HU Protein of Escherichia coli Has a Role in the Repair of Closely Opposed Lesions in DNA*
Mitsumasa Hashimoto
,
Barry Imhoff,
Md Moshi Ali and
Yoke W. Kow
From the
Department of Radiation Oncology, Emory University, Atlanta, Georgia
30303
Received for publication, April 15, 2003
, and in revised form, May 12, 2003.
 |
ABSTRACT
|
---|
Closely opposed lesions form a unique class of DNA damage that is generated
by ionizing radiation. Improper repair of closely opposed lesions could lead
to the formation of double strand breaks that can result in increased
lethality and mutagenesis. In vitro processing of closely opposed
lesions was studied using double-stranded DNA containing a nick in close
proximity opposite to a dihydrouracil. In this study we showed that HU
protein, an Escherichia coli DNA-binding protein, has a role in the
repair of closely opposed lesions. The repair of dihydrouracil is initiated by
E. coli endonuclease III and processed via the base excision repair
pathway. HU protein was shown to inhibit the rate of removal of dihydrouracil
by endonuclease III only when the DNA substrate contained a nick in close
proximity opposite to the dihydrouracil. In contrast, HU protein did not
inhibit the subsequent steps of the base excision repair pathway, namely the
DNA synthesis and ligation reactions catalyzed by E. coli DNA
polymerase and E. coli DNA ligase, respectively. The nick-dependent
selective inhibition of endonuclease III activity by HU protein suggests that
HU could play a role in reducing the formation of double strand breaks in
E. coli.
 |
INTRODUCTION
|
---|
Ionizing radiation generates a wide spectrum of DNA damage, including
strand breaks, abasic
(AP)1 sites, base
damages, and cross-links
(12).
The energy of ionizing radiation is not dispersed uniformly in the absorbing
medium but as packets of energy along the tracks of the charge particles. Each
deposition of energy can generate multiple ionization events that can lead to
the generation of a unique class of lesion called clustered lesion
(38).
In addition, when ionizing radiation interacts directly with DNA, multiple DNA
ionizations can occur at the site of interaction that can also lead to the
formation of clustered lesion. Thus, both the direct and indirect action of
ionizing radiation on DNA can lead to the generation of clustered lesions
(38).
The nature of clustered lesions is complex. They can consist of a
combination of DNA breaks, AP sites, and base damages, either present on the
same or opposing strands of DNA
(47).
A double strand break can be considered as a clustered lesion consisting of
two single strand breaks located in close proximity on opposing strands of
DNA. Much effort has been directed to understanding the repair of ionizing
radiation-induced lesions such as strand breaks, AP sites, and base lesions;
however, the biological process involved in the repair of these lesions within
a clustered site is poorly understood. Strand breaks, AP sites, and base
damages are repaired predominantly via the base excision repair (BER) pathway
(9,
10). When AP sites and base
damages are processed via BER, intermediary strand breaks are generated. For
clustered lesions consisting of DNA damage located on opposing strands of DNA,
a double strand break can be generated if the excision of the second lesion is
initiated before the first lesion is completely repaired. The formation of a
double strand break can be more lethal and mutagenic than the original closely
opposed lesions. To reduce the formation of double strand breaks during
cellular processing of clustered lesions, it is likely that there are
mechanisms within cells that help to reduce the probability of initiating the
repair of a second lesion before the first lesion is fully repaired.
It is interesting to note that many of the DNA repair glycosylases that
initiate the repair of base lesions and AP sites are intrinsically inhibited
by nearby nicks. Escherichia coli endonuclease III and
formamidopyrimidine N-glycosylase activities are inhibited by nicks
that are in close proximity and opposite to the base lesion
(1114).
The observed inhibition could be due to the fact that a nick increases the
flexibility of DNA around the remaining lesion. The increased flexibility
could lead to a decrease in the affinity of these DNA glycosylases for lesions
that are located close to a nick. However, prolonged incubation of DNA
containing clustered damage with either endonuclease III or
formamidopyrimidine N-glycosylase can still lead to the generation of
double strand breaks. Because a nearby nick can only slow down the action of a
repair glycosylase, it is therefore likely that additional mechanisms are
necessary to further inhibit the action of DNA glycosylases so that generation
of double strand breaks can be reduced during the repair of clustered lesions.
We had show earlier that KU, a human double-stranded DNA-binding protein, can
bind to a nick opposite dihydrouracil (DHU), leading to a significant
inhibition of the human endonuclease III activity
(15). The increased inhibition
of human endonuclease III activity by KU protein will help to reduce the
possibility of generating double strand breaks during the repair of closely
opposed lesions. Furthermore, even if a double strand break is formed, KU can
bind to the ends of a newly generated double strand break and tether the two
ends together as a protein-DNA complex, preventing the possibility of
mis-joining (15).
