Identification of nuclear proteins that interact with platinum-modified DNA by photoaffinity labeling

Article abstract of DOI:10.1021/ja049533o

Interactions between cellular proteins and cisplatin-modified DNA are important in determining the anticancer activity of the drug. To develop a general approach for identifying proteins that mediate cellular responses to cisplatin, photoreactive cisplati

Full text of DOI:10.1021/ja049533o

Published on Web 05/08/2004
Identification of Nuclear Proteins that Interact with Platinum-Modified DNA by
Photoaffinity Labeling
Christiana Xin Zhang, Pamela V. Chang, and Stephen J. Lippard*
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Received January 26, 2004; E-mail: lippard@lippard.mit.edu
Cisplatin is widely used in the clinical treatment of various
malignancies and has especially high potency against testicular
cancer. The therapeutic mechanism of cisplatin involves DNA
binding, with the formation of 1,2-intrastrand cross-links being the
major platinum-DNA reaction. Cisplatin binding to DNA causes
a dramatic distortion in the duplex structure, which serves as a
1
recognition signal for repair and other cellular processes. Much
effort has focused on isolating and identifying specific proteins that
mediate the cellular responses to cisplatin. By screening a human
cDNA expression library with cisplatin-modified DNA probes, our
laboratory identified a gene that encodes one such structure-specific
recognition protein, SSRP1.2,3 This protein contains a high-mobility
Figure 1. (A) Photoreactive cisplatin analogues with a tethered benzophe-
none moiety. (B) Oligonucleotide sequence used in the photocross-linking
studies. The platinated bases are shown in boldface font.
group (HMG) domain, a DNA-binding element that recognizes
distorted DNA structures including that afforded by Pt 1,2-in-
trastrand cross-links. This method, however, cannot identify proteins
that interact with cisplatin-modified DNA as a consequence of post-
translational modification or as part of a multiprotein complex, in
which the protein of interest does not directly bind to the damaged
DNA.
radiolabeled DNA probes were incubated with the HMGB1 proteins
at 0 °C for 30 min to promote the formation of noncovalent DNA-
protein binding. Following irradiation with 365 nm light, the
reaction mixture was analyzed by gel electrophoresis. The results
indicated the formation of the protein-DNA cross-linked adducts
(Figure S1). Under the same conditions, a cisplatin-modified DNA
probe did not afford any DNA-protein cross-links, consistent with
To probe more generally the binding of proteins to cisplatin-
DNA adducts, a series of platinum(II) complexes containing a
tethered, photoreactive aryl azide group were prepared, and their
ability to form protein photocross-links when bound to DNA was
investigated. During the study, DNA modified with cisplatin,
initially examined in a control experiment, exhibited higher
photocross-linking efficiency when irradiated at 302 nm than did
the complexes containing the aryl azide group.4 The mechanism
proposed for the formation of the protein cross-links to the cisplatin-
modified DNA involves photosubstitution of one platinum-
coordinated guanine base by an amino acid in the vicinity of the
platinum center. A serious limitation of this method is the
requirement of a coordinating and well-positioned protein side chain
in near contact with the platinum atom. In our continuing effort to
develop a more general photocross-linking approach to identify
cellular proteins that are involved in the cisplatin mechanism of
action, we describe here the synthesis of cisplatin analogues having
a tethered photoreactive benzophenone moiety (Figure 1A). Upon
irradiation at 365 nm, a wavelength that does not labilize Pt-
guanine bonds, the appended benzophenone group generates a
highly reactive species that binds irreversibly to cellular proteins
in the vicinity of the platinum-modified DNA probe. With the use
of this method, several nuclear proteins were isolated and identified.
A 25 base-pair oligonucleotide duplex containing a single d(GpG)
platination site and a biotin moiety at the 3′ end of the bottom
strand was modified with PtBPn complexes (Figure 1). The
4
previous work. Of the PtBPn complexes, DNA modified with
PtBP6 afforded the highest cross-linking efficiency. Thus, further
studies were conducted with the PtBP6-modified DNA.
Since the platinum coordination sphere in PtBPn is unsym-
metrical, two orientational isomers can form upon DNA binding,
in which the benzophenone is positioned toward either the 3′- or
8
5
′-end of the DNA top strand. In the case of PtBP6, the two isomers
,5
can be resolved by ion exchange HPLC (Figure S2). The orientation
of the benzophenone group relative to DNA in each isomer has
not been determined and is currently under investigation. The two
isomers exhibited different photocross-linking efficiencies, however,
both with the HMGB1 and with proteins in nuclear extracts (Figure
S3). Presumably, there is a more favorable orientation of the
benzophenone moiety relative to the DNA-binding proteins in the
isomer with higher photocross-linking efficiency.
The isomer with better photocross-linking ability was used to
isolate DNA damage-specific binding proteins from a HeLa nuclear
cell extract. Preincubation of the modified DNA probe with the
HeLa nuclear extract followed by irradiation at 365 nm for 2 h at
0
°C led to the formation of several DNA-protein cross-linked
products (Figure 2A). To identify the cross-linked proteins, a large-
scale photocross-linking reaction was carried out, and the DNA-
protein cross-links were isolated by immobilization onto streptavidin-
coated magnetic beads. The cross-linked adducts were then resolved
on an SDS-PAGE gel, and three of the proteins were identified by
peptide fingerprint mass spectrometric mapping and/or Western blot
analysis (Figure 2A,B). The identities of the other cross-linked
proteins are currently under investigation.
3′-biotin moiety was introduced to facilitate isolation of the DNA-
protein cross-linked adducts by immobilization on a streptavidin-
coated solid support. To test the photocross-linking ability of the
modified DNA probes, full-length HMGB1 and its HMG-domain
constituents were chosen because of their established binding
affinity for 1,2-intrastrand platinum-DNA cross-links.6 The
Among the cross-linked proteins are HMGB1 and HMGB2
(Figure 2), both anticipated because of their well-established binding
6
,7,9
ability to the 1,2-intrastrand platinum-DNA cross-links.
A
,7
protein with ∼120 kDa molecular mass was identified as poly-
6536
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J. AM. CHEM. SOC. 2004, 126, 6536-6537
10.1021/ja049533o CCC: $27.50 © 2004 American Chemical Society

