Title: The L1Tc, Long Interspersed Nucleotide Element from Trypanosoma cruzi, Encodes a Protein with 3′-Phosphatase and 3′-Phosphodiesterase Enzymatic Activities
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. 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. long interspersed nucleotide element methyl methanesulfonate t-butyl hydroperoxide open reading frame apurinic/apyrimidinic single-stranded DNA isopropyl-1-thio-β-d-galactopyranoside 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 (1Gabriel A. Boeke J.D. Skalka A.M. Goff S.P. Reverse Transcriptase. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1993: 275-327Google Scholar). These elements, originally described in mammalian genomes, have been detected in a wide variety of species from protozoa to fungi, plants, and animals (2Hutchison III, C.A. Hardies S.C. Loeb D.D. Sehee W.R. Edgell M.H. Berg D.E. Howe M.M. Mobile DNA. American Society for Microbiology, Washington D. C.1989: 593-617Google Scholar). Evidence exists that these elements are capable of transposition mediated by an RNA intermediate (3Eickbush T.H. New Biol. 1992; 4: 430-440PubMed Google Scholar). 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 (3Eickbush T.H. New Biol. 1992; 4: 430-440PubMed Google Scholar). 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 (4Martín F. Marañón C. Olivares M. Alonso C. López M.C. J. Mol. Biol. 1995; 247: 49-59Crossref PubMed Scopus (124) Google Scholar), and is actively transcribed in the parasite Trypanosoma cruzi (4Martín F. Marañón C. Olivares M. Alonso C. López M.C. J. Mol. Biol. 1995; 247: 49-59Crossref PubMed Scopus (124) Google Scholar, 5Requena J.M. Martín F. Soto M. López M.C. Alonso C. Gene (Amst.). 1994; 146: 245-250Crossref PubMed Scopus (16) Google Scholar). This element encodes enzymes that are probably involved in their own transposition (4Martín F. Marañón C. Olivares M. Alonso C. López M.C. J. Mol. Biol. 1995; 247: 49-59Crossref PubMed Scopus (124) Google Scholar). 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 (4Martín F. Marañón C. Olivares M. Alonso C. López M.C. J. Mol. Biol. 1995; 247: 49-59Crossref PubMed Scopus (124) Google Scholar, 6Martín F. Olivares M. Alonso C. López M.C. Trends Biochem. Sci. 1996; 248: 283-285Google Scholar). We have also described the existence of an endonuclease activity specific for AP sites, in the NL1Tc recombinant protein encoded by the ORF1 (7Olivares M. Alonso C. López M.C. J. Biol. Chem. 1997; 272: 25224-25228Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The potential biological role of the NL1Tc protein was shown by its ability to complement lethal Escherichia coli BW286, Δxth and dut-1 genotype, double mutant bacteria lacking the coding gene for the exonuclease III enzyme (7Olivares M. Alonso C. López M.C. J. Biol. Chem. 1997; 272: 25224-25228Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar).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 (6Martín F. Olivares M. Alonso C. López M.C. Trends Biochem. Sci. 1996; 248: 283-285Google Scholar, 7Olivares M. Alonso C. López M.C. J. Biol. Chem. 1997; 272: 25224-25228Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Feng et al. (8Feng Q. Moran J.V. Kazazian Jr., H. Boeke J.D. Cell. 1996; 87: 905-916Abstract Full Text Full Text PDF PubMed Scopus (845) Google Scholar) 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 (9Demple B. Harrison L. Annu. Rev. Biochem. 1994; 63: 915-948Crossref PubMed Scopus (1286) Google Scholar, 10Barzilay G. Hickson I.D. Bioessays. 1995; 17: 713-719Crossref PubMed Scopus (196) Google Scholar). The 3′-phosphatase and 3′-phosphodiesterase activities have been described to contribute to the repair of oxidative DNA damage (11Demple B. Johnson A. Fung D. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7731-7735Crossref PubMed Scopus (199) Google Scholar). 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.DISCUSSIONIn previous studies we reported that the amino acid sequences from the ORF1 of L1Tc and the consensus sequence of the AP nuclease family (17Seki S. Hatsushika M. Watanabe K. Nagao K. Tsutsui K. Biochim. Biophys Acta. 1992; 1131: 287-299Crossref PubMed Scopus (112) Google Scholar) show 30% identity, which extends to all nonsite-specific LINEs described (4Martín F. Marañón C. Olivares M. Alonso C. López M.C. J. Mol. Biol. 1995; 247: 49-59Crossref PubMed Scopus (124) Google Scholar, 6Martín F. Olivares M. Alonso C. López M.C. Trends Biochem. Sci. 1996; 248: 283-285Google Scholar). 