In this study, we showed that the E. coli DNA-binding protein HU
also inhibits the activity of E. coli endonuclease III when a nick is
present in close proximity and opposite to a base damage. In contrast, HU
protein did not inhibit significantly the subsequent steps of the repair
process, namely DNA repair synthesis and DNA ligation. Thus HU could
potentially mediate the sequential repair of a clustered lesion consisting of
closely opposed DNA damage. These data further suggest that DNA-binding
proteins that have high affinity for nicks, such as poly (ADP-ribose) DNA
polymerase, could also mediate similar processes in the cells.
 |
EXPERIMENTAL PROCEDURES
|
---|
DNA SubstratesAll oligonucleotides were obtained from
Operon and purified by polyacrylamide gel (15%) electrophoresis as described
previously (16).
Oligonucleotides were 5'-end-labeled with [
-32P]ATP
(Amersham Biosciences) using T4 polynucleotide kinase, following the
instructions provided by the supplier. 32P-labeled oligonucleotides
containing DHU were annealed to the appropriate complementary strands at 1:1.5
ratios in a buffer containing 10 mM Tris-HCl, pH 7.5, 0.1
M NaCl, and 2 mM mercaptoethanol by heating the mixture
to 90 °C and cooling down slowly to room temperature. The oligonucleotide
duplexes in Sequence I were
used in this study (Q represents dihydrouracil). Duplex N contains a
nick and a DHU. The nick is two nucleotides 3' from DHU on the
complementary strand. In contrast, duplex L contains only a single DHU.
To determine the effect of distances between the nick and DHU on the
inhibition of endonuclease III activity by HU protein, the substrates were
prepared as in Sequence II.
Duplex DNA containing a 5' flap was constructed by hybridizing a
5' 32P-labeled 33-mer and an unlabeled 16-mer with the
complementary 30-mer. This will generate a duplex containing a
5'-labeled flap of 19 nucleotides long deriving from the unhybridized
5' sequences of the 33-mer. The sequence of the three oligonucleotides
for preparing the flap DNA is as follows: 33-mer,
5'-ATGTGGAAAATCTCTAGCAGGCTGCAGGTCGAC; 16-mer, 5'-CAGCAACGCAAGCTTG;
30-mer, 5'-GTCGACCTGCAGCCCAAGCTTGCGTTGCTG; and
Sequence III, where (Fp) =
flap, 5'-ATGTGGAAAATCTCTAGCA.
Enzymes and ProteinsE. coli endonuclease III was purified
from an overproducing E. coli strains employing MonoS, MonoQ and
phenyl-Sepharose column as described previously
(17). Endonuclease V was
purifed from an E. coli overproducing strain employing MonoS, MonoQ,
and phenyl-Sepharose (18). A
small amount of HU protein was initially obtained as a gift from Dr. Roger
McMaken (John Hopkins University), and was later purified from E.
coli strains overproducing HU
and HU
subunits following
published procedures (19). HU
protein is an 18-kDa heterodimeric protein consist of HU
and HU
subunits, each subunit has a molecular mass of 9 kDa
(19). Briefly, hupA
and hupB genes were PCR amplified from E. coli genomic DNA.
The hupA gene was PCR-amplified using primers
5'-CCCCCCCCATATGAACAAGACTCAACTGATTGATGTAATT and
5'-CCCCCCCCTCGAGCTTAACTGCGTCTTTCCAGTCCTTGCCA, which introduce
NdeI and XhoI restriction sites near the ends of the PCR
fragment. Similarly, the hupB gene was PCR-amplified using primers
5'-CCCCCCCCATATGAAATAAATCTCAATTGATCGACAAGATT and
5'-CCCCCCCCTCGAGGTTTACCGCGTCTTTCAGTGCTTTACCT. Each of the PCR products
were restricted with NdeI and XhoI, and ligated into
pET22b(+) that was previously restricted with NdeI and XhoI,
using T4 DNA ligase (16 °C for 16 h) and electroporated into E.
coli BL21(pLysS), producing ampicillin-resistant colonies. The constructs
thus generated (pHuA and pHuB) code for HU
and HU
modified to
contain six C-terminal histidines. BL21(pLysS) cells harboring either pHuA or
pHuB were grown to 0.7 OD, and overproduction of HU
and Hu
were
achieved by the addition of 0.5 mM of
isopropyl-1-thio-
-D-galactopyranoside and continued to grow
the cells for an additional 16 h at room temperature. HU
and Hu
proteins were then purified individually using nickel-nitrilotriacetic acid
columns, following instructions supplied by the manufacturer (Novagen). E.
coli DNA polymerase I and E. coli DNA ligase were purchased from USB
biochemicals.
Enzyme AssaysEndonuclease III activity was assayed in a
standard reaction mix (10 µl) containing 0.1 M KCl, 10
mM Tris-HCl, pH 7.5, 50 fmol of labeled DNA substrates, and 20 fmol
of endonuclease III. The reaction was performed at 37 °C for 10 min.
Nick translation catalyzed by E. coli DNA polymerase I was
performed in a buffer solution (10 µl) containing 67 mM
potassium phosphate, pH 8.0, 6.7 mM MgCl2, 1
mM EDTA, 50 mM NaCl, 33 µM dATP, 33
µM dCTP, 33 µM dGTP, and 33 µM dTTP.
