J Biol Chem, Vol. 274, Issue 34, 23883-23886, August 20, 1999
The L1Tc, Long Interspersed Nucleotide Element from
Trypanosoma cruzi, Encodes a Protein with
3'-Phosphatase and 3'-Phosphodiesterase Enzymatic Activities*
Mónica
Olivares
§,
M. Carmen
Thomas¶,
Carlos
Alonso
, and
Manuel C.
López**
From the Departamento de Biología Molecular,
Instituto de Parasitología y Biomedicina "López
Neyra," Consejo Superior de Investigaciones Científicas,
Calle Ventanilla 11, 18001 Granada, Spain and the Centro de
Biología Molecular "Severo Ochoa," Consejo Superior de
Investigaciones Científicas-Universidad Autónoma,
Madrid, Spain 28049
 |
ABSTRACT |
The presence of a long interspersed nucleotide
element, named L1Tc, which is actively transcribed in the parasite
Trypanosoma cruzi, has been recently described. The open
reading frame 1 of this element encodes the NL1Tc protein, which has
apurinic/apyrimidinic endonuclease activity and is probably implicated
in the first stage of the transposition of the element. In the present
paper we show that NL1Tc effectively removes 3'-blocking groups
(3'-phosphate and 3'-phosphoglycolate) from damaged DNA substrates.
Thus, both 3'-phosphatase and 3'-phosphodiesterase activities are
present in NL1Tc. We propose that these enzymatic activities would
allow the 3'-blocking ends to function as targets for the insertion of
L1Tc element, in addition to the apurinic/apyrimidinic sites previously
described. The potential biological function of the NL1Tc protein has
also been evidenced by its ability to repair the DNA damage induced by
the methyl methanesulfonate alkylating or oxidative agents such as
hydrogen peroxide and t-butyl hydroperoxide in
Escherichia coli (xth and
xth, nfo) mutants.
| |
INTRODUCTION |
Long interspersed nucleotide elements
(LINE)1 are retrotransposons,
which contain open reading frames similar to those present in
retroviruses and long terminal repeat retrotransposons, that lack long
terminal repeats (1). These elements, originally described in mammalian
genomes, have been detected in a wide variety of species from protozoa
to fungi, plants, and animals (2). Evidence exists that these elements
are capable of transposition mediated by an RNA intermediate (3).
Sequence analysis of LINEs shows that they encode for the enzymes
involved in their own transposition. Several authors have suggested
that integration of LINEs takes place at DNA breaks already
existing in the chromosome produced by host-encoded products, probably
during DNA repair or recombination (3). However, the exact integration
site and the mechanisms of transposition of the LINEs remain unknown.
We have recently described a LINE, named L1Tc, which shares high
homology with the human L1 LINE (4), and is actively transcribed in the
parasite Trypanosoma cruzi (4, 5). This element encodes enzymes that are probably involved in their own transposition (4).
Interestingly, the ORF1 of L1Tc has significant homology in the
catalytic domains with the AP class II endonuclease family of DNA
repair enzymes. This homology seems to be a common general feature of
all nonsite-specific retrotransposon elements (4, 6). We have also
described the existence of an endonuclease activity specific for AP
sites, in the NL1Tc recombinant protein encoded by the ORF1 (7). The
potential biological role of the NL1Tc protein was shown by its ability
to complement lethal Escherichia coli BW286,
xth and dut-1 genotype, double mutant bacteria
lacking the coding gene for the exonuclease III enzyme (7).
In the context of the integration mechanisms postulated for the
nonsite-specific nonlong terminal repeat retrotransposons we proposed
that the AP endonuclease activity of the NL1Tc recombinant protein may
be connected with the formation of free 3'-OH ends into the DNA where
integration of these elements would occur (6, 7). Feng et
al. (8) have reported, on the other hand, that the protein encoded
by the ORF2 NH2 terminus of the human element L1Hs has
nuclease activity but shows no preference for AP sites. The high number
of potential AP sites that could be generated along the chromosomal DNA
by the NL1Tc protein can explain the high copy number and dispersion of
the L1Tc elements throughout the genome. We cannot, however, exclude
the existence of other mechanisms for the generation of potential
integration sites.
