RESUMO
Escherichia coli DNA ligase (EcoLigA) repairs 3'-OH/5'-PO4 nicks in duplex DNA via reaction of LigA with NAD(+) to form a covalent LigA-(lysyl-Nζ)-AMP intermediate (step 1); transfer of AMP to the nick 5'-PO4 to form an AppDNA intermediate (step 2); and attack of the nick 3'-OH on AppDNA to form a 3'-5' phosphodiester (step 3). A distinctive feature of EcoLigA is its stimulation by ammonium ion. Here we used rapid mix-quench methods to analyze the kinetic mechanism of single-turnover nick sealing by EcoLigA-AMP. For substrates with correctly base-paired 3'-OH/5'-PO4 nicks, kstep2 was fast (6.8-27 s(-1)) and similar to kstep3 (8.3-42 s(-1)). Absent ammonium, kstep2 and kstep3 were 48-fold and 16-fold slower, respectively. EcoLigA was exquisitely sensitive to 3'-OH base mispairs and 3' N:abasic lesions, which elicited 1000- to >20000-fold decrements in kstep2. The exception was the non-canonical 3' A:oxoG configuration, which EcoLigA accepted as correctly paired for rapid sealing. These results underscore: (i) how EcoLigA requires proper positioning of the nick 3' nucleoside for catalysis of 5' adenylylation; and (ii) EcoLigA's potential to embed mutations during the repair of oxidative damage. EcoLigA was relatively tolerant of 5'-phosphate base mispairs and 5' N:abasic lesions.
Assuntos
Monofosfato de Adenosina/química , DNA Ligases/química , Reparo do DNA , DNA/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , NAD/química , Monofosfato de Adenosina/metabolismo , Sulfato de Amônio/química , Sulfato de Amônio/farmacologia , Pareamento Incorreto de Bases , Sequência de Bases , DNA/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , DNA Ligases/genética , Ensaios Enzimáticos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Expressão Gênica , Cinética , Dados de Sequência Molecular , NAD/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Especificidade por SubstratoRESUMO
DNA3'pp5'G caps synthesized by the 3'-PO4/5'-OH ligase RtcB have a strong impact on enzymatic reactions at DNA 3'-OH ends. Aprataxin, an enzyme that repairs A5'pp5'DNA ends formed during abortive ligation by classic 3'-OH/5'-PO4 ligases, is also a DNA 3' de-capping enzyme, converting DNAppG to DNA3'p and GMP. By taking advantage of RtcB's ability to utilize certain GTP analogs to synthesize DNAppN caps, we show that aprataxin hydrolyzes inosine and 6-O-methylguanosine caps, but is not adept at removing a deoxyguanosine cap. We report a 1.5 Å crystal structure of aprataxin in a complex with GMP, which reveals that: (i) GMP binds at the same position and in the same anti nucleoside conformation as AMP; and (ii) aprataxin makes more extensive nucleobase contacts with guanine than with adenine, via a hydrogen bonding network to the guanine O6, N1, N2 base edge. Alanine mutations of catalytic residues His147 and His149 abolish DNAppG de-capping activity, suggesting that the 3' de-guanylylation and 5' de-adenylylation reactions follow the same pathway of nucleotidyl transfer through a covalent aprataxin-(His147)-NMP intermediate. Alanine mutation of Asp63, which coordinates the guanosine ribose hydroxyls, impairs DNAppG de-capping.
Assuntos
DNA/química , Exodesoxirribonucleases/química , Guanosina/química , Proteínas de Schizosaccharomyces pombe/química , Domínio Catalítico/genética , DNA/metabolismo , Primers do DNA , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Guanosina/análogos & derivados , Modelos Moleculares , Mutação , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Moldes GenéticosRESUMO
When DNA breakage results in a 3'-PO4 terminus, the end is considered 'dirty' because it cannot prime repair synthesis by DNA polymerases or sealing by classic DNA ligases. The noncanonical ligase RtcB can guanylylate the DNA 3'-PO4 to form a DNA3'pp5'GOH cap. Here we show that DNA capping precludes end joining by classic ATP-dependent and NAD(+)-dependent DNA ligases, prevents template-independent nucleotide addition by mammalian terminal transferase, blocks exonucleolytic proofreading by Escherichia coli DNA polymerase II and inhibits proofreading by E. coli DNA polymerase III, while permitting templated DNA synthesis from the cap guanosine 3'-OH primer by E. coli DNA polymerase II (B family) and E. coli DNA polymerase III (C family). Human DNA polymerase ß (X family) extends the cap primer predominantly by a single templated addition step. Cap-primed synthesis by templated polymerases embeds a pyrophosphate-linked ribonucleotide in DNA. We find that the embedded ppG is refractory to surveillance and incision by RNase H2.
