We found that, in human whole cell extracts, end-damage-specific proteins apurinic⁄ apyrimidinic endo-nuclease 1 and polynucleotide kinase in the case of 3¢ ends containing phosphoglycola
Trang 1stability of X-ray cross-complementing protein 1 at the sites of DNA single-strand break repair
Jason L Parsons1, Irina I Dianova1, Emma Boswell1, Michael Weinfeld2and Grigory L Dianov1
1 MRC Radiation and Genome Stability Unit, Harwell, Oxon, UK
2 Cross Cancer Institute, University of Alberta, Edmonton, Alberta, Canada
DNA single-strand breaks (SSBs) arising as a result
of the disruption of phosphodiester bond linkage of
nucleotides in a polymer are dangerous, because, if left
unrepaired, they may block vital processes such as
DNA transcription and DNA replication SSBs arise
by several distinct mechanisms including direct energy
deposition by ionizing radiation, attack by reactive
oxygen species, during enzymatic processing of
endo-genous DNA lesions, and as a result of aberrant DNA
topoisomerase I activity (reviewed in [1,2]) Many of
the SSBs arise as a consequence of loss of the DNA
base and subsequent sugar fragmentation, which
should be considered as a single nucleotide gap with
modified 5¢ and ⁄ or 3¢ ends For example, SSBs
pro-duced by ionizing radiation or by attack from reactive
oxygen species often contain 3¢-phosphoglycolate or
3¢-phosphate groups [3] Similarly, some
radiation-induced SSBs, as well as those arising during base
excision repair (BER), contain a 5¢-sugar phosphate
residue [4–6] Thus, SSBs produced by several geno-toxic agents include a variety of termini that have to
be converted into conventional 3¢-OH ⁄ 5¢-phosphate nicks before the gap can be filled by a DNA polym-erase and the DNA ends resealed by a DNA ligase Several BER enzymes have been shown to possess
‘end cleaning’ activities The major mammalian endo-nuclease that processes abasic sites [apurinic⁄ apyri-midinic endonuclease 1 (APE1)] [7] was also shown
to be the major 3¢-phosphoglycolate activity in human cell extracts [8–11] Human polynucleotide kinase (PNK) is the major 3¢-phosphatase [12,13], and DNA polymerase b (Pol b) is the major activity in human cell extracts catalysing removal of 5¢-sugar phosphate residues [14] Although most of the enzymes involved
in end processing have been identified and this pro-cess plays a key role in maintaining genome stability, the precise mechanism governing recognition and processing of such a variety of SSBs is unclear To
Keywords
apurinic ⁄ apyrimidinic endonuclease 1; DNA
polymerase b; DNA repair; polynucleotide
kinase; X-ray cross-complementing protein 1
Correspondence
G L Dianov, Radiation and Genome
Stability Unit, Medical Research Council,
Harwell, Oxfordshire OX11 0RD, UK
Fax: +44 1235 841200
Tel.: +44 1235 841134
E-mail: g.dianov@har.mrc.ac.uk
(Received 6 July 2005, revised 2 September
2005, accepted 8 September 2005)
doi:10.1111/j.1742-4658.2005.04962.x
Ionizing radiation, oxidative stress and endogenous DNA-damage pro-cessing can result in a variety of single-strand breaks with modified 5¢ and⁄ or 3¢ ends These are thought to be one of the most persistent forms of DNA damage and may threaten cell survival This study addresses the mechanism involved in recognition and processing of DNA strand breaks containing modified 3¢ ends Using a DNA–protein cross-linking assay, we followed the proteins involved in the repair of oligonucleotide duplexes containing strand breaks with a phosphate or phosphoglycolate group at the 3¢ end We found that, in human whole cell extracts, end-damage-specific proteins (apurinic⁄ apyrimidinic endo-nuclease 1 and polynucleotide kinase in the case of 3¢ ends containing phosphoglycolate and phosphate, respectively) which recognize and pro-cess 3¢-end-modified DNA strand breaks are required for efficient recruitment of X-ray cross-complementing protein 1–DNA ligase IIIa heterodimer to the sites of DNA repair
Abbreviations
SSB, single-strand break; BER, base excision repair; APE1, apurinic ⁄ apyrimidinic endonuclease 1; PARP-1, poly(ADP)-ribose polymerase-1; PNK, polynucleotide kinase; Pol b, DNA polymerase b; XRCC1, X-ray cross-complementing protein 1; WCE, whole cell extract.