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 (7Olivares M. Alonso C. López M.C. J. Biol. Chem. 1997; 272: 25224-25228Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). 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 (8Feng Q. Moran J.V. Kazazian Jr., H. Boeke J.D. Cell. 1996; 87: 905-916Abstract Full Text Full Text PDF PubMed Scopus (845) Google Scholar). 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 (18Cost G.J. Boeke J.D. Biochemistry. 1998; 37: 18081-18093Crossref PubMed Scopus (188) Google Scholar). 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 (4Martín F. Marañón C. Olivares M. Alonso C. López M.C. J. Mol. Biol. 1995; 247: 49-59Crossref PubMed Scopus (124) Google Scholar, 7Olivares M. Alonso C. López M.C. J. Biol. Chem. 1997; 272: 25224-25228Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 8Feng Q. Moran J.V. Kazazian Jr., H. Boeke J.D. Cell. 1996; 87: 905-916Abstract Full Text Full Text PDF PubMed Scopus (845) Google Scholar). 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 (9Demple B. Harrison L. Annu. Rev. Biochem. 1994; 63: 915-948Crossref PubMed Scopus (1286) Google Scholar). 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 (9Demple B. Harrison L. Annu. Rev. Biochem. 1994; 63: 915-948Crossref PubMed Scopus (1286) Google Scholar,19Gu L. Huang S-M. Sander M. J. Biol. Chem. 1994; 269: 32685-32692Abstract Full Text PDF PubMed Google Scholar). 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 (13Sander M. Huang S-M. Biochemistry. 1995; 34: 1267-1274Crossref PubMed Scopus (23) Google Scholar, 14Izumi I. Ishizaki K. Ikenaga M. Yonei S. J. Bacteriol. 1992; 174: 7711-7716Crossref PubMed Google Scholar): 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 (11Demple B. Johnson A. Fung D. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7731-7735Crossref PubMed Scopus (199) Google Scholar). 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 (7Olivares M. Alonso C. López M.C. J. Biol. Chem. 1997; 272: 25224-25228Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). 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 (8Feng Q. Moran J.V. Kazazian Jr., H. Boeke J.D. Cell. 1996; 87: 905-916Abstract Full Text Full Text PDF PubMed Scopus (845) Google Scholar) 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 (7Olivares M. Alonso C. López M.C. J. Biol. Chem. 1997; 272: 25224-25228Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). 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) (20Teng S-C. Kim B. Gabriel A. Nature. 1996; 383: 641-644Crossref PubMed Scopus (202) Google Scholar, 21Moore J.K. Haber J.E. Nature. 1996; 383: 644-646Crossref PubMed Scopus (201) Google Scholar) has recently been described. 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 (1Gabriel A. Boeke J.D. Skalka A.M. Goff S.P. Reverse Transcriptase. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1993: 275-327Google Scholar). These elements, originally described in mammalian genomes, have been detected in a wide variety of species from protozoa to fungi, plants, and animals (2Hutchison III, C.A. Hardies S.C. Loeb D.D. Sehee W.R. Edgell M.H. Berg D.E. Howe M.M. Mobile DNA. American Society for Microbiology, Washington D. C.1989: 593-617Google Scholar). Evidence exists that these elements are capable of transposition mediated by an RNA intermediate (3Eickbush T.H. New Biol. 1992; 4: 430-440PubMed Google Scholar). 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 (3Eickbush T.H. New Biol. 1992; 4: 430-440PubMed Google Scholar). 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 (4Martín F. Marañón C. Olivares M. Alonso C. López M.C. J. Mol. Biol. 1995; 247: 49-59Crossref PubMed Scopus (124) Google Scholar), and is actively transcribed in the parasite Trypanosoma cruzi (4Martín F. Marañón C. Olivares M. Alonso C. López M.C. J. Mol. Biol. 1995; 247: 49-59Crossref PubMed Scopus (124) Google Scholar, 5Requena J.M. Martín F. Soto M. López M.C. Alonso C. Gene (Amst.). 1994; 146: 245-250Crossref PubMed Scopus (16) Google Scholar). This element encodes enzymes that are probably involved in their own transposition (4Martín F. Marañón C. Olivares M. Alonso C. López M.C. J. Mol. Biol. 1995; 247: 49-59Crossref PubMed Scopus (124) Google Scholar). 