(Received for publication, August 3, 1995; and in revised form, October 11, 1995)
From the
Saccharomyces cerevisiae Rad2 protein functions in the
incision step of the nucleotide excision repair of DNA damaged by
ultraviolet light. Rad2 was previously shown to act endonucleolytically
on circular single-stranded M13 DNA and also to have a 5` 3`
exonuclease activity (Habraken, Y., Sung, P., Prakash, L., and Prakash,
S.(1993) Nature 366, 365-368; Habraken, Y., Sung, P.,
Prakash, L., and Prakash, S.(1994) J. Biol. Chem. 269,
31342-31345). Using two different branched DNA structures, pseudo
Y and flap, we have determined that Rad2 specifically cleaves the
5`-overhanging single strand in these DNAs. Rad2 nuclease is more
active on the flap structure than on the pseudo Y structure. Rad2 also
acts on a bubble structure that contains an unpaired region of 14
nucleotides, but with a lower efficiency than on the pseudo Y or flap
structure. The incision points occur at and around the single
strand-duplex junction in the three classes of DNA structures.
Nucleotide excision repair (NER) ()of UV-damaged DNA
in eukaryotes occurs by dual incision of the damaged DNA
strand(1) . Genetic and biochemical studies in the yeast Saccharomyces cerevisiae have indicated the requirement for a
large number of protein factors in NER(2, 3) .
Reconstitution of the incision step of NER requires the DNA damage
binding protein Rad14, the Rad4-Rad23 complex, the Rad1-Rad10 nuclease,
the Rad2 nuclease, the three-subunit replication protein A, and the
six-subunit pol II transcription factor TFIIH(3) . The
Rad1-Rad10 and Rad2 nucleases mediate dual incision of the damaged DNA
strand, releasing excision DNA fragments of 24-27 nucleotides in
length(3) .
The Rad2 nuclease is related to the Escherichia coli pol I 5` 3` exonuclease in that these
enzymes share homology in domains conserved in pol I and related
microbial nucleases. The E. coli 5`
3` nuclease
functions in the removal of RNA primers from the newly synthesized DNA
strand. The temperature-sensitive polA ex1 mutant of E.
coli is defective in the 5`
3` nuclease activity, and
joining of Okazaki fragments is retarded in this mutant(4) . In
addition to containing a 5`
3` exonuclease activity, the pol I
nuclease exhibits a structure-specific activity that cleaves the 5`-end
single-stranded DNA or RNA at its junction with the duplex
DNA(5) .
A mammalian 45-kDa 5` 3` exonuclease that is
related to E. coli pol I 5`
3` nuclease and that shares
homology with yeast Rad2 is required for lagging strand DNA synthesis
in reconstituted DNA replication
systems(6, 7, 8, 9, 10) .
Following the RNase H1-catalyzed cleavage of primer RNA one nucleotide
5` of the RNA-DNA junction, the 5`
3` exonuclease removes the
remaining monoribonucleotide of the RNA primer (9) . Like the
pol I nuclease, the mammalian enzyme also has a similar
structure-specific activity(11) . The RTH1 gene
encodes the S. cerevisiae counterpart of this mammalian 45-kDa
exonuclease(12) . Genetic studies with the rth1
mutant strain have indicated a role of RTH1 in DNA replication as
well as in DNA mismatch repair(12, 13) .
The
protein encoded by RTH1 and its mammalian counterpart contains
380 amino acids. Rad2, by contrast, is a much larger protein,
containing 1031 residues. The homology between yeast Rad2, RTH1, and
their mammalian counterparts is restricted to three regions (for
references, see (12) ). Moreover, whereas RTH1 and its
mammalian counterparts have a role in DNA replication and in mismatch
repair, Rad2 is required for NER, but has no apparent involvement in
DNA replication and mismatch
repair(2, 3, 12) . (
)Thus, Rad2
and RTH1 proteins have diverged functionally. Here, we examine the
action of Rad2 nuclease on various DNA structures.
Figure 1:
DNA substrates used. Asterisks indicate the position of the 5`-P label, and the
oligonucleotides that carry the label are underlined. The
conditions used for annealing the oligonucleotides and other
experimental details that pertain to the construction of the substrates
are described under ``Materials and
Methods.''
Figure 2: Rad2 nuclease specifically cleaves 5`-overhanging single-stranded tail in pseudo Y structure. Radiolabeled oligonucleotide A (lanes 1 and 2), pseudo Y-2 (lanes 3 and 4), and pseudo Y-1 (lanes 5-8) (75 fmol each) were incubated without (lanes 1, 3, and 5) or with Rad2 protein (10 ng in lane 6, 15 ng in lane 7, and 20 ng in lanes 2, 4, and 8) for 10 min at 30 °C. The reaction mixtures were run on a 11% polyacrylamide gel followed by autoradiography to reveal the radiolabeled DNA species. The numbers to the right of the autoradiogram mark the positions in nucleotides of the mixture of size standards used.
Figure 6: Summary of cleavage sites in DNA substrates. The numbers -1, 0, +1, and +2 indicate cleavage sites 1 base into the duplex region, at the single strand-duplex junction, 1 base into the single-stranded region, and 2 bases into the single-stranded region, respectively.
The cleavage reaction mediated by Rad2 protein is specific for the 5`-overhanging single-stranded tail in the pseudo Y structure because (i) no cleavage of oligonucleotide A used in the construction of pseudo Y-1 occurred at the same (Fig. 2, lane 2) and higher (data not shown) concentrations of Rad2 protein, and (ii) the 3`-overhanging single strand in a similar DNA structure (pseudo Y-2) that was obtained by hybridizing radiolabeled oligonucleotide E to nonlabeled oligonucleotide A (see Fig. 1and ``Materials and Methods'') was not cleaved by Rad2 (Fig. 2, lane 4).
