(Received for publication, August 7, 1995)
From the
In Saccharomyces cerevisiae, the RAD1 and RAD10 genes are involved in DNA nucleotide excision repair (NER) and in a pathway of mitotic recombination that occurs between direct repeat DNA sequences. In this paper, we show that purified Rad1 and Rad10 interact with a synthetic bubble structure and incise the DNA at the 5`-side of the centrally unpaired region. When Rad1-Rad10 and purified XPG protein (the human homolog of yeast Rad2 protein) were co-incubated with the DNA substrate, we observed incisions at both ends of the bubble. This reaction mimics the dual incision step in nucleotide excision repair in vivo. In addition, the recent suggestion that Rad1 can act to resolve Holliday junctions (Habraken, Y., Sung, P., Prakash, L., and Prakash, S.(1994) Nature 371, 531-534), explaining the recombination defect observed in rad1 mutants, has been further investigated. However, using proteins purified in two different laboratories we were unable to show any interaction between Rad1 and synthetic Holliday junctions. The role that Rad1-Rad10 plays in recombination is likely to resemble its activity in NER by acting upon partially unpaired DNA intermediates such as those formed by recombination mechanisms involving single-strand DNA annealing.
The process of nucleotide excision repair plays a major role in the removal of DNA lesions following DNA damage. In bacteria, excision repair is initiated by three proteins, UvrA, UvrB, and UvrC, which promote a dual incision reaction by cleaving the 3rd to 5th phosphodiester bond 3` to the lesion and the 8th bond at the 5`-side of the lesion. The 12-13-nucleotide-long oligomer containing the lesion is then removed from the DNA, and the single-stranded gap is filled and sealed by DNA polymerases and DNA ligase(1, 2) . Dual incision is also observed in eukaryotes, and higher organisms excise a fragment of 27-29 nucleotides in length(3) .
The isolation of UV-sensitive mutants has helped to define the genetic complexity of nucleotide excision repair in Saccharomyces cerevisiae, and it is now known that many genes are involved in the process. These include RAD1, RAD2, RAD3, RAD4, RAD7, RAD10, RAD14, RAD16, RAD23, TFB1, SSL1, and SSL2(1, 4) . Remarkably, the sequences of excision repair genes in yeast and mammalian cells are highly homologous, and the enzymology of the two systems is very similar.
The RAD1 and RAD10 genes of S.
cerevisiae encode polypeptides that interact to form a stable
complex (5, 6, 7) that exhibits a
structure-specific endonuclease activity. The endonuclease cleaves
single-stranded DNA tails with 3`-ends at the junction with duplex
DNA(8) , indicating that Rad1-Rad10 promotes the 5`-incision
event in NER. ()Genetic studies show that rad1 and rad10 mutants also exhibit defects in mitotic
recombination(9, 10, 11, 12, 13, 14) .
In particular, RAD1 and RAD10 are required for
intrachromosomal recombination between direct repeats and affect the
integration of linear DNA molecules and circular plasmids into genomic
sequences. RAD1 is also required for double-strand break
repair between two direct repeats when the double-strand break occurs
within a region of heterology(12) . Recently it was suggested
that Rad1 can resolve Holliday junctions(15) , thus explaining
the recombination defect observed in rad1 mutants.
In this paper we have further investigated the specific endonuclease activity of Rad1-Rad10 using substrates that mimic intermediates thought to occur during nucleotide excision repair and recombination. We show that the purified Rad1 and Rad10 proteins cut a synthetic bubble structure at the 5`-side of the centrally unpaired region and that Rad1-Rad10 together with purified XPG protein (the human homolog of yeast Rad2 protein) catalyze a dual incision reaction. In contrast to the report by Habraken et al.(15) , we did not detect any cleavage of Holliday junctions by Rad1 or Rad1-Rad10.
A key event in NER in eukaryotic cells is the introduction of
dual incisions flanking a damaged site in DNA, leading to the excision
of a DNA fragment about 30 nucleotides long(3) . To investigate
the incision step in more detail, we prepared a synthetic bubble
structure, which contains a 30-nucleotide-long unpaired region flanked
by duplex regions of similar length (Fig. 1). This model
substrate is likely to resemble an intermediate in NER, after repair
proteins have unwound and separated the DNA strands around the damaged
region(19) . When purified S. cerevisiae Rad1 and
Rad10 proteins were incubated with a synthetic bubble structure in
which one strand was 5`-P-end labeled, we observed
cleavage of the labeled strand at the 5`-side of the unpaired region,
2 bases into the adjacent duplex DNA (Fig. 1, lanes b and c). No products were observed that corresponded to
cleavage within the unpaired region or on the 3`-side. The polarity of
cleavage is therefore the same as that observed previously with model
DNA structures with single-stranded DNA tails with 3`-ends(8) .
