(Received for publication, February 5, 1996; and in revised form, March 12, 1996)
From the
Replication protein A (RPA), a heterotrimeric protein of 70-,
32-, and 14-kDa subunits, is an essential factor for DNA replication.
Biochemical studies with human and yeast RPA have indicated that it is
a DNA-binding protein that has higher affinity for single-stranded DNA.
Interestingly, in vitro nucleotide excision repair studies
with purified protein components have shown an absolute requirement for
RPA in the incision of UV-damaged DNA. Here we use a mobility shift
assay to demonstrate that human RPA binds a UV damaged duplex DNA
fragment preferentially. Complex formation between RPA and the
UV-irradiated DNA is not affected by prior enzymatic photo-reactivation
of the DNA, suggesting an affinity of RPA for the (6-4) photoproduct.
We also show that Mg in the millimolar range is
required for preferential binding of RPA to damaged DNA. These findings
identify a novel property of RPA and implicate RPA in damage
recognition during the incision of UV-damaged DNA.
Nucleotide excision repair (NER) ()represents the
most important cellular mechanism for repairing DNA damaged by
ultraviolet (UV) light. Defects in NER result in failure to remove UV
lesions from DNA and cause the cancer-prone disease xeroderma
pigmentosum (XP) in humans. Cell fusion studies have so far identified
seven XP genes, A through G.
Extensive genetic studies in Saccharomyces cerevisiae have indicated the requirement of the RAD1, RAD2, RAD3, RAD4, RAD10, RAD14, and RAD25 genes in the incision step of NER(1) . In addition to their role in NER, RAD3 and RAD25 are essential for RNA polymerase II (Pol II) transcription(2, 3, 4) , and their encoded proteins are constituents of Pol II transcription factor TFIIH(5, 6) . The incision step of NER has been reconstituted using components purified to near homogeneity, and these studies have indicated the requirement of Rad14, the Rad4-Rad23 complex, Rad2 nuclease, Rad1-Rad10 nuclease, replication protein A (RPA), and TFIIH in dual incision of UV-damaged DNA(6) . The structure and function of NER proteins have been conserved to a remarkable degree from yeast to humans, and a combination of human equivalents of these yeast proteins also mediates dual incision of UV-damaged DNA(7) .
Biochemical studies in yeast have indicated the following roles of Rad proteins in the incision phase of NER. Rad14 is a DNA damage recognition protein(8) . Rad3 and Rad25 encode the two DNA helicase subunits of TFIIH, with a likely role in unwinding the damaged DNA(3, 9) . Rad23 exists in a tight complex with Rad4(6) , and Rad23 has been shown to promote complex formation between Rad14 and TFIIH(10) . Thus, Rad4-Rad23 proteins may function in tethering the different components of the incision machinery at the damage site. Rad1-Rad10 and Rad2 endonucleases mediate the dual incision of the damage-containing DNA strand(11, 12, 13) . The human homologs of the yeast NER proteins share similar biochemical activities. XPA binds damaged DNA, XPD and XPB are DNA helicases, and XPG and ERCC1-XPF encode the two nucleases (14, 15, 16, 17, 18) .
Interestingly, in both the yeast and human systems, incision of UV-damaged DNA is absolutely dependent upon RPA(6, 7) . However, the precise role of RPA in the incision process remains unclear. RPA is a highly conserved heterotrimeric protein, composed of three subunits of 70, 32, and 14 kDa in humans. In DNA replication, RPA is required for origin-dependent unwinding by the SV40 large T antigen (19) . In incision, RPA could function in an analogous manner by binding and stabilizing ssDNA created as a result of unwinding by the two DNA helicases present in TFIIH. Alternatively, RPA could function in another step, such as in damage recognition. Here, we show that RPA binds specifically to UV-damaged DNA, thus identifying RPA as a damage recognition factor.