Figure 3: Rad2 also acts on 5`-overhanging single-stranded tail in flap DNA. A, cleavage of the flap structure. Flap-1 (lanes 1-4) and Flap-2 (lanes 5 and 6) DNAs (75 fmol each) were incubated for 10 min at 30 °C without (lanes 1 and 5) and with 10 ng (lane 2), 15 ng (lane 3), and 20 ng (lanes 4 and 6) of Rad2 protein. The radiolabeled cleavage products were revealed by autoradiography after electrophoresis of the reaction mixtures. B, inhibition of flap cleavage by Rad2 antibodies. The Flap-1 substrate (75 fmol; lanes 1-5) was incubated with 20 ng of Rad2 protein for 10 min (lanes 2-5) in the presence of 1 µg of affinity-purified antibodies specific for Rad2 (lane 3), Rad1 (lane 4), or Rad10 (lane 5). The positions in nucleotides of the size markers are shown in lane M.
The Flap-1 and pseudo Y-1 cleaving activities are intrinsic to Rad2 protein because (i) the Rad2 protein used in this study is essentially homogeneous, and (ii) the cleavage of Flap-1 (Fig. 3B) and pseudo Y-1 (data not shown) was strongly inhibited by affinity-purified antibodies raised against Rad2 protein (compare lanes 3 and 2) expressed in and purified from E. coli(14) , but it was unaffected by antibodies specific for Rad1 and Rad10 proteins (lanes 4 and 5).
Figure 4:
Rad2 cleaves flap structures more
efficiently than pseudo Y. Pseudo Y-1 and Flap-1 DNAs (75 fmol each)
were incubated for 10 min at 30 °C with the indicated amounts of
Rad2 protein. The gel containing the radiolabeled DNA substrates and
cleavage products was analyzed in the PhosphorImager to obtain data for
a graphical representation of the results. , pseudo Y-1;
,
Flap-1.
The DNA structure-specific nuclease
activity of Rad2, as assayed using Flap-1 DNA as substrate, requires
Mg, which cannot be replaced by Ca
,
Co
Cu
, or Zn
,
although Mn
is partially effective (Table 1).
The flap cleavage activity is not affected by KCl concentrations up to
50 mM, but higher amounts of the salt result in significant
inhibition of the activity (data not shown), and the activity is
abolished by 0.1% SDS (Table 1).
Figure 5:
Cleavage of bubble DNA structure by Rad2.
Flap-1 (lanes 1 and 2), pseudo Y-1 (lanes 3 and 4), and bubble (lanes 5-8) DNAs (75
fmol each) were incubated for 10 min at 30 °C without (lanes
1, 3, and 5) or with 20 ng (lane 6), 30
ng (lanes 2, 4, and 7), and 40 ng (lane
8) of Rad2 protein. PhosphorImager analysis revealed 17% (lane
6), 27% (lane 7), and 33% (lane 8) cleavage of
the bubble DNA, 90% cleavage of the pseudo Y-1 (lane 4),
and >90% cleavage of the Flap-1 (lane 2) substrates. The
positions in nucleotides of the size markers used are shown in the M lanes.
Our work indicates that Rad2 cleaves flap and pseudo Y
structures and that it is more active in cleaving flap structures than
pseudo Y. In this regard, Rad2 resembles the mammalian FEN-1 and S.
cerevisiae RTH1 nucleases, which are also more efficient at
cleaving flap structures than pseudo Y(11) . A variety of
experiments have indicated that the E. coli pol I 5` 3`
exonuclease gains access to the cleavage site by moving from the free
5`-end of single-stranded DNA to the site of cleavage at the junction
with duplex DNA(5) . Biochemical studies of FEN-1 and calf
thymus 5`
3` exonuclease have indicated a similar requirement
for a free 5`-end for strand cleavage to occur. (
)In
agreement with these observations, we find no cleavage of bubble
structure by the S. cerevisiae RTH1 protein that we have
purified to near homogeneity (data not shown). Unlike RTH1 and FEN-1
nucleases, Rad2 cleaves the bubble structure, albeit with a lower
efficiency than the flap or pseudo Y structure. The differential
ability of Rad2 and RTH1 proteins to cleave bubble DNA may reflect the
affinity of these proteins for binding bubble DNA. Rad2 may possess a
domain that confers the ability to bind bubble DNA, and the inability
of RTH1 to cleave bubble DNA may arise from the absence of this domain.
The ability of FEN-1/RTH1 to cleave 5`-end single-stranded DNA at its junction with duplex DNA has led to the suggestion that Rad2 cleaves the damaged DNA strand on the 3`-side of the damage during NER(11) . While the manner of cleavage of model DNA substrates by Rad2 reported in our present work and by Rad1-Rad10 reported by Bardwell et al.(16) is congruent with the proposal that these proteins incise the damaged DNA strand on the 3`- and 5`-sides of the damage, respectively, direct evidence demonstrating this cleavage pattern in NER is as yet unavailable. Since neither the Rad2 nor Rad1-Rad10 nuclease has any affinity for damaged DNA(14, 17, 18) , they must be targeted to the damage site via interaction with the damage recognition factors. The interaction of human XPA with the ERCC1 protein(19, 20) would suggest that the Rad1-Rad10 nuclease is targeted to the damage site via interaction with the damage recognition protein Rad14. Interaction with the other components of the NER machinery may target Rad2 to the damage site. It remains to be determined whether the site of placement of the Rad1-Rad10 and Rad2 nucleases on the damaged DNA strand is coincident with the cleavage pattern of these enzymes on model DNA substrates. The recent reconstitution of the incision step of NER with purified components in yeast (3) should make it feasible to ascertain the manner of assembly and the site of cleavage by these nucleases.