Trang 2address the mechanism involved in DNA SSB
recog-nition and repair, we used a formaldehyde
cross-linking assay to follow the proteins involved in
processing of 3¢-end-modified DNA SSBs in human
cell extracts
Results
Outline of cross-linking assay
We have recently developed a new DNA–protein
cross-linking protocol aimed at revealing the
engage-ment of BER proteins during repair of damaged DNA
[15] In brief, this protocol uses oligonucleotides
con-taining a 3¢-biotinylated end which are used to form a
damage-containing and a control duplex
oligonucleo-tide complete with a hairpin loop to prevent nuclease
digestion of the oligonucleotide during incubation with
cell extracts (Fig 1) The oligonucleotides are
subse-quently bound to streptavidin magnetic beads and
incubated with whole cell extracts (WCEs) in buffer
containing ATP, dNTPs and NAD+to allow repair to
proceed After incubation for the times indicated,
pro-teins involved in repair are cross-linked to the DNA
and to each other by the addition of formaldehyde
The beads are subsequently washed before reversal of
the cross-links, and released proteins are analysed by
gel electrophoresis and immunoblotting with the
cor-responding antibodies We used this assay to follow
repair protein cross-linking during incubation with
human WCE for three different oligonucleotide duplexes containing a one-nucleotide gap marked with 3¢-hydroxyl, 3¢-phosphate or 3¢-phosphoglycolate ends
in comparison with a control undamaged duplex (Fig 1)
XRCC1 is not essential for Pol b binding to a gap-containing DNA
Although it is obvious that DNA SSBs containing modified 3¢-end lesions will require specific proteins for
‘cleaning’ the ends and preparing them for DNA repair, the time and mechanism of entry of these pro-teins and other BER partners into the repair process is unknown This mechanism should include identifica-tion of these lesions as a strand break, verificaidentifica-tion of the nature of the 3¢ end, and identification of a satis-factory pathway for repair The XRCC1 component of the XRCC1–DNA ligase IIIa heterodimer is thought
to be a protein providing such a mechanism by acting
as a nick sensor and a docking platform for formation
of a multiprotein complex capable of repairing various strand breaks [16] Consequently, to fulfil these func-tions, the XRCC1–DNA ligase IIIa heterodimer should be the first protein to bind to a variety of strand breaks, and recruitment of other repair proteins should depend on it We tested this model in a direct experiment by analysing the interdependence of BER protein binding⁄ cross-linking to different DNA sub-strates
Fig 1 Structures of oligonucleotides used To construct 3¢-biotinylated hairpin substrates for cross-linking, oligonucleotides were designed to contain the complementary sequence with a TTTT hairpin loop and a 3¢-biotinylated moiety (designated *) Substrates (2–4) contain a single-nucleotide gap with a 3¢ end containing an hydroxyl (OH), phosphate (P) or phosphoglycolate (PG) residue, respectively.
Trang 3Two proteins are required to repair a
single-nucleo-tide gap containing substrate with a 3¢-hydroxyl end:
Pol b, which will add one nucleotide to the 3¢ end and
fill the gap, and XRCC1–DNA ligase IIIa
hetero-dimer, which will seal the DNA ends In agreement
with this, using HeLa WCE, we were able to cross-link
these proteins to the gap-containing substrate to a
greater extent than to the control substrate (Fig 2A),
indicating damage specificity of cross-linking When
5¢-labelled gap-containing oligonucleotide attached to the
beads was incubated with WCE, gap filling by Pol b
can be observed as early as 0.25–0.5 min, at a point
