 min after addition of PCNA, and about half in 5 min (Figure 5, Figure 6, and Figure 7). The rate is similar to that measured in vivo in UV-irradiated normal human cells (Erixon and Ahnstrijm, 1979). Role of XP-A Protein in Incision of Damaged DNA The proteins (other than HSSB) that are essential for incision of damaged DNA appear to be in CFII. One component in this fraction is the XPA protein (Robins et al., 1991) afactorthat has acentral role in nucleotide excision repair. Cells from XP complementation group A show defective incision of UV-irradiated genomic DNA (Fornace et al., 1976; Erixon and Ahnstrdm, 1979; Kaufmann and Briley, 1990; Thielmann et al., 1991) but are competent in the second stage of nucleotide excision repair. For example, XP-A cells can complete repair if incisions are introduced in UV-irradiated DNA by simple pyrimidine dimerspecific endonucleases from procaryotes (Tanaka et al., 1977; de Jonge et al., 1985). Similarly, XP-A extracts in vitro can perform repair synthesis at gaps and incisions generated in DNA by procaryotic repair enzymes (Wood et al., 1988; Hansson et al., 1990). The results shown in Figure 7 provide evidence that the generation of nicked intermediates in damaged DNA requires XP-A protein. Indeed, the ability of the XP-A protein to bind damaged DNA suggests that it is involved in recognition of DNA damage
XP and ERCC proteins
(V) I
DNA polymerase
(Vi) Figure
8. Schematic
6 /&
DNA ligase
Model for Nucleotide
Excision
Repair
and may form part of an endonucleolytic complex (Robins et al., 1991). It is generally assumed that the repair defects in the most characterized XP complementation groups are confined to the incision stage, but experiments similar to those in Figure 7, using other XP extracts, will permit a direct analysis. Sequence of Events in DNA Nucleotide Excision Repair A model for nucleotide excision repair in mammalian cells, based on the data reported here, is illustrated in Figure 8. In the first stage, incisions are introduced near a damaged site by the cooperative action of a number of proteins (i). These include the XP-A polypeptide, as well as up to eight or more XP and excision repair cross-complementing (ERCC) gene products implicated by genetic evidence and in vivo experiments (Hoeijmakers, 1991). Stable incised intermediates are detected only if the HSSB protein is present. HSSB might help to displace an oligonucleotide containing DNA damage, recycling the incision proteins and thus effectively increasing the number of incision events (ii). The HSSB could then serve to protect a nicked or gapped region from being processed or enzymatically degraded (iii). Upon addition of PCNA to the incised DNA, gap-filling synthesis takes place. Since PCNA can bind to primer-template junctions in cooperation with the RF-C protein, a similar interaction may take place at the incision generated in the first stage of the reaction (iv). Once bound, repair synthesis is mediated by a DNA polymerase that interacts with PCNA (v), and the repair patch is sealed by a DNA ligase (vi). The introduction of a helix-distorting mutagenic lesion into DNA poses a considerable challenge to the cell, and it appears that the number of components required for DNA nucleotide excision repair may be as great or even
PCNA-Dependent 373
DNA Repair
Synthesis
greater than the number required for replication. Repair requires not only the many proteins involved in incision and a DNA polymerase, but also additional replication factors such as PCNA and HSSB as shown here. Experimental
Procedures
Preparation and Fractionation of Whole Cell Extracts HeLa cells were grown in suspension in RPM1 1640 medium containing 5% fetal calf serum. The lymphoblastoid XP-A cell line GM2345 was obtained from the Human Genetic Mutant Cell Repository(Coriell Institute, Camden, N. J.) and was grown in RPM 1640 medium containing 10% fetal calf serum. Splicing mutations that inactivate theXPACgene have been identified in the patient (XPPOS) from whom this line was derived (Satokata et al., 1990). All cells were confirmed to be free of Mycoplasma. Nineteen liters of HeLa cells at 1 x lo6 cells per milliliter were washed in phosphate-buffered saline A and collected to give a packed cell volume of 24 ml. A whole cell extract was prepared as described (Wood et al., 1968) except that the 165,000 x g