The AP endonuclease activity contributes to the repair of
apurinic/apyrimidinic sites and carries 3'-phosphodiesterase and 3'-phosphatase activities as well (9, 10). The 3'-phosphatase and
3'-phosphodiesterase activities have been described to contribute to
the repair of oxidative DNA damage (11). In the present paper we have
analyzed whether those enzymatic activities are present in the
recombinant protein NL1Tc. Thus, the existence of these activities in
NL1Tc can contribute to a better understanding of the mechanisms by
which these elements are integrated into the genome as well as their
putative role in DNA repair processes. We show that both 3'-phosphatase
and 3'-phosphodiesterase activities are associated with the
endonuclease NL1Tc encoded by the nonlong terminal repeat
retrotransposon L1Tc. The biological function of the NL1Tc protein was
examined by expression of the NL1Tc protein in E. coli null
mutants lacking both exonuclease III and endonuclease IV coding
sequences after treating them with alkylating and oxidative agents.
| |
EXPERIMENTAL PROCEDURES |
Bacterial Strains and Plasmids--
The bacterial strains BW9109
((xth-pncA)) and BW528 ((xth-pncA),
nfo-1::kan) were provided by Dr. B. Weiss
(University of Michigan, Ann Arbor). The plasmids used were pTrcHisA
(Stratagene), pHisNL1Tc (constructed by inserting the ORF1 of L1Tc into
the pTrcHisA vector (7)), and pSPFM55 containing a 5.0-kilobase cDNA insert called L1Tc (4).
Complementation Assays--
Bacteria (100 µl,
A600 = 0.4) transformed with pHisNL1Tc and
pTrcHisA vectors were incubated with varying concentrations of different DNA damaging agents (hydrogen peroxide
(H2O2), t-butyl hydroperoxide
(t-BuO2H), and methyl methanesulfonate (MMS)) at 37 °C for 15 min. 0.5 µCi of
[methyl-3H]dTMP were added and incubated at
37 °C for 15 min. Incorporation was terminated by adding 12.5%
trichloroacetic acid. After 15 min at 4 °C, samples were transferred
to a fiberglass filter and washed twice with 10% trichloroacetic acid.
Thymidine incorporation was measured in a liquid scintillation counter.
The expression level of the NL1Tc protein in transformed strains was
determined using SDS-polyacrylamide gel electrophoresis and Western
blot analysis (12).
3'-Phosphodiesterase Activity--
125 ng/ml relaxed circular
plasmid DNA (pSPFM55) was incubated for 10 min at 37 °C with 0.05 µM phleomycin in 25 mM sodium phosphate (pH
7.2), 10 mM NaCl, 5 mM MgCl2, and
10 µM iron ammonium sulfate. This treatment produces
ssDNA breaks with 3'-phosphoglycolate termini (13). The reaction was
terminated by the addition of a volume of phenol/chloroform (1:1) equal
to the volume of the reaction mixture. After extraction, it was ethanol
precipitated. The primer activation reaction was carried out by
incubation of the DNA substrate with 5 µM purified NL1Tc
for 30 min at 30 °C in 50 mM Tris-HCl (pH 7.5), 50 µg/ml bovine serum albumin, and 5 mM MgCl2,
followed by 10 min at 65 °C. Exonuclease III enzyme was added to the
positive control reaction. As a negative control no enzyme was added to
the reaction. Samples were cooled on ice. The nick translation reaction
was carried out in buffer (l50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 100 mM NaCl, 1 mM dithiothreitol) in the presence of 100 µM
each dATP, dTTP, and dGTP, 1 µM dCTP, 0.5 µCi/µl
[
-32P]dCTP, and 0.01 unit of Klenow polymerase. The
mixture was incubated for 30 min at 37 °C. The reaction was stopped
by adding 5 µl of 2 mg/ml bovine serum albumin, 2 µl of 10 mg/ml
herring sperm DNA, and 1 ml of ice-cold 10% trichloroacetic acid.