where substantial cross-linking of Pol b is observed,
and was nearly completed within 2 min (Fig 2B)
Ligation was mostly accomplished between 1 and
2 min, and the XRCC1–DNA ligase IIIa heterodimer
can be efficiently cross-linked to the substrate during
this period Approximately 80% of the repair of the substrate is achieved within 4 min at a point where the proteins are dissociating from the DNA Therefore, cross-linking of Pol b and the XRCC1–DNA ligase IIIa heterodimer correlates well with the kinetics of repair
We next immunodepleted the XRCC1–DNA ligase IIIa heterodimer from WCE using XRCC1 antibodies and tested whether immunodepletion will affect Pol b binding⁄ cross-linking to the gapped DNA As Pol b interacts with and partially coprecipitates XRCC1, immunodepleted extracts contained slightly less Pol b (20%; Fig 3A), and therefore slightly reduced (10%) cross-linking was observed (Fig 3B) However, we found that, when normalized to the protein amount in the cell extract, Pol b retained full ability to recognize and bind a single-nucleotide gap in the absence of XRCC1–DNA ligase IIIa (Fig 3C), suggesting that Pol b probably binds first, processes the gapped DNA
to the ligatable stage by filling the gap, and then recruits XRCC1–DNA ligase IIIa heterodimer to accomplish the repair To test this, we immunodepleted Pol b from WCE and monitored efficiency of XRCC1–DNA ligase IIIa binding⁄ cross-linking to the gap-containing substrate We found that, although the amount of XRCC1 remains the same in mock or Pol b-depleted cell extracts (Fig 3D), cross-linking of XRCC1 in the latter was reduced by approximately twofold (Fig 3E,F), indicating that Pol b is required for efficient XRCC1–DNA ligase IIIa binding to a one-nucleotide gap containing substrate As Pol b pro-cessing of a single-nucleotide gap containing the 3¢-hydroxyl end was required for efficient XRCC1– DNA ligase IIIa binding, we speculate that all dam-age-processing proteins bind to the SSB before XRCC1–DNA ligase IIIa
Processing of the modified 3¢ end is required for efficient XRCC1–DNA ligase IIIa binding
We further explored this model in which a damage-specific protein binds first, processes the lesion, and then recruits the end-joining machinery According
to this model, repair of the 3¢-phosphoglycolate end would involve sequential processing by APE1 (the major 3¢-phosphoglycolate activity in human cell extracts [11]), and Pol b before XRCC1–DNA ligase IIIa would seal the strand break Therefore, recruit-ment of the XRCC1–DNA ligase IIIa heterodimer should depend on APE1 To test this model we immu-nodepleted APE1 from HeLa WCE The amounts of XRCC1 and Pol b were unaffected by the immuno-depletion protocol (Fig 4A) Furthermore, removal of
A
B
Fig 2 Damage-specific cross-linking of proteins involved in the
repair of a 5¢-phosphate gap substrate and the correlation with
repair kinetics (A) A gap-containing (left panel) or the control (right
panel) biotinylated hairpin substrate was bound to magnetic
strept-avidin beads and incubated with 100 lg HeLa cell extract for the
times indicated After being cross-linked with formaldehyde, the
beads were washed, the cross-links were reversed, and proteins
were separated by SDS ⁄ PAGE (10% gel), transferred to
poly(viny-lidene difluoride) membranes and analysed by immunoblotting with
the indicated antibodies (B) Alternatively, a 5¢-labelled
gap-contain-ing substrate was used After incubation with cell extract, the
beads were washed, the DNA was stripped from the beads by
heating at 90 C for 3 min and separated on a 20% acrylamide gel
before exposure to storage phosphor screens at 4 C and analysis
by phosphorimaging.