Duplex N (see Sequence I) is
composed of a 56-mer containing a DHU hybridized to two complementary DNA, a
21-mer and a 35-mer. The 35-mer was 5'-end-labeled with 32P
and used as the primer for E. coli polymerase I-dependent nick
translation DNA synthesis. The rate of formation of a labeled full-length
product was used as an estimate for the E. coli polymerase I
activity.
DNA ligation was also performed with duplex N in a ligation buffer (10
µl) containing 30 mM Tris, pH 8.0, 4 mM
MgCl2, 50 mM NaCl, 1 mM dithiothreitol, 20
µM NAD+, and 5 µg of bovine serum albumin. In this
case, the 21-mer was 5'-end-labeled to provide the 5'-phosphoryl
group that is required for the ligation reaction. The rate of formation of a
full-length, 5'-end-labeled 56-mer was used for estimation for the DNA
ligase activity.
Endonuclease III, nick translation and DNA ligation reactions were stopped
with 5 µl of a stop buffer containing 90% formamide, 10 mM EDTA,
0,.1% xylene, and 0.1% bromphenol blue. After the addition of a stop buffer,
the reaction mixture was immediately heated at 90 °C for 10 min. A 5-µl
sample was loaded onto a 12.5% denaturing polyacrylamide gel and
electrophoresed at 2000 V for 1.5 h. The polyacrylamide gel was then dried and
the amount of products formed was estimated by using the STORM PhosphoImager
(Amersham Biosciences).
 |
RESULTS
|
---|
Effect of HU Protein on Endonuclease III ActivityHU protein
binds tightly to various DNA replication and recombination intermediate
structures
(2022).
In addition, it has a high affinity for DNA containing nicks and small gaps.
Castaing et al. (23)
showed that HU bound to DNA containing one base gap generated by DNA
glycosylases. These data suggest that HU might play a role in preventing the
untimely exonuclease digestion of DNA in cells by inhibiting the binding of
exonuclease III to nicks and small gaps. Kamashev and Rouviere-Yaniv
(20) showed that inhibition of
exonuclease III activity by HU protein was only observed with DNA containing
nicks. However, HU protein did not protect intact linear DNA from exonuclease
III digestion, even at very high HU protein concentrations
(20). Earlier, we showed that
the human KU70/80 complex inhibited the rate of removal of DHU by human
endonuclease III (15).
Inhibition was only observed when the DNA substrate contained a DHU that was
close to and opposite a nick. Because HU protein also binds tightly to DNA
containing a nick, it is thus expected that, like human KU protein, HU protein
might also inhibit the endonuclease III activity.
Fig. 1 shows the effect of
increasing HU protein concentrations on the endonuclease III activity. Two DNA
substrates were used in this experiment: duplex N, which contained a nick
opposite and in close proximity to a DHU, and duplex L, which contains only a
DHU. When duplex L was used as a substrate, increasing the amount of HU
protein had no effect on the activity of E. coli endonuclease III
(Fig. 1, panel A).
However, when duplex N was used as the substrate, increasing the amount of HU
protein led to a significant inhibition of the endonuclease III activity
(Fig. 1, panel B). At
400 nM of HU protein, greater than 75% inhibition of endonuclease
III activity was observed. This is comparable with the inhibition of E.
coli exonuclease III activity by HU; at 400 nM HU inhibited
50% of the exonuclease activity of exonuclease III on nicked plasmid DNA
(20).
To determine the effect that distances between the nick and DHU have on the
inhibitory effect of HU on endonuclease III activity, five additional
substrates were prepared with distances between the nick and DHU varied from
2, 4, 6, and 8 nucleotides lengths. Fig.
2 showed that HU exerted its maximum inhibitory effect when the
nick was 2 nucleotides from the base lesion DHU. At 400 nM of HU
protein, 50% of endonuclease III activity was inhibited when the nick was 2
nucleotides away, either 3' or 5' from DHU. When the nick was 4,
6, and 8 nucleotides away from DHU, inhibition of endonuclease III activity by
HU was observed to be about 35, 22, and 12%, respectively.

View larger version (18K):
[in this window]
[in a new window]
|
FIG. 2. Effect of distance between the nick and DHU on the inhibitory effect of
HU on endonuclease III activity. 20 fmols of duplex DNA containing nicks
that are 0, 2, 4, 6, and 8 nucleotides from DHU on the complementary strand
were incubated with 10 fmols of E. coli endonuclease III and 400
nM of HU in a standard endonuclease III buffer at 37 °C for 10
min. The amount of endonuclease III-induced nicks was measured by
electrophoresis on a 12.5% denaturing polyacrylamide gel. Under the reaction
condition used, endonuclease III activity on control DNA (CON) was
14.8 fmols of nick per 10 min (100%).
|
|
Effect of HU on DNA Polymerase I ActivityTo have sequential
repair of closely opposed lesions, it is necessary that repair synthesis
catalyzed by DNA polymerase I (Pol I) be carried out unimpeded on the DNA
strand containing the newly generated nick, whereas the cleavage activity of
endonuclease III on the opposing lesion is inhibited by HU protein. It is
therefore expected that concentrations of HU protein that inhibit endonuclease
III activity will have little effect on the activity of Pol I. The effect of
increasing concentrations of HU protein on Pol I activity was examined by
using duplex N with the 35-mer 5'-end-labeled with 32P. DNA
synthesis, or DNA polymerase activity was measured by the rate of extension of
the 5'-end-labeled 35-mer to the full-length 56-mer. Because E.
coli Pol I contains both 3'-5' and 5'-3'
exonuclease activity, the amount of Pol I used for this experiment was chosen
by determining the amount of Pol I that will give a good rate of DNA synthesis
but little degradation of labeled primer.