Samples were washed with 1 ml of 10% trichloroacetic acid. The
incorporated radioactivity present in the acid-insoluble fractions was
measured in a liquid scintillation counter.
3'-Phosphatase Activity--
125 ng/µl pSPFM55 DNA was
incubated for 30 min at 37 °C with 2 milliunits of micrococcal
nuclease in 150 mM Tris-HCl (pH 8.8), 1 mM
CaCl2, and 50 mM NaCl. The treatment produces
ssDNA breaks with phosphorylated 3'-termini (13, 14). The samples were extracted with phenol-chloroform once and then precipitated with ethanol. The conditions of the reactivation assay as well as the positive and negative control reactions were carried out as described above for the phleomycin-treated DNA substrate.
| |
RESULTS |
Complementation of the DNA Repair-deficient Strains BW9109 and
BW528--
To determine whether the NL1Tc protein can functionally
complement the repair deficiency in vivo generated by
oxidative and alkylating agents, pHisNL1Tc-transformed BW9109 and BW528
strains were made. As a negative control both strains were transformed with pTrcHisA. Expression analysis of the NL1Tc recombinant protein in
the transformed bacteria was checked by Western blot. High expression
of the NL1Tc recombinant protein was observed in both strains, even in
the absence of the IPTG-inducing agent (Fig. 1). The E. coli strain BW9109,
deficient in exonuclease III (xth), is described as a mutant
sensitive to the alkylating agent MMS and to oxidative agents, such as
H2O2, or to t-BuO2H (9,
15). The E. coli strain BW528, deficient in both exonuclease
III (xth) and endonuclease IV (nfo), is known to
be hypersensitive to the described oxidative and alkylating agents (9).
The strain XL1-blue was used as a positive control. The results
obtained show that NL1Tc expression in the mutant strains confers
significant resistance to the damage caused by the alkylating agent MMS
(Fig. 2A). MMS alkylates
mainly the N groups of purine-generating AP sites via spontaneous and
enzymatic hydrolysis of glycosydic bonds (16). In both mutant strains
transformed with pHisNL1Tc, the survival levels following treatment
with 10 mM MMS were very similar to the values observed for
the control strain XL1-blue. The resistance to MMS of both NL1Tc
transformed mutant strains did not require IPTG and was not enhanced by
IPTG induction (data not shown).
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Fig. 1.
Expression of NL1Tc in BW9109 and BW528
E. coli strains using pHisNL1Tc expression
constructs. Western blot of protein samples. The proteins were
separated on 10% SDS-polyacrylamide gels, transferred to
nitrocellulose membrane, and probed with anti-6-histidine antiserum.
Lane MW, the sizes of protein standards (in kDa); lane
1, E. coli strains without IPTG; and lane 2,
E. coli strains induced with 1 mM IPTG for
2 h. The arrow indicates the localization of the NL1Tc
protein.
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Fig. 2.
Sensitivity of mutant strains BW9109 and
BW528 to alkylating and oxidative agents carrying pTrcHisA or
pHisNL1Tc. Survival percentage of mutant strains to MMS
(A), H2O2 (B), and
t-butyl hydroperoxide (TBHP) (C). The
XL1-blue strain was used as a positive control ( ). BW9109
transformed with pTrcHisA ( ) and pHisNL1Tc ( ) is shown. BW528
transformed with pTrcHisA ( ) and pHisNL1Tc ( ) is shown. Values
shown are the average of three experiments.