centrifugation was done in a Ti60 rotor (Beckman). Supernatant (158 ml) was collected. Protein was precipitated with (NH&SO, and dialyzed overnight at 4OC. Whole cell extract protein (660 mg (27.5 ml]) was loaded onto a 300 ml column (5 x 15 cm) of phosphocellulose (Whatman Pll) that been equilibrated in Buffer A (25 mM Hepes-KOH [pH 7.81, 1 mM EDTA, 0.01% NP40, 10% glycerol, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride)containing 0.1 M KCI. Fractions(l0 ml) werecollected at a flow rate of 90 ml/hr. After collection of flow-through fractions, bound protein was eluted with Buffer A containing 1 .O M KCI. The peak protein fractions from the flow-through (CFI) and the 1 .O M KCI elution (CFII) were used. Fractionation of XP-A cells to produce CFY and CFIIxp,A was done in the same way, starting with 10 liters of cells. CFI (300 mg in 40 ml) was fractionated on a 150 ml column (5 x 6 cm) of DEAE Biogel (BioRad) that had been equilibrated in Buffer A containing 0.15 M KCI. The flow-through was collected at 90 mllhr, and bound protein was eluted from the column in Buffer A containing 1.0 M KCI. Peak fractions from the flow-through (CFIA) and the 1.0 M elution (CFIB) were used. CFII (250 mg; 50 ml), CFIA (165 mg; 55 ml) and CFIB (80 mg; 20 ml) were dialyzed against 25 mM Hepes-KOH (pH 7.9) 1 mM EDTA, 17% glycerol: 1 mM dithiothreitol, 12 mM MgCI,, and 0.1 M KCI. Aliquots of CFI, II, IA. and IB were frozen at -60% Purification of HSSB, PCNA, and XP-A Proteins HSSB was purified to homogeneity from HeLa cells as previously described (Kenny et al., 1990). PCNA was purified from an E. coli strain (a generous gift from B. Stillman) that expressed the human PCNA cDNA from an inducible T7 promoter by a procedure similar to that of Fien and Stillman (1992). The protein was subjected to sequential chromatography on Q-Sepharose, S-Sepharose, hydroxyapatite, and phenyl-Sepharose. At the end of this procedure, a single homogeneous band of 36 kd was detected by Coomassie blue staining of an SDS-polyacrylamide gel. For immunoblotting, the anti-HSSB monoclonalantibody34A(Kennyetal., 1990)and theanti-PCNAmonoclonal antibody PC10 (Waseem and Lane, 1990) were utilized. XP-A protein was isolated from calf thymus as described (Robins et al., 1991). The most purified fraction (Fraction VII, 1 nglml) was used in these experiments Repair Synthesis Plasmids pBluescript KS+ and pHM14 were grown in E. coli strain JM109. pBluescript KS+ was UV irradiated (450 J/mz). Both plasmids were treated with E. coli Nth protein, and closed-circular DNA was isolated from cesium chloride and sucrose gradients (Wood et al., 1988; Biggerstaff et al., 1991). Reaction mixtures (50 ul) contained 250 ng of nonirradiated pHM14 and 250 ng of irradiated pBluescript KS, 45 mM C(P-Hydroxyethyl)-1-piperazineethanesulfonic acid-KOH (pH 7.8) 70 mM KCI, 7.4 mM MgCI,, 0.9 mM dithiothreitol, 0.4 mM EDTA, 20 uM each dGTP, dCTP, and TTP, 8 uM dATP, 74 kBq of (@P]dATP (110 TBq/mmol), 2 mM ATP, 40 mM phosphocreatine, 2.5 ug of creatine phosphokinase, 3.4% glycerol, and 18 ng of bovine serum albumin, with human cell extract protein as indicated. Reactions were incubated at 30% for the times indicated. Plasmid DNA was purified from
the reaction mixtures, linearized with BamHl (if appropriate for the analysis), and electrophoresed overnight on a 1% agarose gel containing 0.5 uglml ethidium bromide. Datawere analyzed byautoradiography, densitometry. and scintillation counting of excised DNA bands (Wood et al., 1988) taking account of the ~1.6-fold greater fluorescence of nicked-circular DNA over closed-circular DNA. Acknowledgments We are grateful to N. Waseem and D. P. Lane (University of Dundee) and to D. Coverley for valuable and productive discussions, for suggesting and participating in initial cooperative experiments, and for general encouragement. N. Waseem also contributed to the purification of PCNA. We thank P. Robins for XP-A protein, M. Biggerstaff for preparing purified DNA substrates, C. J. Jones for Nth protein, and the ICRF Cell Production Unit for growing large numbers of cells. The helpful comments of J. Diffley, T. Hunt, T. Lindahl, D. Szymkowski, and S. West are appreciated. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received
December
23, 1991; revised
January
24, 1992
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