For examining the DNA damage binding properties of RPA, it was purified from E. coli strain BL21(DE3) harboring plasmid p11d-tRPA, which co-expresses the three RPA subunits(20) . RPA thus purified is fully active in the in vitro replication of SV40 DNA(20) , in nucleotide excision repair in a reconstituted system(7) , and exhibits an affinity for ssDNA indistinguishable from that observed with RPA purified from HeLa cell extract(20) . Based on the chromatographic procedures described by Henricksen et al.(20) that yield biologically active RPA protein, we subjected clarified bacterial extract to fractionation in columns of Affi-Gel Blue, hydroxyapatite, and Mono Q. As shown in Fig. 1A, the RPA pool (fraction IV) from the final step of purification on Mono Q contained stoichiometric amounts of the 70-, 32-, and 14-kDa subunits of RPA, and a minor species migrating below the largest subunit, which, as has been noted before(20) , is a proteolytic product of the 70-kDa RPA subunit. The fraction IV RPA (Fig. 1A) was used in all of the DNA binding experiments described below.
Figure 1:
RPA binds UV damaged DNA. A, the Mono Q (fraction IV) RPA pool, 1.5 µg of protein,
was run in a 12% SDS-polyacrylamide gel and stained with Coomassie
Blue; the three subunits of RPA (lane 2) are indicated by the arrows. Aside from the minor proteolytic product that migrates
below the largest subunit, no other protein species was detected at
this loading and when 3 µg of fraction IV RPA was analyzed (data
not shown). B, RPA, 100 ng, was incubated with undamaged DNA (lane 1) and with DNA irradiated with 0.5, 1, 3, 5, 7, 9, 12,
and 15 kJ/m UV (lanes 2-9, as indicated),
for 30 min at 30 °C, as described under ``Materials and
Methods.'' F, unbound DNA probe; C, RPA-DNA
complex. A minor slower migrating protein-DNA complex (not marked) is
also detected above the main complex. This slower migrating form may
correspond to RPA binding to multiple sites on damaged DNA. C,
graphical representation of the results in B.
The lesions induced
by UV light occur predominantly at pyrimidine residues in DNA. As DNA
probe for damage binding, a 130-base pair DNA fragment high in
pyrimidine content was therefore chosen, and it was generated by
restriction digest of plasmid DNA, purified, and 3`-end-labeled with P, as described under ``Materials and Methods.''
The radiolabeled DNA fragment was then irradiated with an ultraviolet
source for increasing lengths of time, giving final dosages ranging
from 0.5 to 15 kJ/m
. In the UV damage binding reaction, RPA
was incubated with the DNA fragment which had received increasing UV
dosage in the presence of 3 mM MgCl
and 30 mM KCl at 30 °C for 30 min, and the reaction mixtures were mixed
with loading buffer and then applied onto 3.5% polyacrylamide gels.
After electrophoresis at 4 °C, the gels were dried and exposed to
x-ray films to visualize the free DNA probe and any retarded form of
the DNA probe, which would be indicative of an RPA-DNA complex. As
shown in Fig. 1B, a slow migrating form of the UV
irradiated DNA fragment, designated as C, was seen upon
incubation with RPA, indicating that RPA binds UV damage. The amount of
the RPA-DNA complex increased with the UV dose, and densitometric
scanning of the autoradiogram indicated that, at the highest dose (15
kJ/m
) used, greater than 85% of the input DNA probe was
bound by RPA (Fig. 1, B and C). We next
examined complex formation as a function of RPA concentration, using
DNA fragment that had been irradiated with 15 kJ/m
of UV.
As expected, the amount of RPA-damaged DNA complex increased with the
RPA concentration (Fig. 2, A and B). In these
( Fig. 1and Fig. 2) and other experiments (data not
shown), only a low level (
2%) of binding of the nonirradiated
counterpart of the DNA fragment was detected, even at the highest
amount of RPA used (Fig. 2A, lane 3). Taken
together, our results indicate that RPA binds preferentially to duplex
DNA containing UV-induced lesions.
Figure 2:
RPA concentration dependence of damage
binding. A, undamaged DNA (lanes 1-3) either
incubated alone (lane 1) or with 40 and 100 ng of RPA (lanes 2 and 3). UV-irradiated DNA (15
kJ/m) was incubated alone (lane 4) and with 20,
40, 60, 80, and 100 ng of RPA (lanes 5-9), as indicated,
at 30 °C for 30 min. F, unbound DNA; C, RPA-DNA
complex. B, graphical representation of the results in A.