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The possible DNA repair activity of NL1Tc against damage by oxidative
agents H2O2 and t-BuO2H,
which mainly cause ssDNA breaks with 3'-blocking groups, was also
determined by complementation assays in E. coli BW9109 and
BW528 mutant strains. The repair activity was measured by determining
the level of [3H]thymidine incorporation in the bacteria
transformed with pHisNL1Tc or pTrcHisA (negative control) after
treatment with H2O2 and
t-BuO2H, in a similar way to the complementation
assays mentioned above. The results obtained, shown in Fig. 2,
B and C, reveal that expression of NL1Tc in the
transformed bacteria produces a significant fall in the sensitivity to
both oxidative agents. At 1 mM H2O2
concentrations, an oxidative agent that induces 3'-phosphate as
terminal blocking groups, NL1Tc fully protects against the damage
obtaining survival levels similar to those of the control bacteria
XL1-blue (Fig. 2B). When the H2O2
concentration was increased, the complementation effect was more
clearly observed in the double mutant strain, which is considerably
more sensitive to the action of this oxidative agent. In the case of
bacteria treated with t-BuO2H, an agent that
mainly generates 3'-phosphoglycolate as terminal blocking groups,
expression of the NL1Tc protein effectively complements the absence of
the gene coding for exonuclease III in the BW9109 strain. The survival
indices were very similar to those of the wild-type strain. However, in
the double strain mutant BW528, NL1Tc only marginally complements the
repair activity (Fig. 2C).
The comparison of the DNA damage resistance in strains expressing NL1Tc
relative to strains not expressing NL1Tc are shown in Table
I. Expression of NL1Tc protein in BW9109
and BW528 increases the resistance to the DNA damage caused by the
alkylating agent MMS to 120 and 390%, respectively. The expression of
NL1Tc in the BW9091 mutant induces a survival increase of 290 and 120% in the BW258 double mutant strain exposed to 1 mM
H2O2. The increase was 79 and 18% in both
strains, respectively, after exposure to 3.5 mM
t-BuO2H. In summary, the NL1Tc protein repairs
the DNA damage caused by the MMS alkylating agent and the
H2O2 oxidative agent more efficiently than the
DNA damage caused by t-BuO2H.
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Table I
Comparison of the sensitivity to alkylating and oxidative agents of AP
mutant strains expressing NL1Tc against to the strains not expressing
NL1Tc
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3'-Phosphatase and 3'-Phosphodiesterase Activities on
NL1Tc--
To determine the presence of 3'-phosphatase and
3'-phosphodiesterase activities associated with the recombinant protein
NL1Tc, DNA samples treated with specific drugs that cause strand breaks with 3'-blocking lesions (3'-phosphate and 3'-phosphoglycolate) were
used as substrates. The ability of NL1Tc to eliminate the 3'-blocking
ends generated in the DNA template was observed indirectly by measuring
the stimulation of DNA synthesis following pretreatment of the
substrate with the recombinant protein. Hydrolysis carried out by the
repair enzyme generated 3'-OH ends suitable for being used as primers
by the Klenow fragment of DNA polymerase I, which was immediately added
to the medium. The enzymatic activity was determined by measuring dCTP
32P incorporation to the DNA template used as substrate. As
a positive control exogenous exonuclease III was added to a sample.
Micrococcal nuclease and phleomycin were used to create 3'-phosphate
and 3'-phosphoglycolate-blocked ssDNA breaks, respectively. In each
case, the conditions were those previously described (13, 14), which
favor ssDNA breaks over double-stranded DNA breaks producing substrates
having as average 1-4 nicks/molecule. The results obtained reveal that
NL1Tc effectively removes 3'-blocking groups (3'-phosphate and
3'-phosphoglycolate) from the damaged DNA substrates, as shown in Fig.
3, A and B. Thus,
similar to the exonuclease III enzyme, our data indicate that NL1Tc has
the potential to hydrolyze DNA when the substrate is
phosphate-blocked.
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Fig. 3.
Phosphatase and phosphodiesterase activity
assays. DNA treated with micrococcal nuclease (A) and
phleomycin (B). NL1Tc, reactivation assays in the
presence of NL1Tc at 2 µM concentration;
ExoIII, reactivation assays in the presence of 10 units of
exonuclease III; NC, reactivation assays in the absence of
the enzyme. 30 min of incubation (black bar) and at time
zero (white bar) are shown. The DNA synthesis was carried
out by Klenow polymerase in the presence of
[-32P]dCTP. Values shown are the average of three
experiments.