Fig. 3 shows a titration of the
amount of Pol I used in the competing reactions of primer extension and
degradation. It was found that under the reaction conditions used,
0.010.05 units of Pol I gave an optimum rate of primer extension with
little degradation of the primer (Fig.
3, lanes 68). However, higher concentrations of
Pol I led to substantial degradation of the labeled 35-mer
(Fig. 3, lanes
25). Based on these data, the effect of HU protein on the nick
translation was studied with 0.01 units of DNA Pol I.
Fig. 4 shows that increasing
concentrations of HU protein (up to 400 nM) had little inhibitory
effect on the Pol I nick translation-DNA synthesis activity
(Fig. 4, panel A, lanes
58). Using 0.01 units of Pol I, a 10 min reaction converted 45% of
the substrate to the expected 56-mer full-length product
(Fig. 4, panel B). The
extent of nick translation catalyzed by Pol I was little affected, even in the
presence of 400 nM of HU protein
(Fig. 4, panel B).
Recent studies showed that in the presence of poly (ADP-ribose) DNA
polymerase, the human polymerase
favors a strand displacement synthesis
(24). Because Pol I can also
carry out strand displacement DNA synthesis, it is thus of interest to find
out whether, in the presence of protein, Pol I can shift from nick translation
to strand displacement DNA synthesis. To do this, both the 35-mer and the
21-mer of duplex N were 5'-end-labeled with 32P.
Fig. 5 shows the time course of
the reaction with Pol I. At an early time interval (1 min after the addition
of Pol I), 400 nM of HU showed a slight inhibition on the rate of
formation of the full-length 56-mer (panels A and B, lane
1). In the presence of 400 nM HU, the 1 min reaction generated
predominantly primer extension products that are shorter than the full-length
product (panel B, lane 1). However, as the reaction progressed, most
of the extended products were extended to become full-length 56-mer (panel
B, lanes 29). However, the rate of degradation of the 5' DNA
strand (the labeled 21-mer) was not inhibited (the faster migrating species of
all lanes of panels A and B). These data suggest
that high concentration of HU only slightly slowed down the initial extension
of the 35-mer and appeared to have little effect on the progression of the
nick translation reaction. This was indicated by the rapid formation of the
full-length product and the extent of the degradation of the labeled 21-mer.
These data thus suggest that the presence of HU protein did not shift Pol I
from the nick translation mode of DNA synthesis to strand displacement mode of
DNA repair synthesis.

View larger version (62K):
[in this window]
[in a new window]
|
FIG. 3. Titration of E. coli DNA polymerase I activity. 20 fmols of
duplex N containing a 5'-labeled 35-mer was incubated in a standard nick
translation buffer with decreasing E. coli DNA Pol I concentration.
The nick translation reaction was performed at 37 °C for 10 min. At the
end of 10 min, the reaction mix was assayed with a denaturing 12.5%
polyacrylamide gel for the formation of full-length nick translation product.
Lane 1, control duplex N showing the 5'-labeled 35-mer
(primer). Lane 28, duplex N was incubated with 1 unit
(lane 2), 0.5 units (lane 3), 0.25 units (lane 4),
0.1 units (lane 5), 0.05 units (lane 6), 0.025 units
(lane 7), and 0.01 units (lane 8) of Pol I.
|
|

View larger version (18K):
[in this window]
[in a new window]
|
FIG. 4. Effect of HU on DNA polymerase I activity. Panel A, DNA
polymerase I activity was measured by its nick translation activity. 20 fmols
of duplex N containing a 5'-labeled 35-mer was incubated in a standard
nick translation buffer with 0.01 unit of E. coli DNA Pol I and an
increasing amount of HU protein. The nick translation reaction was performed
at 37 °C for 10 min. At the end of 10 min, the reaction mix was assayed
with a denaturing 12.5% polyacrylamide gel for the formation of full-length
nick translation product. Lanes 14, duplex N was incubated
with 0 nM (lane 1), 100 nM (lane 2),
200 nM (lane 3), and 400 nM (lane 4)
of HU protein. Lanes 58, duplex N was incubated with 0.01 unit
of Pol I and 0 nM (lane 5), 100 nM (lane
6), 200 nM (lane 7), and 400 nM (lane
8) of HU protein. Panel B, nick translation assays performed in
panel A were quantified using the Storm PhsophoImager (Amersham
Biosciences). Duplex N was incubated with increasing amounts of HU ( ) or
with 0.01 unit of Pol I and increasing amounts of HU ( ).