Assuntos
Quebras de DNA , Reparo do DNA , Ribonucleotídeos/metabolismo , Aminoacil-tRNA Sintetases/metabolismo , Animais , Bovinos , Reparo do DNA por Junção de Extremidades , DNA Ligases/metabolismo , DNA Nucleotidiltransferases/metabolismo , DNA Polimerase II/metabolismo , DNA Polimerase III/metabolismo , DNA Polimerase beta/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Guanosina Difosfato/metabolismo , Humanos , Cinética , Ribonuclease H/metabolismo , Ribonucleotídeos/químicaRESUMO
Many biological scenarios generate "dirty" DNA 3'-PO4 ends that cannot be sealed by classic DNA ligases or extended by DNA polymerases. The noncanonical ligase RtcB can "cap" these ends via a unique chemical mechanism entailing transfer of GMP from a covalent RtcB-GMP intermediate to a DNA 3'-PO4 to form DNA3'pp5'G. Here, we show that capping protects DNA 3' ends from resection by Escherichia coli exonucleases I and III and from end-healing by T4 polynucleotide 3' phosphatase. By contrast, the cap is an effective primer for DNA synthesis. E. coli DNA polymerase I and Mycobacterium DinB1 extend the DNAppG primer to form an alkali-labile DNApp(rG)pDNA product. The addition of dNTP depends on pairing of the cap guanine with an opposing cytosine in the template strand. Aprataxin, an enzyme implicated in repair of A5'pp5'DNA ends formed during abortive ligation by classic ligases, is highly effective as a DNA 3' decapping enzyme, converting DNAppG to DNA3'p and GMP. We conclude that the biochemical impact of DNA capping is to prevent resection and healing of a 3'-PO4 end, while permitting DNA synthesis, at the price of embedding a ribonucleotide and a pyrophosphate linkage in the repaired strand. Aprataxin affords a means to counter the impact of DNA capping.
Assuntos
Reparo do DNA , DNA/metabolismo , Guanosina/metabolismo , Capuzes de RNA/metabolismo , Sequência de Bases , DNA/biossíntese , DNA Polimerase Dirigida por DNA/metabolismo , Escherichia coli/enzimologia , Exodesoxirribonucleases/metabolismo , Exonucleases/metabolismo , Dados de Sequência Molecular , Nucleotídeos/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Moldes GenéticosRESUMO
T4 RNA ligase 2 (Rnl2) repairs 3'-OH/5'-PO4 nicks in duplex nucleic acids in which the broken 3'-OH strand is RNA. Ligation entails three chemical steps: reaction of Rnl2 with ATP to form a covalent Rnl2-(lysyl-Nζ)-AMP intermediate (step 1); transfer of AMP to the 5'-PO4 of the nick to form an activated AppN- intermediate (step 2); and attack by the nick 3'-OH on the AppN- strand to form a 3'-5' phosphodiester (step 3). Here we used rapid mix-quench methods to analyze the kinetic mechanism and fidelity of single-turnover nick sealing by Rnl2-AMP. For substrates with correctly base-paired 3'-OH nick termini, kstep2 was fast (9.5 to 17.9 sec(-1)) and similar in magnitude to kstep3 (7.9 to 32 sec(-1)). Rnl2 fidelity was enforced mainly at the level of step 2 catalysis, whereby 3'-OH base mispairs and oxoguanine, oxoadenine, or abasic lesions opposite the nick 3'-OH elicited severe decrements in the rate of 5'-adenylylation and relatively modest slowing of the rate of phosphodiester synthesis. The exception was the noncanonical A:oxoG base pair, which Rnl2 accepted as a correctly paired end for rapid sealing. These results underscore (1) how Rnl2 requires proper positioning of the 3'-terminal ribonucleoside at the nick for optimal 5'-adenylylation and (2) the potential for nick-sealing ligases to embed mutations during the repair of oxidative damage.
Assuntos
RNA Ligase (ATP)/química , RNA de Cadeia Dupla/química , Proteínas Virais/química , Adenina/análogos & derivados , Adenina/química , Ácido Apurínico/química , Pareamento de Bases , Sequência de Bases , Escherichia coli , Guanina/análogos & derivados , Guanina/química , Cinética , RNA de Cadeia Dupla/genéticaRESUMO
It has long been suggested that the import of nuclease colicins requires protein processing; however it had never been formally demonstrated. Here we show that two RNase colicins, E3 and D, which appropriate two different translocation machineries to cross the outer membrane (BtuB/Tol and FepA/TonB, respectively), undergo a processing step inside the cell that is essential to their killing action. We have detected the presence of the C-terminal catalytic domains of these colicins in the cytoplasm of target bacteria. The same processed forms were identified in both colicin-sensitive cells and in cells immune to colicin because of the expression of the cognate immunity protein. We demonstrate that the inner membrane protease FtsH is necessary for the processing of colicins D and E3 during their import. We also show that the signal peptidase LepB interacts directly with the central domain of colicin D in vitro and that it is a specific but not a catalytic requirement for in vivo processing of colicin D. The interaction of colicin D with LepB may ensure a stable association with the inner membrane that in turn allows the colicin recognition by FtsH. We have also shown that the outer membrane protease OmpT is responsible for alternative and distinct endoproteolytic cleavages of colicins D and E3 in vitro, presumably reflecting its known role in the bacterial defense against antimicrobial peptides. Even though the OmpT-catalyzed in vitro cleavage also liberates the catalytic domain from colicins D and E3, it is not involved in the processing of nuclease colicins during their import into the cytoplasm.