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| |
DISCUSSION |
In previous studies we reported that the amino acid sequences from
the ORF1 of L1Tc and the consensus sequence of the AP nuclease family
(17) show 30% identity, which extends to all nonsite-specific LINEs
described (4, 6). It was also shown that the recombinant protein
encoded by the ORF1 of the L1Tc LINE is capable of hydrolyzing a 37-mer
double-stranded DNA fragment containing an internal AP site and nicking
supercoiled plasmids containing apurinic/apyrimidinic sites (7). The
NH2-terminal end of the ORF2 of the human L1 element, which
has high sequence homology with the ORF1 of the T. cruzi
L1Tc element, has also nuclease activity but there is no evidence for
AP endonuclease activity (8). Recent studies have shown that L1
endonuclease is specific for the unusual DNA structural features found
at the TpA junction of the
5'-(dTn-dAn)·5'-(dTn-dAn) tracts (18). We believe that the endonuclease activity encoded by LINEs
might be involved in the integration mechanisms of these LINEs into the
host genome as it would be responsible for generating free 3'-OH sites
required as primers for integration (4, 7, 8). The present paper
reveals that the NL1Tc protein encoded by the ORF1 of the mobile LINE
L1Tc from T. cruzi has the ability to repair the DNA damage
induced by alkylating and oxidative agents in E. coli
(xth and xth, nfo) mutants. NL1Tc
expression in these repair-deficient cells provides resistance to both
alkylating (MMS-induced) and oxidative (H2O2-
and t-BuO2H-induced) DNA damage. Quantitative
analysis of the repair capacity of NL1Tc shows that NL1Tc expression in
BW9109 (xth) and BW528 (xth and nfo)
completely reversed the MMS sensitivity of the mutants. NL1Tc had a
moderate effect on sensitivity to H2O2 and only
a very modest effect on sensitivity to t-BuO2H.
It was interesting to observe that the endonuclease activity encoded in
a LINE of T. cruzi could substitute for the prokaryotic
enzyme of E. coli, demonstrating that NL1Tc is endowed with
potent AP endonuclease activity.
The AP endonuclease family is made up of a group of multifunctional
proteins with four principal nuclease functions, AP endonuclease, 3'-exonuclease, 3'-phosphodiesterase, and 3'-phosphatase. The most
distinctive feature of the members of this protein family is to have an
efficient AP endonuclease activity (9). Studies with
Drosophila Rrp1 mutants have established a strong
correspondence between sensitivity to one of these chemical compounds
(H2O2, t-BuO2H, or MMS)
and deficiency in one of the tested enzymatic functions
(3'-phosphatase, 3'-phosphodiesterase, or AP endonuclease). H2O2 sensitivity corresponds to a deficiency in
phosphatase activity, t-BuO2H sensitivity
corresponds to a deficiency in phosphodiesterase activity, and MMS
sensitivity corresponds to a deficiency in AP endonuclease activity (9,
19). The ability of NL1Tc to repair 3'-terminal damage in DNA has also
been demonstrated using two distinct activity assays similar to those
reported for AP repair enzymes (13, 14): a 3'-phosphodiesterase assay
that directly measures the removal of terminal phosphoglycolate and a
3'-phosphatase assay that directly measures the removal of terminal
phosphate. It has been demonstrated that 3'-phosphatase and
3'-phosphodiesterase activities are essential for the repair of the
oxidative damage that causes 3'-blocking ends (11). The results
obtained showed that NL1Tc efficiently repairs oxidative damage that
includes 3'-phosphatase-blocked termini but only a small amount of the damage that includes 3'-phosphoglycolate-blocked termini. These results
are consistent with those obtained in the complementation assays where
a significantly higher repair index was observed for
H2O2-induced damage than for
t-BuO2H-induced damage. The higher 3'-phosphatase activity relative to the 3'-phosphodiesterase activity detected in the NL1Tc protein together with the ability to repair H2O2-induced damage to a higher extent than to
repair t-BuO2H-induced damage in mutant bacteria
in repair enzymes cause the NL1Tc protein to be more similar to the
exonuclease III enzyme than to other endonucleases such as RrpI protein
from Drosophila or endonuclease IV from E. coli.