|
|

View larger version (67K):
[in this window]
[in a new window]
|
FIG. 5. Effect of HU on the 5'-3' exonuclease activity of DNA
polymerase I. 20 fmols of duplex N containing a 5'-labeled 35-mer
and 5'-end-labeled 21-mer was incubated in a standard nick translation
buffer with 0.01 unit of E. coli DNA Pol I and 400 nM of
HU protein. The nick translation was assayed by the formation of a full-length
56-mer and the 5'-3 exonuclease activity by the formation of labeled
nucleotide shorter than the 21-mer. Lane C, control duplex N
containing the 5'-labeled 35-mer and 21-mer. Panel A, nick
translation was performed in the absence of HU protein. Panel B, nick
translation was performed in the presence of 400 nM HU protein. For
both panels A and B, 1 min reaction (lane 1), 2 min
reaction (lane 2), 3 min reaction (lane 3), 4 min reaction
(lane 4), 5 min reaction (lane 5), 8 min reaction (lane
6), 10 min reaction (lane 7), 12 min reaction (lane 8),
and 15 min reaction (lane 9).
|
|
In addition to being a DNA polymerase, Pol I is also a flap endonuclease
that is capable of cleaving the 5' flap from a flap DNA structure
(25). Flap DNA structures are
thought to be formed on the DNA lagging strand during processing of Okazaki
fragment (26). HU was shown to
bind to flap DNA with high affinity
(22). It is thus of interest
to find out whether the flap endonuclease activity of Pol I is affected by HU
protein. 5'-Labeled flap DNA was prepared by hybridizing 5'
32P-labeled 33-mer and unlabeled 16-mer with the complementary
30-mer, generating a duplex DNA having a 5'-labeled flap of 19
nucleotides long. We have shown earlier that both the E. coli
endonuclease V and Pol I cleave the 5' flap at the second phosphodiester
bond 3' to the flap junction
(27), generating a
5'-end-labeled 20-mer. Fig.
6 showed that incubating the labeled 5' flap DNA with E.
coli endonuclease V generated predominantly a 20-mer and a small amount
of 19-mer, an observation that is consistent with the earlier findings (Ref.
26;
Fig. 6, lane 5).
Furthermore, increasing the amount of HU protein led to inhibition of the flap
endonuclease activity of endonuclease V
(Fig. 6, lanes
68). The inhibition of the flap endonuclease activity by HU is
most likely the result of HU binding to the flap junction because HU binds
tightly to a flap junction (Kd = 1.8
nM; Ref. 20),
Similarly, incubating the flap DNA with Pol I led to the generation of a
5'-end-labeled 20-mer (Fig.
6, lane 9), and the flap endonuclease activity of Pol was
also inhibited by HU protein (Fig.
6, lanes 1012). In contrast, the 5'-3'
exonuclease activity of Pol I was not affected by increasing the amount of HU
protein (Fig. 5).

View larger version (42K):
[in this window]
[in a new window]
|
FIG. 6. Effect of HU on the flap endonuclease activity of E. coli
endonuclease V and DNA polymerase I. Flap endonuclease activity of
endonuclease V and Pol I were assayed as described under "Experimental
Procedures." 20 fmols of flap DNA containing a 5'-end-labeled flap
was incubated with increasing amounts of HU (lane 1,0nM;
lane 2, 100 nM; lane 3, 200 nM;
lane 4, 400 nM), with 5 ng of E. coli
endonuclease V and increasing amounts of HU (lane 5, 0 nM;
lane 6, 100 nM; lane 7, 200 nM;
lane 8, 400 nM), and with 0.05 units of Pol I and
increasing amounts of protein HU (lane 9, 0 nM; lane
10, 100 nM; lane 11, 200 nM; lane
12, 400 nM).
|
|
Effect of HU Protein on DNA Ligase ActivityIt was shown
earlier that KU 70/80 protein complex increases the ligation efficiency of
human DNA ligase III (28). It
is also interesting to note that HU is essential for DNA ligase to effectively
ligate short DNA fragments into circles
(29). Therefore, we expect
that HU protein will have little or no effect on the rate of ligation
catalyzed by E. coli DNA ligase. To examine this, duplex N was used
as the ligation substrate, and the 21-mer was 5'-end-labeled with
32P. This will provide the 5' phosphate that is necessary for
the DNA ligase reaction. At the two DNA ligase concentrations (0.1 and 0.01
units), HU protein showed little or no inhibitory effect on the extent of
ligation (Fig. 7, panel A,
lanes 512). In the absence of HU, a 10 min reaction with 0.1 or
0.01 units of E. coli DNA ligase produced 74.6 and 6.5% of ligation,
respectively (Fig. 7,
panel B). HU protein showed little or no inhibitory effect on the
extent of ligation even at 400 nM of HU, a concentration that led
to greater than 75% inhibition of endonuclease III activity
(Fig. 7, panel
B).