Trang 4APE1 did not affect binding⁄ cross-linking of XRCC1
to the gapped DNA substrate where Pol b, but not
APE1 processing, is required (Fig 4B) However
bind-ing⁄ cross-linking of XRCC1 to the
3¢-phosphoglyco-late-containing substrate was approximately twofold
reduced in APE1-immunodepleted but not
mock-immunodepleted cell extracts (Fig 4C,D) To
demon-strate that deficient XRCC1 cross-linking was
exclusively due to the immunodepletion of APE1, we
complemented depleted cell extracts with purified
recombinant human APE1 which stimulated XRCC1
binding⁄ cross-linking (Fig 4E) Taken together these
experiments indicate that APE1 is required for efficient
XRCC1–DNA ligase IIIa heterodimer binding to the
3¢-phosphoglycolate-containing substrate
In a similar set of experiments, we immunodepleted PNK and investigated cross-linking of XRCC1 and PNK to a 3¢-phosphate-containing substrate Immuno-depletion of PNK notably reduced the 3¢-phosphatase activity of cell extracts (Fig 5A) and further demon-strates that PNK is the major 3¢-phosphate-processing activity in human cell extracts as previously described [13] Furthermore, immunodepletion of PNK did not affect the amounts of XRCC1 and Pol b in the extract (Fig 5B) Using the same extract, we found efficient cross-linking of XRCC1 to the 3¢-OH gapped substrate (Fig 5C) for which no processing by PNK but only processing by Pol b, which remains in the immuno-depleted extract, is required Using these extracts,
we were able to efficiently cross-link both XRCC1
E
F
Fig 3 XRCC1–DNA ligase IIIa heterodimer
is not essential for Pol b binding to a single-nucleotide gap-containing substrate Western blot analysis of WCE, mock immunodepleted (Mock) and XRCC1 (A) or Pol b (D) immuno-depleted cell extracts (ID) with XRCC1 and Pol b antibodies A gap-containing or the con-trol biotinylated hairpin substrate was bound
to magnetic streptavidin beads and incubated with 100 lg HeLa mock immunodepleted cell extract, XRCC1 or Pol b immunodepleted HeLa cell extract (B and E, respectively) for the times indicated and cross-linked with for-maldehyde The beads were subsequently washed, the cross-links were reversed, and proteins were separated by SDS ⁄ PAGE (10% gel), transferred to poly(vinylidene diflu-oride) membranes, and analysed by immuno-blotting with the indicated antibodies The total amounts of Pol b (C) or XRCC1 (F) cross-linked throughout the time course using the XRCC1 and Pol b immunodepleted HeLa cell extract, respectively, were quantified from three independent experiments by densito-metry.
Trang 5and PNK in mock-immunodepleted cell extracts, but
a threefold reduction in XRCC1 cross-linking was
observed using the PNK-depleted cell extracts
(Fig 5D,E), even though the two extracts contained
equal amounts of XRCC1 Finally, addition of purified
human PNK to the PNK-immunodepleted cell extracts
stimulated XRCC1 cross-linking (Fig 5F) These
experiments suggest that XRCC1–DNA ligase IIIa
het-erodimer would not efficiently bind to the
3¢-phos-phate-containing substrate before removal of the
phosphate group by PNK Although immunodepletion
of XRCC1–DNA ligase IIIa heterodimer reduced the
level of endogenous PNK (data not shown), when
PNK was brought to the mock immunodepletion level
by addition of the recombinant human protein, it was efficiently cross-linked to the 3¢-phosphate-containing substrate in XRCC1-depleted extracts (data not shown), indicating that, as in the case of Pol b, PNK binding is not dependent on XRCC1–DNA ligase IIIa heterodimer
Interaction between PNK and XRCC1 is required for effective XRCC1–DNA ligase IIIa recruitment
to the site of DNA damage PNK interacts with the phosphorylated form of XRCC1–DNA ligase IIIa through its FHA domain, and a mutation (R35A) in this domain disrupts this
A
B
C
Fig 4 APE1 is required for efficient XRCC1–DNA ligase IIIa binding to a 3¢-end phosphoglycolate-containing substrate Western blot analysis
of WCE, mock immunodepleted (Mock) and APE1-immunodepleted HeLa cell extract (ID) with the indicated antibodies (A) A 3¢-end hydro-xyl-containing substrate (B) or a 3¢-end phosphoglycolate-containing substrate (C and E) bound to magnetic streptavidin beads was incubated with 100 lg mock immunodepleted, APE1-immunodepleted or APE1-immunodepleted extract complemented with 30 ng APE1 (E, last two lanes) for the times indicated and cross-linked with formaldehyde The beads were subsequently washed, the cross-links were reversed, and proteins were separated by SDS⁄ PAGE (10% gel), transferred to poly(vinylidene difluoride) membranes, and analysed by immunoblotting with the indicated antibodies The total amounts of XRCC1 cross-linked throughout the time course using the 3¢-end phosphoglycolate-containing substrate were quantified from three independent experiments by densitometry (D).
Trang 6interaction [17] Using site-directed mutagenesis, we
generated the R35A PNK mutant and demonstrate that
it is deficient in its interaction with XRCC1 compared
with the wild-type protein (Fig 6A) However, the mutant retains full 3¢-phosphatase activity (Fig 6B) Using the cross-linking assay, we demonstrate that
A
B
D
C
Fig 5 PNK is required for efficient XRCC1–DNA ligase IIIa binding to a 3¢-end phosphate-containing substrate A 5¢-labelled oligonucleotide substrate containing a 3¢-phosphate gap was incubated with mock immunodepleted or PNK-immunodepleted HeLa cell extract (0–5 lg) for
20 min before the addition of formamide loading dye, and the DNA separated on a 6% acrylamide gel and analysed by phosphorimaging (A) Western blot analysis of WCE, mock immunodepleted (Mock) and PNK-immunodepleted cell extracts (ID) with the indicated antibodies (B).