The reported phylogenetic analysis made by comparison of the conserved
domains of the AP proteins and those of LINEs showed that the L1 (L1hs,
L1ms, L1mm, L1m, and L1md), the cin4 and the Tad1-1 are closer in
evolution to the AP family proteins than to the rest of the LINEs (7).
Interestingly, the exonuclease III protein is clearly closer in
evolution to the LINEs than to endonuclease IV.
Given the potential involvement of the nucleases encoded by the LINEs
in their own integration mechanism (8) we propose that the
3'-phosphatase and 3'-phosphodiesterase enzymatic activities detected
in NL1Tc would allow the 3'-blocking ends to function as targets for
the insertion of L1Tc element, in addition to the AP sites previously
described (7). On the other hand, it should not be excluded that the
presence of the 3'-repair activities associated with NL1Tc could be
indicative of a possible repair role of the L1Tc element. In fact,
repair of double-stranded DNA breaks because of the insertion of the
Ty1 element from Saccharomyces cerevisiae in the presence of
functional reverse transcriptase (from human L1, yeast Ty1, or
Crithidia CRE1) (20, 21) has recently been described.
| |
ACKNOWLEDGEMENTS |
We are grateful to Dr. Bernard Weiss for
providing the BW528 and BW9109 strains.
| |
FOOTNOTES |
*
This work was supported by Grant PB96-0829 from
Dirección General de Ensenanta Superior e Investigación
Cien (Promocion General del Conocimiento), Spain.The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) X83098.
§
Supported by a Plan Andaluz de Investigación Predoctoral
Fellowship (Formación de Personal Investigador), Junta de
Andalucía.
¶
Supported by Fordo de Investigaciones Sanitarías 97/4207
Postdoctoral Fellowship, Instituto de Salud Carlos III.
**
To whom correspondence should be addressed. Dept. de
Biología Molecular, Inst. de Parasitología y
Biomedicina López Neyra, Consejo Superior de Investigaciones
Científicas, Ventanilla 11, 18001 Granada, Spain. Tel.:
34-958-203802; Fax: 34-958-203323; E-mail:
mclopez@ipb.csic.es.
| |
ABBREVIATIONS |
The abbreviations used are:
LINE, long
interspersed nucleotide element;
MMS, methyl methanesulfonate;
t-BuO2H, t-butyl hydroperoxide;
ORF, open reading frame;
AP, apurinic/apyrimidinic;
ssDNA, single-stranded
DNA;
IPTG, isopropyl-1-thio-
-D-galactopyranoside.
| |
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S. R. Heras, M. C. Lopez, J. L. Garcia-Perez, S. L. Martin, and M. C. Thomas
The L1Tc C-Terminal Domain from Trypanosoma cruzi Non-Long Terminal Repeat Retrotransposon Codes for a Protein That Bears Two C2H2 Zinc Finger Motifs and Is Endowed with Nucleic Acid Chaperone Activity
Mol. Cell. Biol.,
November 1, 2005;
25(21):
9209 - 9220.
[Abstract]
[Full Text]
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M. Olivares, J. L. Garcia-Perez, M. C. Thomas, S. R. Heras, and M. C. Lopez
The Non-LTR (Long Terminal Repeat) Retrotransposon L1Tc from Trypanosoma cruzi Codes for a Protein with RNase H Activity
J. Biol. Chem.,
July 26, 2002;
277(31):
28025 - 28030.
[Abstract]
[Full Text]
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Copyright © 1999 by the American Society for Biochemistry and Molecular Biology.
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