View larger version (15K):
[in this window]
[in a new window]
|
FIG. 7. Effect of HU on E. coli DNA ligase
activity. Panel A, 20 fmols of duplex N containing a
5'-labeled 21-mer was incubated in a standard ligation without DNA
ligase (lanes 14), with 0.1 units of DNA ligase (lanes
58), and with 0.01 units of DNA ligase (lanes
912). DNA ligase activity was assayed by the formation of a
full-length 56-mer. Varying amounts of HU were added to each reaction mix: 0
nM (lanes 1, 5, and 9); 100 nM
(lanes 2, 6, and 10); 200 nM (lanes 3,
7, and 11); 400 nM (lanes 4, 8, and
12). Panel B, DNA ligase assays performed in panel
A were quantified using the Storm PhsophoImager (Amersham Biosciences).
Duplex N was incubated with increasing amounts of HU ( ), with 0.1 unit of
DNA ligase and increasing amounts of HU ( ), or with 0.01 units of DNA
ligase and increasing amounts of HU ( ).
|
|
 |
DISCUSSION
|
---|
After exposure to ionizing radiation, the individual damage in each
clustered lesion is expected to be repaired via the BER pathway. The
processing of DNA damage via the BER pathway generates single strand breaks or
small gaps as repair intermediates. These repair intermediates require DNA
polymerase and DNA ligase to complete the repair. Therefore, if a nick
generated from the initial repair of one of the lesion within the clustered
lesion site is not sealed before initiating the repair of additional lesions,
a DNA double strand break will be generated. The uncoordinated repair of
clustered lesions will lead to an increase in the cellular level of double
strand breaks.
In vitro experiments from this laboratory showed that the human
DNA-binding protein KU helps to reduce the formation of frank double strand
breaks by inhibiting the activity of DNA glycosylase (endonuclease III) in
removing the base lesion opposite to a nick
(15). Furthermore, KU has a
high affinity for DNA ends and will tether the two ends of a double strand
break, thus reducing the possibility of misjoining. It is interesting to note
that despite the fact that KU inhibits the incision activity of endonuclease
III on DNA containing a nick in close proximity opposite to a dihydrouracil
(15), KU has little or no
inhibitory activity on DNA ligation
(28). However, it is not known
whether the binding of KU to a nick will lead to any appreciable inhibition of
repair synthesis by human polymerase
or other polymerases such as Pol
and Pol µ that might be involved in the non-homologous rejoining
of double strand breaks
(3031).
To further understand the role of DNA nick-binding proteins on the repair
of closely opposed lesions, we decided to examine the role of the HU protein
of E. coli. HU is a basic DNA-binding protein that constitutes one of
the 12 capsid proteins of E. coli that bind to E. coli
genome (32). In addition to
its nonspecific double strand DNA-binding activity, HU protein has a high
affinity for various replicative and recombinational DNA structures
(2021).
HU also exhibits high affinity for DNA nicks and small gaps, and the
Kd for binding to nick and small gap is around
24 nM (20,
23). DNA nicks and small gaps
are intermediates generated during DNA repair via the base excision repair
pathway. In this study we showed that similar to KU, increasing concentrations
of HU led to an increased inhibition of endonuclease III activity on DHU.
Inhibition of endonuclease III activity was only observed when an opposing
nick was close to DHU. The nick-dependent inhibition exerted by HU on
endonuclease III activity thus suggests that HU plays a novel role in limiting
the ability of endonuclease III to produce additional nick within a clustered
lesion site before the intermediary nick is fully repaired. It is proposed
that the nick-dependent inhibition of DNA glycosylase activity (in this study,
endonuclease III activity) by HU will allow DNA polymerase I and DNA ligase to
fully repair the nick or gap before endonuclease III or other DNA glycosylases
begin to initiate the repair of additional lesions. These data therefore
suggest that in a cell, when the repair of one of the base damage within a
cluster lesion site is initiated, the generation of a one base gap or nick
should lead to substantial increase in the affinity of HU protein for this
damage site. Normally HU has a low affinity for double-stranded DNA
(Kd = 25,000 nM); however, the
affinity of HU for DNA containing a nick is four orders of magnitude higher
than a double-stranded DNA with a Kd around 2 to
8 nM (20,
23). HU is involved in several
other cellular processes including the formation of the nucleoid structure,
DNA replication, and recombination. HU is present in E. coli at high
abundance, and it was estimated that E. coli has
30,000
molecules of HU protein per cell
(33). In comparison, there are
only about 200 molecules of endonuclease III per
cell.2 In this study,
we showed that HU binding to a nick did not inhibit the activities of both Pol
I and DNA ligase. The binding of HU to a nick thus allows the Pol I and DNA
ligase to fully repair the nick while inhibiting the activity of endonuclease
III on other lesions that are located in close proximity
(Fig. 8). Once the DNA is
completely repaired, affinity of HU at the clustered site will decrease
substantially, thus leading to the rapid dissociation of HU from the DNA. The
dissociation of HU from the damaged site will allow endonuclease III to
initiate the repair of the remaining lesion. The sequential repair of closely
opposed lesions thus avoids the formation of a potentially lethal and
mutagenic double strand break intermediate
(Fig. 8).