A 3¢-end phosphate-containing substrate (D and F) or a 3¢-end hydroxyl-containing substrate (C) bound to magnetic streptavidin beads was incubated with 100 lg mock immunodepleted or PNK-immunodepleted HeLa cell extract or PNK-immunodepleted extract complemented with 20 ng PNK (F, last two lanes) for the times indicated and cross-linked with formaldehyde The beads were subsequently washed, the cross-links were reversed, and proteins were separated by SDS ⁄ PAGE (10% gel), transferred to poly(vinylidene difluoride) membranes, and analysed by immunoblotting with the indicated antibodies The total amounts of XRCC1 cross-linked throughout the time course using the 3¢-end phosphate-containing substrate were quantified from three independent experiments by densitometry (E).
Trang 7the complementation of PNK-depleted cell extracts
with wild-type PNK restores XRCC1–DNA ligase IIIa
heterodimer binding⁄ cross-linking (Fig 6C) However,
complementation with the R35A PNK mutant has very
little effect on XRCC1–DNA ligase IIIa heterodimer
binding⁄ cross-linking (Fig 6C), indicating that
inter-action between PNK and XRCC1 is important
for recruitment of XRCC1–DNA ligase IIIa
hetero-dimer
Discussion
Poly(ADP)-ribose polymerase-1 (PARP-1) has a very
high affinity for strand breaks and binds them before
any other repair proteins [18] Binding of PARP-1 to
the SSB stimulates formation of poly(ADP)-ribose
polymers and dissociation of PARP-1 from the DNA
[19] A recent study from our group showed that
PARP-1 is always involved in BER of DNA base
lesions and SSBs and is important for preventing deg-radation of excessive SSBs by cellular nucleases as previously proposed [20] Blocking of poly(ADP)-ribosylation and consequent dissociation of PARP-1 from SSB results in inhibition of SSB repair and repair foci formation [18,21] However, when there are suffi-cient amounts of repair enzymes present, they effi-ciently substitute PARP-1 from the nicked DNA [18] Therefore, when repair enzymes are in excess over the amount of DNA SSBs, neither the cross-linking effi-ciency of XRCC1–DNA ligase IIIa heterodimer and Pol b nor the rate of repair are affected in PARP-1-deficient cells [22,23], suggesting that in this situation PARP-1 is not essential for recruitment of other BER proteins That is why XRCC1–DNA ligase IIIa het-erodimer plays a central role in the current models for SSB repair As XRCC1–DNA ligase IIIa heterodimer interacts with APE1 [24], Pol b [25] and PNK [26] it was proposed that XRCC1 serves as a docking
plat-B
A
C
Fig 6 Interaction between PNK and XRCC1 is required for efficient XRCC1–DNA ligase IIIa binding to a 3¢-end phosphate-containing sub-strate (A) Pull-down of XRCC1 from WCE by His-tagged wild-type (WT) or R35A mutant PNK was performed as described in Experimental procedures (B) The activities of wild-type and R35A mutant PNK were analysed by incubation for 20 min with a 3¢-end phosphate-containing substrate followed by the addition of formamide loading dye The DNA was separated on a 6% acrylamide gel and analysed by phosphor-imaging (C) A 3¢-end phosphate-containing substrate bound to magnetic streptavidin beads was incubated with 100 lg
PNK-immunodeplet-ed HeLa cell extract or PNK-immunodepletPNK-immunodeplet-ed extract complementPNK-immunodeplet-ed with 20 ng wild-type or R35A mutant PNK for the times indicatPNK-immunodeplet-ed and cross-linked with formaldehyde The beads were subsequently washed, the cross-links were reversed, and proteins were separated by SDS ⁄ PAGE (10% gel), transferred to poly(vinylidene difluoride) membranes, and analysed by immunoblotting with the indicated antibodies.