However, the results presented here provide evidence that a free
single-stranded DNA end is not required for structure-specific cleavage
by Rad1-Rad10, indicating that the duplex
-single-strand junction
is directly recognized by the endonuclease. Cleavage of the bubble
structure required both proteins (Fig. 2, lane d),
since neither Rad1 (lane b) nor Rad10 (lane c) alone
had any effect on the bubble substrate.
Figure 1:
Rad1-Rad10 and XPG incise DNA at
opposite sides of a bubble. The line diagram shows the bubble substrate (19) formed by annealing two 90-mer oligonucleotides to form a
centrally unpaired region of 30 nucleotides. The
poly(T)-containing strand (oligo 2) was labeled with
P at the 5`-end (asterisk). Arrows indicate cleavage positions on the labeled strand. The bubble
substrate (0.5 ng) was incubated alone (lane a) or with the
indicated combinations of proteins. Reaction conditions for Rad1-Rad10
were 50 mM Tris-HCl, pH 8.5, 20 mM NaCl, and 5
mM MgCl
(lane b) (16) and for XPG
were 20 mM Hepes, pH 6.8, 10% glycerol, 2 mM MgCl
, 1 mM dithiothreitol, and 50 µg/ml
BSA (lane f)(18) . All other reactions (lanes c,
d, and e) were carried out in a compromise buffer (50
mM Tris-HCl, pH 7.5, 20 mM NaCl, 10% glycerol, 5
mM MgCl
, 1 mM dithiothreitol, and 50
µg/ml BSA). Amounts of proteins were: Rad1 (100 ng), Rad10 (20 ng),
and XPG (100 ng). Reaction mixtures (20 µl) were incubated for 90
min at 37 °C, and
P-labeled DNA products were analyzed
by 12% denaturing polyacrylamide gel electrophoresis and visualized by
autoradiography. Lane g, oligonucleotide markers of defined
length.
Figure 2:
Both
Rad1 and Rad10 are required for incision of the bubble structure. The
bubble substrate (0.5 ng; P-labeled at the 5`-end of oligo
2) was incubated alone (lane a) with 62 ng of Rad1 (lane
b), 12 ng of Rad10 (lane c), 62 ng of Rad1, and 12 ng of
Rad10 (lane d) or 50 ng of RuvC (lane e). Incubation
was for 60 min at 30 °C (37 °C for RuvC) in a 20-µl
reaction mix containing 50 mM Tris-HCl, pH 7.5, 25 mM NaCl, 5 mM MgCl
, 1 mM dithiothreitol, and 50 µg/ml BSA. Electrophoretic analysis was
as described in the legend to Fig. 1.
In human cells, the XPG endonuclease makes the 3`-incision during nucleotide excision repair(19) . When XPG was compared with Rad1-Rad10, we observed that the bubble substrate was nicked with a polarity opposite to that seen in reactions with the Rad1-Rad10 complex (Fig. 1, lanes e and f). When the bubble substrate was incubated with both XPG and the Rad1-Rad10 protein complex (Fig. 1, lane d), incision products were observed that corresponded to cleavage at the 3`- and 5`-sides of the bubble.
The S. cerevisiae homologue of the human XPG protein is Rad2 (21) .
Consistent with this, a truncated form of Rad2 has been shown to cleave
model DNA structures with the same polarity as XPG(22) . The
dual incision reaction of NER in yeast is therefore likely to involve
Rad2 and the Rad1-Rad10 complex, with the corresponding functions
performed in humans by XPG and the ERCC1-ERCC4 protein complex (where
ERCC1 is the homologue of Rad10 (23) and ERCC4 is the homologue
of Rad1()). Although the exact mechanics of damage
recognition, DNA unwinding, and incision remain to be elucidated, the
results presented here strengthen the view that a partially unwound or
open complex around a DNA lesion is an intermediate in the NER process.
Genetic evidence indicates that, in addition to their role in NER, the Rad1 and Rad10 proteins are involved in a pathway of mitotic recombination(1, 4) , possibly by playing a role in the resolution of recombination intermediates (such as Holliday junctions). Recent studies reported that the Rad1 protein (1-5 µg/ml) could resolve synthetic Holliday junctions by endonucleolytic cleavage(15) . Since the structure of a Holliday intermediate is quite different from intermediates that occur during NER, we compared the activities of Rad1 and Rad1-Rad10 with RuvC protein, the E. coli Holliday junction resolvase(24, 25) .