View larger version (16K):
[in this window]
[in a new window]
|
FIG. 8. Proposed role of E. coli HU protein in the repair of closely
opposed lesions. Endo III, endonuclease III; Exo III,
exonuclease III.
|
|
We showed that in this study, significant inhibition of endonuclease III
activity was observed only when the nick was within two nucleotides from the
base lesion. When the distance of the nick was 4 and 6 nucleotides away,
inhibition of endonuclease III activity by HU decreased to about 35 and 14%,
respectively. Because the inhibition of endonuclease III activity by HU was
observed to be similar when the nick is two nucleotides, either 3' or
5' from DHU, it is therefore likely that the binding of HU to the nick
is symmetrical. These observations thus suggest that HU heterodimer probably
covers about five nucleotides footprint, which is consistent with its small
size of only 18 kDa. KU 70/80 complex was shown to have a footprint of about
25 nucleotides (34), and
considering the molecular mass of KU, which has a molecular mass of 150 kDa, a
footprint of 5 nucleotides for HU is thus reasonable. It has been estimated
that damage within a clustered lesion can have a spread of up to 20
nucleotides (7), and at
physiological pH, two breaks within a distance of 1520 nucleotides can
lead to double strand breaks. The relatively small footprint of HU when it
binds to a nick thus suggests that other proteins might be interacting with HU
to generate a larger footprint that can contribute more effective inhibition
of the glycosylase activity when the nick is further away from the opposing
base damage.
In human cells, double strand breaks are repaired predominantly by
nonhomologous end joining activity. In contrast, in bacteria such as E.
coli, a double strand break is repaired through homologous recombination.
Because bacteria are haploid; the ability to prevent the generation of
excessive double strand breaks is crucial for their survival. It is
interesting to point out that E. coli mutants lacking HU protein are
hyper-sensitive to both UV and ionizing radiation
(3637).
Because HU is required for homologous recombination, the increased sensitivity
of mutants to ionizing radiation could be due to a reduced recombination
activity. However, the increased radio-sensitivity could also be due to the
result of increased formation of double strand breaks generated from the
uncoordinated repair of clustered lesion. The lack of cellular HU protein will
lead to a significant increase in the uncoordinated repair of clustered
lesions. It is important to point out that E. coli triple mutant
cells (fpg nth nei) lacking the major DNA glycosylases, exhibit
increased survival toward ionizing radiation as compared with either the
single mutants or the wild type cells
(35). The increased survival
in the triple mutant could be due to a decrease in the ability of the mutant
cell to process base lesion within the clustered lesion, thus led to decrease
in formation of double strand breaks. However, the increased survival might be
accompanied by an increase in mutation frequency, possibly the result of
mutagenic bypass of the unrepaired base lesions. Because HU is involved in
many cellular processes and is also a constituent of the nucleoid capsid
complex, the amount of HU protein available for sequential repair might be
rate-limiting. At higher doses of ionizing radiation, significant amounts of
HU might be tied up with nicks generated by ionizing radiation, thus leading
to a significant reduction of HU that is available for sequential repair of
clustered lesions. It is therefore interesting to find out whether
overproduction of HU protein in E. coli wild type cells will lead to
increased resistance toward ionizing radiation. Furthermore, it is also
expected that the increased radioresistance will not be accompanied by a
significant increase in the overall mutation frequency.
 |
FOOTNOTES
|
---|
* This work was supported by National Institutes of Health Grant CA 90860.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact. 
Present address: Frontier Collaborative Research Center, Tokyo Institute of
Technology, Yokohama 226, 8503, Japan. 
To whom correspondence should be addressed: Division of Cancer Biology, Dept.
of Radiation Oncology, Emory University School of Medicine, 145 Edgewood Ave.,
Atlanta, GA 30303. Tel.: 404-616-6951; Fax: 404-616-5689; E-mail:
ykow{at}emory.edu.
1 The abbreviations used are: AP, abasic; BER, base excision repair; DHU,
dihydrouracil; Pol I, polymerase I. 
2 Y. W. Kow, unpublished data. 
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. Bernard Weiss for critical reading of the manuscript.
 |
REFERENCES
|
---|
- von Sonntag, C. (1987) The Chemical Basis
of Radiation Biology, Taylor and Francis, London
- Hall, E. J. (1994) Radiobiology for the
Radiobiologist, J. B. Lippincott Company,
Philadelphia
- Ward, J. F. (1988) Progr. Nucl. Acids Mol.
Biol. 35,
95125
- Sutherland, B. M., Bennett, P. V., Sidorkina, O., and Laval, J.
(2000) Biochemistry
39,
80268031[CrossRef][Medline]
[Order article via Infotrieve]
- Sutherland, B. M., Bennett, P. V., Sidorkina, O., and Laval, J.
(2000) Proc. Natl. Acad. Sci. U. S. A.
97,
103108[Abstract/Free Full Text]
- Chaudhry, M. A., and Weinfeld, M. (1995) J.
Mol. Biol. 249,
914922[CrossRef][Medline]
[Order article via Infotrieve]
- Ward, J. F. (1981) Radiat.
Res. 86,
185195[Medline]
[Order article via Infotrieve]
- Hall, E. J. (1993) Radiology for the
Radiologist, J. B. Lippincott Company, Philadelphia
- Freidberg, E. C., Walker, G. C., and Siede, W.