C O MMU N I C A T I O N S
protein (Csb), an important protein in the transcription-coupled
repair (TCR) pathway. This observation suggests a role of PARP-1
in TCR. Both the NER and TCR pathways are implicated for
cellular repair of cisplatin-DNA damage.1 Our results providing
direct evidence that PARP-1 contacts cisplatin-DNA 1,2-intrastrand
cross-links in nuclear cell extracts are thus consistent with the
observations in the literature and indicate that poly(ADP-ribosyl)-
ation activity may be crucial for cellular repair of platinated DNA.
In conclusion, we have discovered a general approach to isolate
and identify cellular proteins that interact with platinum-modified
DNA based on photoaffinity labeling. With this method, several
nuclear proteins have been identified that are likely to be involved
in processing cisplatin-DNA adducts in cancer cells.
,16
Figure 2. Photocross-linking reactions of PtBP6-modified DNA with HeLa
nuclear extract. (A) 10% SDS-PAGE gel demonstrating the formation of
several DNA-protein cross-links. Lane 1: radiolabeled DNA probe alone
in BSA-containing buffer; lane 2: with HeLa nuclear extract. (B) Western
blot analysis showing that HMGB1, HMGB2, and PARP-1 are the cross-
linked proteins. Lane 1: photocross-linking reactions; lane 2: HeLa nuclear
extract as positive control. (C) The DNA-protein cross-links formed from
the photocross-linking reactions in the absence (lanes 1, 3) or in the presence
Acknowledgment. This work was supported by Grant CA34992
from the National Cancer Institute. C. X. Z. acknowledges the Anna
Fuller Foundation for a postdoctoral fellowship. We also thank the
MIT Biopolymers Laboratory and the Department of Chemistry
Instrumentation Facility for mass spectrometric analysis, Y. Jung
for providing HMG proteins, and D. Wang for preparing the nuclear
extract.
+
(lanes 2, 4) of NAD . Lanes 1 and 2: with HeLa nuclear extract; lanes 3
and 4: with purified PARP-1.
Supporting Information Available: A detailed experimental
section, a PAGE gel showing the formation of the DNA-HMGB1 cross-
links, an HPLC trace of the two orientational isomers of PtBP6-modified
(ADP-ribose)polymerase-1 (PARP-1) by peptide fingerprint mass
mapping method. The identity of PARP-1 was confirmed by
Western blot analysis (Figure 2B), as well as by a photocross-linking
reaction with purified PARP-1 protein. The latter led to the
formation of the same DNA-PARP-1 cross-link, as demonstrated
by SDS-PAGE (Figure 2C, lane 3). PARP-1 is an abundant nuclear
2
5AGGA, an SDS-PAGE gel showing different photocross-linking
efficiencies of the two orientational isomers, and the mass spectrum
of the DNA-PARP-1 cross-link (PDF). This material is available free
of charge via the Internet at http://pubs.acs.org.
protein which catalyzes the formation and transfer of poly-
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