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through reduction of Ref-1 as thioredoxin did not
directly stimulate the DNA-binding activity. A subset
of AP-1 proteins copurified with Ref-1 through several
columns although Ref-1 does not bind DNA in associa-
tion with these proteins. The specificity of AP-1's inter-
action with DNA as determined by DNA footprinting
experiments was not altered by Ref-1; foot-printing
patterns obtained in the presence of DTT or Ref-1
were identical.
24
Based on the N-terminal sequence derived for Ref-1,
primers were designed to complement the deduced 5'
coding sequences.
24
Following mixed primer PCR
amplification, the DNA fragments generated were
used to probe a Jurkat cDNA library and isolate a puta-
tive cDNA clone encoding human Ref-1. A hexa-His
tagged version of the protein was expressed and puri-
fied under denaturing conditions, resolubilized, and
found to stimulate c-Fos/c-Jun DNA-binding activity
in a manner similar to that of the purified Ref-1 from
HeLa cells. Ref-1 is the major AP-1 redox factor in
nuclear extracts as demonstrated by immunodepletion.
In addition to stimulating the DNA-binding activity of
AP-1, Ref-1 was found to stimulate DNA-binding
activity of other transcription factors, including Myb,
CREB, and NF-
k
B. Further analysis of Ref-1 revealed
that it is in fact identical to human apurinic/apyrmi-
dinic endonuclease (HAP1 or APE1).
25,26
Thus, Ref-1
(APE1) is a bifunctional protein including both redox
activity and DNA repair activity.
columns were performed on a HeLa nuclear extract
monitoring stimulation of p53 DNA-binding activity.
27
However, in this case, p53 did not copurify with Ref-1
through chromatographic separations as AP-1 did, and
Ref-1's ability to stimulate the DNA-binding activity of
p53 is more complex, involving both redox regulation
of the DNA-binding domain and redox-independent
mechanisms involving the C-terminal regulatory
domain. While Ref-1 stimulated the DNA-binding
activity of p53 approximately 20-fold, DTT was found
to stimulate the DNA-binding activity approximately
50-fold. Addition of both DTT and Ref-1 resulted in
an approximately 10-fold higher stimulation than the
expected additive effect of the two. Using a C-termi-
nally truncated p53, Jayaraman et al.
27
found that
DTT but not Ref-1 stimulated its DNA-binding activity.
Thus, Ref-1's stimulatory activity results in part
through interaction with the C-terminal domain.
27
In
more recent work, the tetramerization domain of p53
has also been shown to interact with Ref-1 promoting
the formation of tetramers from dimers enhancing the
sequence-specific DNA-binding activity of p53 through
a redox-independent mechanism.
35
NF-
F Activity Is Inhibited by an Upstream
Factor
A third transcription factor whose DNA-binding
activity is regulated by APE1 in a redox-dependent
manner is NF-
k
B, whose protein family members
belong to the immunoglobulin-fold class of transcrip-
tion factors.
28
Genes regulated by NF-
k
B are involved
in the immune function and development.
36
Through
the use of sulfhydryl modifying reagents, the redox
state of NF-
k
B was first implicated in its ability to
bind DNA in 1991.
37
Similar to AP-1, NF-
k
Bcomprises
a family of proteins that form homo- and heterodimers
including p50, p65, p52, c-Rel, and Rel B, which all
include a conserved Rel homology domain;
38
the heter-
odimeric p50/p65 was the heterodimeric form first
described.
39
In contrast to the previous examples, the
role of APE1 in enhancing the DNA-binding activity
of NF-
k
Redox Regulation of the DNA-binding
Activity of p53
In later work, Ref-1 was identified as the redox factor
that stimulated the DNA-binding activity of the tumor
suppressor p53.
27
In response to stress such as DNA
damage or hypoxia, p53 acts at a transcriptional level
to induce cell cycle arrest or apoptosis in order to main-
tain the genetic stability of a cell. Like a number of
diverse transcription factors
28
such as N-FAT, NF-
k
B,
STAT, and NDT80, a meiosis-specific transcription factor
from S. cerevisiae,
29,30
p53 belongs to the immunoglobulin-
fold class of proteins, which lack sequence similarity
but are structurally related. DNA-binding is mediated
by connecting loops from within the Ig-fold and is
distinct for each transcription factor. In response to
DNA-damage, p53 functions as a sequence-specific
transcription factor activating expression of p21,
Waf1, Cip1, mdm2, Gadd45, cyclin G, Bax, IGFBp3, and
others.
31
Similar to AP-1, oxidized p53 does not bind
DNA, suggesting that it is under redox regulation.
32
e
34
A similar strategy to that employed by Xanthoudakis
et al.
24
was used to purify the nuclear redox factor
for p53. Chromatographic separations on phosphocel-
lulose P-11, heparin, Mono-S, and Superdex 75
B was elucidated through characterization
of an inhibitor of NF-
k
B's transcriptional activity.
The inhibitor, a synthetic quinone derivative, (2E)-3-[5-
(2,3-dimethoxy-6-methyl-1,4- benzoquninoyl)]2-nonyl-
2-propenoic acid (E3330), was originally identified
as an anti-inflammatory drug and an inhibitor of
NF-
k
B.
38,40
In earlier studies, redox regulation of NF-
k
B's DNA-binding activity was reported to specifically
depend on the redox state of Cys 62 of the p50 subunit.
41
In this study, the authors concluded that thioredoxin
stimulated the DNA-binding activity of p50/p65
through reduction of a disulfide bond involving Cys
62. NF-
k
B's transcriptional activity has also been
k