Trang 8form to accommodate proteins required for repair of
SSBs [1] It was further suggested that XRCC1–DNA
ligase IIIa heterodimer nucleates assembly of the
‘multitask’ repair complex that would be able to repair
SSBs of any complexity [16], although some data
indi-cates that efficient recruitment⁄ stability of PNK or
Pol b at sites of SSB repair in cells would involve not
only interaction with XRCC1, but also the recognition
of the DNA substrate by PNK⁄ Pol b [26]
Using cell extracts immunodepleted of individual
BER proteins and monitoring binding⁄ cross-linking of
the remaining proteins to the substrate DNA,
contain-ing SSBs with various 3¢-end modifications, we
investi-gated which proteins initiate recognition and repair
of such lesions We found that, after PARP-1
bind-ing⁄ dissociation, repair of the SSBs is always followed
by a specific protein that is required to progress the
particular lesion to the next stage of repair Therefore,
Pol b initiates repair of single-nucleotide gaps, PNK is
required for the initiation of repair of
3¢-phosphate-containing SSBs, and APE1 initiates repair of
3¢-phos-phoglycolate-containing SSBs Removal of these
proteins by immunodepletion extensively decreased
binding⁄ cross-linking of XRCC1–DNA ligase IIIa
het-erodimer to the corresponding substrates However,
removal of Pol b had a less pronounced effect, because
to a certain extent XRCC1 is able, although less
effi-ciently than in the presence of Pol b, to bind gapped
DNA with 3¢-hydroxyl and 5¢-phosphate ends because
it resembles nicked DNA [27] We have also recently
demonstrated that Pol b stimulates binding⁄ cross-linking of XRCC1–DNA ligase IIIa heterodimer during the repair of incised AP sites that are intermediates of BER [28] Conversely, immunodepletion of XRCC1– DNA ligase IIIa heterodimer had little or no effect on the amount of binding⁄ cross-linking of the damage-specific proteins required for repair initiation Taken together, our data do not support the model in which XRCC1–DNA ligase IIIa heterodimer binding follows PARP-1 Alternatively, they support the idea that damage-specific proteins bind before XRCC1–DNA li-gase IIIa and direct repair via a pathway that will include only a subset of proteins required for specific SSB processing
On the basis of our findings, we propose a model in which any of the BER proteins mentioned in this study (APE1, PNK, Pol b and XRCC1–DNA ligase IIIa) are able to independently initiate the repair process by binding to the corresponding DNA lesion (3¢-phospho-glycolate, 3¢-phosphate, single-nucleotide gap and SSBs containing 3¢-hydroxyl and 5¢-phosphate groups, respectively; Fig 7) After initiation, repair may pro-ceed either by the ‘passing the baton’ mechanism by handing the substrate to the next enzyme required [29,30] or by nucleation of a transient damage-specific complex including a subset of enzymes that are required for repair of a particular lesion Our data do not provide strong evidence in favour of transient complexes (although we can see simultaneous cross-linking of several repair proteins on damaged DNA)
Fig 7 A model for mammalian DNA SSB repair Repair of frank strand breaks contain-ing 3¢-hydroxyl and 5¢-phosphate groups is accomplished by XRCC1–DNA ligase IIIa complex (pathway A) However, a single-nucleotide gap-containing strand break with 3¢-hydroxyl and 5¢-phosphate or 3¢-hydroxyl and 5¢-deoxyribose phosphate is recognized
by Pol b, which fills the gap, removes the 5¢-deoxyribose phosphate and recruits XRCC1–DNA ligase IIIa complex to seal the DNA ends (pathway B) Strand breaks con-taining modified 3¢ ends are recognized by the corresponding damage-specific protein (DSP) which converts the modified 3¢ ends into conventional 3¢-hydroxyl ends and fur-ther recruits Pol b and XRCC1–DNA ligase IIIa to accomplish repair Among the known DSPs are APE1 and PNK which recognize and process 3¢ ends containing phosphogly-colate and phosphate groups, respectively.