In the experiments described thus
far, wherein preferential binding of RPA to UV damaged DNA occurred, 3
mM MgCl was present during incubation of DNA with
RPA. Interestingly, when MgCl
was omitted from the
reaction, the undamaged DNA fragment was bound by RPA nearly as well as
the UV irradiated (15 kJ/m
) DNA (Fig. 3, compare lanes 2 and 8). Consistent with the results obtained
in prior experiments ( Fig. 1and Fig. 2), nonspecific
binding of RPA to undamaged DNA was essentially eliminated by the
inclusion of 3 mM Mg
, such that only <2%
of the unirradiated DNA was bound by RPA in the presence of 3 mM Mg
, as compared to binding of >85% of the DNA
in the absence of Mg
(Fig. 3, lanes 8 and 9). As shown in Fig. 3, binding of RPA to the
UV-irradiated fragment was also reduced from 100% to 50% by 3 mM Mg
(lanes 2 and 3). However,
because RPA binds nonspecifically to undamaged DNA in the absence of
Mg
(Fig. 3, lane 8), the reduction
observed (Fig. 3, lanes 2 and 3) was very
likely due to abolition of binding of RPA to sites other than those
containing UV lesions in the target DNA. In support of this notion,
efficient binding to UV damaged DNA still occurred when the
Mg
concentration was further increased to 12 mM (Fig. 3, lane 6). These results therefore indicate
that nonspecific, low affinity binding of RPA to nondamaged duplex DNA
is overcome by Mg
in the millimolar range, allowing
detection of specific binding to the UV damage.
Figure 3:
Effect of Mg on RPA
binding to UV-damaged DNA. UV-damaged DNA (15 kJ/m
) (lanes 1-6) was incubated alone (lane 1) or
with 20 ng of RPA (lanes 2-6) either in the absence of
Mg
(lane 2) or in the presence of 3, 6, 9,
and 12 mM Mg
(lanes 3-6, as
indicated) at 30 °C for 30 min. Undamaged DNA (lanes
7-12) was incubated alone (lane 7) and with 20 ng
of RPA (lanes 8-12) either in the absence of
Mg
(lane 8) or in the presence of 3, 6, 9,
and 12 mM Mg
(lanes 9-12, as
indicated) at 30 °C for 30 min.
The binding of UV damaged DNA by RPA is not affected by KCl up to 100 mM, and the addition of 1 mM ATP has no effect on binding, but the inclusion of 0.2% SDS at the beginning of reaction abolished damage binding (data not shown).
Two major classes of DNA lesions are
induced by exposure to UV light; namely, the cyclobutane pyrimidine
dimers (CPDs) and the pyrimidine(6-4)pyrimidone photoproducts ((6-4)
photoproducts). The kinetics of formation of these two types of UV
lesions, however, differ considerably. At UV doses up to 2
kJ/m, CPDs are introduced into DNA at
5 times the
frequency of that of the(6-4) photoproducts, but formation of the
former reaches photochemical equilibrium at
4 kJ/m
,
whereas the yield of the(6-4) photoproducts continues to increase
linearly with the UV dose above 4
kJ/m
(22, 23) . An examination of the RPA
damage binding profile as a function of the UV dose (Fig. 1, B and C) revealed that the amount of RPA-DNA complex
is relatively insignificant at low UV doses where the CPD represents
the predominant lesion, suggesting that the primary target for RPA in
UV-damaged DNA is the(6-4) photoproduct. We used enzymatic
photoreactivation, a procedure that selectively and quantitatively
removes CPDs from UV-damaged DNA without affecting the content of
the(6-4) photoproducts, to test the notion that RPA has little affinity
for the former class of UV lesion. To do this, DNA which had been
irradiated with a UV dose of either 3 kJ/m
or 15 kJ/m
was incubated with E. coli photolyase under
photoreactivating light, which provides the energy for photolyase to
catalyze the monomerization of the pyrimidine dimers(24) . To
verify that CPDs in the UV-irradiated DNA had been removed, after
incubation with photolyase, the DNA was treated with the bacteriophage
T4 pyrimidine-dimer endonuclease, which nicks DNA at CPD sites. As
shown in Fig. 4A, incubation of UV-irradiated DNA with
photolyase resulted in the removal of T4 endonuclease-sensitive sites,
indicating that the UV-damaged DNA had now been freed of CPDs. We then
examined the ability of the photoreactivated, UV-irradiated DNA to form
a complex with RPA. As shown in Fig. 4, B and C, complex formation between UV irradiated DNA and RPA was
refractory to enzymatic photoreactivation, indicating that RPA does not
detectably bind CPD and suggesting that RPA binds the(6-4) photoproduct
in target DNA. At the UV dose of 12 kJ/m
, where we observed
50-fold preferential binding to UV-damaged DNA (Fig. 1),
there are
1.8(6-4) photoproducts per DNA probe. From these
results, it can be estimated that RPA binds the UV damage with an
affinity
3600-fold over undamaged nucleotides.