(1995) DNA Repair and
Mutagenesis, American Society of Microbiology Press, Washington,
D. C.
- Wilson. D. W., Engelward, B. P., and Samson, L. (1998)
in DNA Repair in Prokaryotes and Lower Eukaryotes
(Nickoloff, J. A., and Hoekstra, M. F., eds.) pp.
2964, Humana Press, Totowa, New
Jersey
- Harrison, L., Hatahet, Z., Purmal, A. A., and Wallace, S. S.
(1998) Nucleic Acids Res.
26,
932941[Abstract/Free Full Text]
- David-Cordonnier, M-H., Laval, J., and O'Neill, P.
(2000) J. Biol. Chem.
275,
1186511873[Abstract/Free Full Text]
- Takeshita, M., Chang, C. N., Johnson, F., Will, S., and Grollman,
A. P. (1987)) J. Biol. Chem.
262,
1017110179[Abstract/Free Full Text]
- Bourdat, A. G., Gasparutto, D., and Cadet, J. (1999)
Nucleic Acids Res. 27,
10161024
- Hashimoto, M., Donald, C. D., Yannone, S. M., Chen, D. J., Roy, R.,
and Kow, Y. W. (2001) J. Biol. Chem.
276,
1282712831[Abstract/Free Full Text]
- Yao, M., and Kow, Y. W. (1995) J. Biol.
Chem. 270,
2860928616[Abstract/Free Full Text]
- Asahara, H., Wistort, P. M., Bank, J. F., Bakerian, R. H., and
Cunningham, R. P. (1989) Biochemistry
10,
44444449
- Yao, M., and Kow, Y. W. (1997) J. Biol.
Chem. 272,
3077430779[Abstract/Free Full Text]
- Pellegrini, O., Oberb, J., Pinson, V., and Rouviere-Yaniv, J.
(2000) Biochimie (Paris)
82,
113[Medline]
[Order article via Infotrieve]
- Kamashev, D., and Rouviere-Yaniv, J. (2000)
EMBO J. 19,
65276535[Abstract/Free Full Text]
- Kamashev, D., Balandina, A., and Rouviere-Yaniv, J.
(1999) EMBO J.
18,
54345444[Abstract/Free Full Text]
- Bonnefoy, E., Takahashi, M., and Rouviere-Yaniv, J.
(1994)) J. Mol. Biol.
242,
116129[CrossRef][Medline]
[Order article via Infotrieve]
- Castaing, B., Zelwer, C., Laval, J., and Boiteux, S.
(1995) J. Biol. Chem.
270,
1029110296[Abstract/Free Full Text]
- Prasad, R., Lavrik, O. I., Kim, S. J., Kedar, P., Yang, X. P.,
Vande Berg, B. J., and Wilson, S. H. (2001) J. Biol.
Chem. 276,
24112414[Abstract/Free Full Text]
- Lyamichev, V. D., Brow, M. A., and Dahlberg, J. E.
(1993) Science
260,
778783[Medline]
[Order article via Infotrieve]
- MacNeill, S. A. (2001) Curr.
Biol. 11,
R842-R844[CrossRef][Medline]
[Order article via Infotrieve]
- Yao, M., and Kow, Y. W. (1996) J. Biol.
Chem. 271,
3067230676[Abstract/Free Full Text]
- Ramsden, D. A., and Gellert, M. (1998) EMBO
J. 17,
609614[Abstract/Free Full Text]
- Hodges-Garcia, Y., Hagerman, P. J., and Pettijohn, D. E.
(1989) J. Biol. Chem.
264,
1462114623[Abstract/Free Full Text]
- Ramadan, K., Mega, G., Shevelav, I. V., Villani, G., Blanco, L.,
and Hubschen, U. (2003) J. Mol. Biol.
328,
6372[CrossRef][Medline]
[Order article via Infotrieve]
- NickMcEhinny, S. A., and Ramsden, D. A. (2003)
Mol. Cell. Biol. 23,
23092315[Abstract/Free Full Text]
- Ishihama, A., and Azam, T. A. (1999) J.
Biol. Chem. 274,
3310533113[Abstract/Free Full Text]
- Rouviere-Yaniv, J. (1978) Cold Spring
Harbor Symp. Quant. Biol. 42,
439447[Medline]
[Order article via Infotrieve]
- de Vries, E., van Driel, W., Bergsma, W. G., Arnberg, A. C., and
van der Vliet, P. C. (1989) J. Mol. Biol.
208,
6578[Medline]
[Order article via Infotrieve]
- Jiang, D., Hatahet, Z., Blaisdell, J. O., Melamede, R. J., and
Wallace, S. S. (1997) J. Bacteriol.
197,
37733782
- Boubrik, F., and Rouviere-Yaniv, J. (1995)
Proc. Natl. Acad. Sci. (U. S. A.)
92,
39583962[Abstract/Free Full Text]
- Li, S., and Waters, R. (1998) J.
Bacteriol. 180,
37503756[Abstract/Free Full Text]
Copyright © 2003 by the American Society for Biochemistry and Molecular Biology.