Fig. 2(lane d)
shows that Rad1-Rad10 cut the bubble structure efficiently whereas RuvC
protein was inactive on this substrate (lane e). We also
compared the activities of Rad1-Rad10 and RuvC on a synthetic Holliday
junction X12(20) . Resolution by RuvC is known to occur by the
introduction of symmetrically related nicks at the site of the
crossover(20) . When the junction was 5`-P-labeled
in strand 2, we observed the nick in this strand by denaturing
polyacrylamide gel electrophoresis (Fig. 3, lane h).
However, using preparations of Rad1 produced in two different
laboratories, we were unable to detect any evidence of nicking when the
junction was incubated with 1.2-12.5 µg/ml purified Rad1
protein (Fig. 3, lanes b-e, and data not shown).
Incubation of the junction with the Rad1-Rad10 complex also failed to
yield cleavage products (Fig. 3, lane g). Experiments
carried out with a different junction (X0(20) ) also provided
negative results (data not shown). Furthermore, we could find no
evidence that Rad1 or Rad10 could bind to the synthetic Holliday
junctions, either individually or as a complex, as measured by
bandshift assays (data not shown).
Figure 3:
Rad1 and Rad10 fail to cleave synthetic
Holliday junctions. Synthetic Holliday junction X12 (0.5 ng),
5`-P-labeled in strand 2, was incubated with the indicated
amounts of Rad1, Rad10, or RuvC (lanes a-h). Reaction
conditions were as described in the legend to Fig. 2. Lane
i, control reaction in which the bubble substrate (0.5 ng;
5`-
P-labeled on oligo 2) was incubated with Rad1 and Rad10
to show that the proteins were active. Reaction products were analyzed
by denaturing 12% polyacrylamide gel electrophoresis and visualized by
autoradiography.
Taken together, these results show that under conditions in which the Rad1-Rad10 complex is highly active and cleaves 85% of the bubble structure (Fig. 3, lane i), it fails to nick the synthetic Holliday junction in a manner expected of a junction-specific resolvase. Though we cannot account for the ability of others to demonstrate Holliday junction resolution by Rad1(15) , it is known that synthetic Holliday junctions tend to ``breathe'' during incubation and may be prone to attack by contaminating single-strand endonucleases (26) .
If Holliday junctions do not serve as targets for Rad1-Rad10, what role do these proteins play in recombination? Studies of budding yeast indicate that there are two pathways of double-strand break-induced recombination(27) . One of these pathways (single-strand annealing or SSA) is utilized during recombination between repeat sequences (Fig. 4B), and deletions between repeats are greatly reduced in the absence of either the RAD1 or RAD10 gene products(9, 13, 14, 28, 29, 30) . However, the SSA pathway for recombination is unlikely to proceed via a Holliday junction intermediate. An important clue to the role of Rad1 in recombination was provided by Fishman-Lobell et al.(12) , who observed that rad1 mutants were defective in recombination between two direct repeat sequences when one of the repeats contained a heterologous region in which recombination was initiated by a double-strand break. It was suggested that RAD1 was required for an endonucleolytic activity that acts in the removal of non-homologous 3`-single-stranded ends at an early stage of recombination(12) . An identical defect in double-strand break-induced recombination is also present in rad10 mutants but not in mutants of other NER genes(29) . An absence of Rad1 or Rad10 would thus block SSA between direct repeats.
Figure 4: Rad1-Rad10 act upon similar structures during NER and recombination by SSA. A, model for NER, indicating recognition of the lesion by the excision repair complex leading to DNA unwinding. The unwound region (modeled in the experiments described here by a synthetic bubble structure) is recognized by the Rad1-Rad10 complex and Rad2, which introduce structure-specific incisions at the 5`- and 3`-sides, respectively. In mammalian cells, similar cuts would be made by ERCC1-ERCC4 complex and XPG, respectively. Following incision and removal of the damage-containing oligonucleotide, the gap would be filled by DNA resynthesis. B, model for the recombinational repair of double-strand breaks by SSA. Following the initiation of recombination by the introduction of a double-stranded break, the broken DNA ends are resected by the action of a 5`-3`-exonuclease. Complementary overlapping 3`-ends then anneal, and Rad1-Rad10 trims off the 3`-ended unpaired single strands. The DNA is then repaired by resynthesis and ligation, resulting in a deletion product in which sequences between two direct repeats have been lost.
Duplex/3`-single-strand DNA junctions that model those thought to be present during the SSA pathway (12) are known to be acted upon by the Rad1-Rad10 nuclease ( (8) and this work). The in vitro properties of Rad1-Rad10, indicative of a structure-specific nuclease, can therefore simultaneously account for a role in the cleavage of some recombination intermediates (Fig. 4B), although not Holliday junctions, as well as the specific cleavage of DNA nucleotide excision repair intermediates (Fig. 4A).