Figure 4:
Removal of CPDs by enzymatic
photoreactivation does not affect damage binding by RPA. A, DNA irradiated with 3 kJ/m (lanes 1, 3, and 5) or 15 kJ/m
(lanes 2, 4, and 6) of UV was incubated with (lanes 5 and 6) or without (lanes 1-4) photolyase
as described under ``Materials and Methods.'' The DNA in lanes 3-6 was treated with T4 pyrimidine-dimer
endonuclease before electrophoresis. B, undamaged DNA (lane 1) and DNA irradiated with 3 kJ/m
(lane
2) or 15 kJ/m
(lane 3) were incubated with
100 ng of RPA at 30 °C for 30 min. Photoreactivated UV-irradiated
DNA (3 kJ/m
in lane 4 and 15 kJ/m
in lane 5) was also incubated with 100 ng of RPA at 30 °C for
30 min. C, the autoradiogram was subjected to image analysis
to determine the amount of RPA-DNA complex formed before and after
photoreactivation of the UV irradiated DNA. -PRE, no
photoreactivation; +PRE, enzymatic
photoreactivation.
In this work, we demonstrate a role for human RPA in DNA damage recognition. RPA binds specifically to UV-damaged DNA in a UV dose- and protein concentration-dependent manner. Enzymatic photoreactivation experiments suggest that RPA has affinity for(6-4) photoproducts. It remains to be determined whether RPA recognizes the UV damage per se or the single-strandedness resulting from UV lesions. The damage binding ability of RPA may explain the absolute dependence of the damage-specific incision reaction on RPA.
RPA
binds tightly to ssDNA with an apparent binding constant of
10
(25, 26) , and the ss binding
activity of RPA is necessary for both replication and recombination. In
SV40 origin-dependent DNA replication, RPA assists in DNA unwinding
catalyzed by T antigen by binding and stabilizing the unwound,
single-stranded DNA. RPA also functions as a single strand binding
protein in DNA chain elongation, and interaction with RPA stimulates
the activity of various enzymes assembled at the replication fork
(reviewed in (27) ). Additionally, RPA functions as an
important component in the homologous DNA pairing and strand exchange
reaction catalyzed by the S. cerevisiae Rad51
protein(28) . In this reaction, as a single strand DNA-binding
protein, RPA enhances the efficiency of Rad51 filament formation on
ssDNA(29) . Our study now implicates RPA as a damage
recognition factor in NER.
The structure and function of RPA have
been conserved among eukaryotes; the 70-kDa subunit binds ssDNA and
interacts directly with the 32- and 14-kDa subunits (30 and references
therein). Between residues 481 and 503, human RPA70 contains a putative
C type zinc finger motif, and this motif is conserved in
all known RPA70 homologs. This C
motif does not appear to
be necessary for ssDNA binding activity, because a protein deleted for
the carboxyl-terminal portion of RPA, including the C
zinc
finger motif, retains normal ssbinding activity(30) . The
C
motif may have a role in specific binding of RPA to DNA
damage sites.