Trang 9However, the formation of nuclear foci by BER
pro-teins supports this model [31,32] It is quite clear that
the lifetime of transient repair complexes, and
there-fore their lifetime within DNA damage foci, depends
on multiple interactions between the proteins involved
XRCC1 protein, which is involved in multiple
interac-tions with other BER proteins, may play an essential
role in assembly and stability of such complexes
Therefore, as recently reported, disruption of the
inter-action of XRCC1 with PNK or Pol b leads to
defici-ency in accumulation of PNK and Pol b in nuclear
foci induced by hydrogen peroxide [17,32] and
increased sensitivity of deficient cells to DNA damage
[33] These data indicate that the mechanism involved
in SSB repair in living cells may be more sophisticated
than in cell extracts
In summary, our data support the idea that BER
enzymes participate in the repair of various SSBs
through participation in multiple repair pathways⁄
complexes involving distinct subsets of repair proteins
For example, strand breaks containing 3¢-phosphate
ends would be initiated by PNK, followed by Pol b and
XRCC1–DNA ligase IIIa heterodimer, or alternatively,
binding of PNK would initiate formation of a transient
DNA repair complex containing all three enzymes
required for repair of this substrate However, the
detailed molecular mechanism of repair events after
strand break binding by end-damage-specific protein is
unclear and requires further investigation
Experimental procedures
Materials
Synthetic oligodeoxyribonucleotides were purchased from
Eurogentec (Seraing, Belgium) and purified by
electrophor-esis on a 20% polyacrylamide gel [32P]ATP[cP] (3000 CiÆ
mmol)1) was purchased from PerkinElmer Life Sciences
(Boston, MA, USA) XRCC1 (ab144) and DNA ligase III
(ab587) antibodies were purchased from Abcam Ltd
(Cam-bridge, UK) Antibodies against rat Pol b, human APE1
and human PNK were raised in rabbit and affinity purified
as described [34] His-tagged PNK and R35A PNK
gener-ated by site-directed PCR mutagenesis were purified on a
nickel chelating resin (Novagen, Madison, WI, USA) as
recommended by the manufacturer
Cells
HeLa cell pellets were purchased from Paragon (Aspen, CO,
USA) WCEs were prepared by the method of Manley et al
[35] and dialysed overnight against buffer containing 25 mm
Hepes⁄ KOH, pH 7.9, 100 mm KCl, 12 mm MgCl2, 0.1 mm
EDTA, 17% glycerol and 2 mm dithiothreitol Extracts were divided into aliquots and stored at)80 C
Immunodepletion of WCEs and western blots Immunodepletion of WCEs was performed as described [36] and verified by SDS⁄ PAGE (10% gel) Proteins were trans-ferred to poly(vinylidene difluoride) membranes and immu-noblotted using the corresponding antibodies Blots were visualized using the ECL plus system (Amersham, Chalfont
St Giles, Bucks, UK)
Cross-linking assay The cross-linking assay with hairpin oligonucleotide sub-strates attached to streptavidin magnetic beads was per-formed as described [18] For direct comparison, proteins cross-linked from different substrates or extracts were ana-lysed on the same immunoblot
Repair assays Repair assays of hairpin oligonucleotide substrates attached
to streptavidin magnetic beads were performed as described previously [18]
Pull-down assay
To analyse the interactions of PNK and R35A PNK with XRCC1, the protocol described by Caldecott et al [37] was followed with modifications Briefly, 50 lg HeLa WCE was incubated with 1 lg His-tagged PNK⁄ R35A PNK in 40 lL buffer A (50 mm Tris⁄ HCl, pH 8, 0.1 m NaCl, 2% glycerol,
1 mm dithiothreitol, 25 mm imidazole, pH 7.5) for 30 min
at room temperature, and then 160 lL buffer A and a
25 lL bed volume of nitrilotriacetate⁄ agarose (Novagen) was added Reactions were incubated on ice with frequent gentle mixing, and after 20 min the nitrilotriacetate⁄ agarose was gently pelleted by brief centrifugation The supernatant was removed, and the nitrilotriacetate⁄ agarose beads washed with 3· 190 lL buffer A After the final wash, the beads were boiled in 60 lL SDS⁄ PAGE sample buffer [25 mm Tris⁄ HCl, pH 6.8, 2.5% (v ⁄ v) 2-mercaptoethanol, 1% (v⁄ v) SDS, 5% (v⁄ v) glycerol, 1 mm EDTA, 0.15 mgÆmL)1bromophenol blue], and 20 lL loaded on to
an SDS⁄ 10% polyacrylamide gel Proteins were transferred
to a poly(vinylidene difluoride) membrane and immuno-blotted with the corresponding antibodies
Acknowledgements
We thank Dr Keith Caldecott for critical reading of the manuscript and for communicating his data before publication
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