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 (4Martín F. Marañón C. Olivares M. Alonso C. López M.C. J. Mol. Biol. 1995; 247: 49-59Crossref PubMed Scopus (124) Google Scholar, 6Martín F. Olivares M. Alonso C. López M.C. Trends Biochem. Sci. 1996; 248: 283-285Google Scholar). We have also described the existence of an endonuclease activity specific for AP sites, in the NL1Tc recombinant protein encoded by the ORF1 (7Olivares M. Alonso C. López M.C. J. Biol. Chem. 1997; 272: 25224-25228Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The potential biological role of the NL1Tc protein was shown by its ability to complement lethal Escherichia coli BW286, Δxth and dut-1 genotype, double mutant bacteria lacking the coding gene for the exonuclease III enzyme (7Olivares M. Alonso C. López M.C. J. Biol. Chem. 1997; 272: 25224-25228Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). 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 (6Martín F. Olivares M. Alonso C. López M.C. Trends Biochem. Sci. 1996; 248: 283-285Google Scholar, 7Olivares M. Alonso C. López M.C. J. Biol. Chem. 1997; 272: 25224-25228Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Feng et al. (8Feng Q. Moran J.V. Kazazian Jr., H. Boeke J.D. Cell. 1996; 87: 905-916Abstract Full Text Full Text PDF PubMed Scopus (845) Google Scholar) 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 (9Demple B. Harrison L. Annu. Rev. Biochem. 1994; 63: 915-948Crossref PubMed Scopus (1286) Google Scholar, 10Barzilay G. Hickson I.D. Bioessays. 1995; 17: 713-719Crossref PubMed Scopus (196) Google Scholar). The 3′-phosphatase and 3′-phosphodiesterase activities have been described to contribute to the repair of oxidative DNA damage (11Demple B. Johnson A. Fung D. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7731-7735Crossref PubMed Scopus (199) Google Scholar). 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. DISCUSSIONIn previous studies we reported that the amino acid sequences from the ORF1 of L1Tc and the consensus sequence of the AP nuclease family (17Seki S. Hatsushika M. Watanabe K. Nagao K. Tsutsui K. Biochim. Biophys Acta. 1992; 1131: 287-299Crossref PubMed Scopus (112) Google Scholar) show 30% identity, which extends to all nonsite-specific LINEs described (4Martín F. Marañón C. Olivares M. Alonso C. López M.C. J. Mol. Biol. 1995; 247: 49-59Crossref PubMed Scopus (124) Google Scholar, 6Martín F. Olivares M. Alonso C. López M.C. Trends Biochem. Sci. 1996; 248: 283-285Google Scholar). 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 (7Olivares M. Alonso C. López M.C. J. Biol. Chem. 1997; 272: 25224-25228Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). 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 (8Feng Q. Moran J.V. Kazazian Jr., H. Boeke J.D. Cell. 1996; 87: 905-916Abstract Full Text Full Text PDF PubMed Scopus (845) Google Scholar). 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 (18Cost G.J. Boeke J.D. Biochemistry. 1998; 37: 18081-18093Crossref PubMed Scopus (188) Google Scholar). 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 (4Martín F. Marañón C. Olivares M. Alonso C. López M.C. J. Mol. Biol. 1995; 247: 49-59Crossref PubMed Scopus (124) Google Scholar, 7Olivares M. Alonso C. López M.C. J. Biol. Chem. 1997; 272: 25224-25228Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 8Feng Q. Moran J.V. Kazazian Jr., H. Boeke J.D. Cell. 1996; 87: 905-916Abstract Full Text Full Text PDF PubMed Scopus (845) Google Scholar). 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 (9Demple B. Harrison L. Annu. Rev. Biochem. 1994; 63: 915-948Crossref PubMed Scopus (1286) Google Scholar). 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 (9Demple B. Harrison L. Annu. Rev. Biochem. 1994; 63: 915-948Crossref PubMed Scopus (1286) Google Scholar,19Gu L. Huang S-M. Sander M. J. Biol. Chem. 1994; 269: 32685-32692Abstract Full Text PDF PubMed Google Scholar). 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 (13Sander M. Huang S-M. Biochemistry. 1995; 34: 1267-1274Crossref PubMed Scopus (23) Google Scholar, 14Izumi I. Ishizaki K. Ikenaga M. Yonei S. J. Bacteriol. 1992; 174: 7711-7716Crossref PubMed Google Scholar): 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 (11Demple B. Johnson A. Fung D. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7731-7735Crossref PubMed Scopus (199) Google Scholar). 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 (7Olivares M. Alonso C. López M.C. J. Biol. Chem. 1997; 272: 25224-25228Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). 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 (8Feng Q. Moran J.V. Kazazian Jr., H. Boeke J.D. Cell. 1996; 87: 905-916Abstract Full Text Full Text PDF PubMed Scopus (845) Google Scholar) 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 (7Olivares M. Alonso C. López M.C. J. Biol. Chem. 1997; 272: 25224-25228Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). 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) (20Teng S-C. Kim B. Gabriel A. Nature. 1996; 383: 641-644Crossref PubMed Scopus (202) Google Scholar, 21Moore J.K. Haber J.E. Nature. 1996; 383: 644-646Crossref PubMed Scopus (201) Google Scholar) has recently been described. In previous studies we reported that the amino acid sequences from the ORF1 of L1Tc and the consensus sequence of the AP nuclease family (17Seki S. Hatsushika M. Watanabe K. Nagao K. Tsutsui K. Biochim. Biophys Acta. 1992; 1131: 287-299Crossref PubMed Scopus (112) Google Scholar) show 30% identity, which extends to all nonsite-specific LINEs described (4Martín F. Marañón C. Olivares M. Alonso C. López M.C. J. Mol. Biol. 1995; 247: 49-59Crossref PubMed Scopus (124) Google Scholar, 6Martín F. Olivares M. Alonso C. López M.C. Trends Biochem. Sci. 1996; 248: 283-285Google Scholar). 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 (7Olivares M. Alonso C. López M.C. J. Biol. Chem. 1997; 272: 25224-25228Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). 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 (8Feng Q. Moran J.V. Kazazian Jr., H. Boeke J.D. Cell. 1996; 87: 905-916Abstract Full Text Full Text PDF PubMed Scopus (845) Google Scholar). 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 (18Cost G.J. Boeke J.D. Biochemistry. 1998; 37: 18081-18093Crossref PubMed Scopus (188) Google Scholar). 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 (4Martín F. Marañón C. Olivares M. Alonso C. López M.C. J. Mol. Biol. 1995; 247: 49-59Crossref PubMed Scopus (124) Google Scholar, 7Olivares M. Alonso C. López M.C. J. Biol. Chem. 1997; 272: 25224-25228Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 8Feng Q. Moran J.V. Kazazian Jr., H. Boeke J.D. Cell. 1996; 87: 905-916Abstract Full Text Full Text PDF PubMed Scopus (845) Google Scholar). 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 (9Demple B. Harrison L. Annu. Rev. Biochem. 1994; 63: 915-948Crossref PubMed Scopus (1286) Google Scholar). 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 (9Demple B. Harrison L. Annu. Rev. Biochem. 1994; 63: 915-948Crossref PubMed Scopus (1286) Google Scholar,19Gu L. Huang S-M. Sander M. J. Biol. Chem. 1994; 269: 32685-32692Abstract Full Text PDF PubMed Google Scholar). 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 (13Sander M. Huang S-M. Biochemistry. 1995; 34: 1267-1274Crossref PubMed Scopus (23) Google Scholar, 14Izumi I. Ishizaki K. Ikenaga M. Yonei S. J. Bacteriol. 1992; 174: 7711-7716Crossref PubMed Google Scholar): 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 (11Demple B. Johnson A. Fung D. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7731-7735Crossref PubMed Scopus (199) Google Scholar). 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 (7Olivares M. Alonso C. López M.C. J. Biol. Chem. 1997; 272: 25224-25228Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). 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 (8Feng Q. Moran J.V. Kazazian Jr., H. Boeke J.D. Cell. 1996; 87: 905-916Abstract Full Text Full Text PDF PubMed Scopus (845) Google Scholar) 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 (7Olivares M. Alonso C. López M.C. J. Biol. Chem. 1997; 272: 25224-25228Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). 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) (20Teng S-C. Kim B. Gabriel A. Nature. 1996; 383: 641-644Crossref PubMed Scopus (202) Google Scholar, 21Moore J.K. Haber J.E. Nature. 1996; 383: 644-646Crossref PubMed Scopus (201) Google Scholar) has recently been described. We are grateful to Dr. Bernard Weiss for providing the BW528 and BW9109 strains.