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1.
Biochem J ; 477(7): 1345-1362, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32207815

RESUMO

We report the identification and characterization of a bacteriophage λ-encoded protein, NinH. Sequence homology suggests similarity between NinH and Fis, a bacterial nucleoid-associated protein (NAP) involved in numerous DNA topology manipulations, including chromosome condensation, transcriptional regulation and phage site-specific recombination. We find that NinH functions as a homodimer and is able to bind and bend double-stranded DNA in vitro. Furthermore, NinH shows a preference for a 15 bp signature sequence related to the degenerate consensus favored by Fis. Structural studies reinforced the proposed similarity to Fis and supported the identification of residues involved in DNA binding which were demonstrated experimentally. Overexpression of NinH proved toxic and this correlated with its capacity to associate with DNA. NinH is the first example of a phage-encoded Fis-like NAP that likely influences phage excision-integration reactions or bacterial gene expression.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteriófago lambda/genética , Bacteriófago lambda/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Proteínas de Bactérias/química , Sequência de Bases , Sítios de Ligação , Simulação por Computador , DNA/metabolismo , DNA Viral/metabolismo , Proteínas de Ligação a DNA/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Fator Proteico para Inversão de Estimulação/química , Fator Proteico para Inversão de Estimulação/genética , Expressão Gênica , Proteínas Mutantes/metabolismo , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Multimerização Proteica/genética , Proteínas Virais/química
2.
PLoS One ; 9(8): e102454, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25083707

RESUMO

Genetic and biochemical evidence suggests that λ Orf is a recombination mediator, promoting nucleation of either bacterial RecA or phage Redß recombinases onto single-stranded DNA (ssDNA) bound by SSB protein. We have identified a diverse family of Orf proteins that includes representatives implicated in DNA base flipping and those fused to an HNH endonuclease domain. To confirm a functional relationship with the Orf family, a distantly-related homolog, YbcN, from Escherichia coli cryptic prophage DLP12 was purified and characterized. As with its λ relative, YbcN showed a preference for binding ssDNA over duplex. Neither Orf nor YbcN displayed a significant preference for duplex DNA containing mismatches or 1-3 nucleotide bulges. YbcN also bound E. coli SSB, although unlike Orf, it failed to associate with an SSB mutant lacking the flexible C-terminal tail involved in coordinating heterologous protein-protein interactions. Residues conserved in the Orf family that flank the central cavity in the λ Orf crystal structure were targeted for mutagenesis to help determine the mode of DNA binding. Several of these mutant proteins showed significant defects in DNA binding consistent with the central aperture being important for substrate recognition. The widespread conservation of Orf-like proteins highlights the importance of targeting SSB coated ssDNA during lambdoid phage recombination.


Assuntos
Bacteriófagos/genética , Bacteriófagos/metabolismo , Família Multigênica , Recombinases/genética , Recombinases/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Sequência de Aminoácidos , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA , Ordem dos Genes , Genoma Viral , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Recombinases/química , Alinhamento de Sequência , Proteínas Virais/química
3.
PLoS One ; 8(11): e78869, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24244379

RESUMO

The Red system of bacteriophage λ is responsible for the genetic rearrangements that contribute to its rapid evolution and has been successfully harnessed as a research tool for genome manipulation. The key recombination component is Redß, a ring-shaped protein that facilitates annealing of complementary DNA strands. Redß shares functional similarities with the human Rad52 single-stranded DNA (ssDNA) annealing protein although their evolutionary relatedness is not well established. Alignment of Rad52 and Redß sequences shows an overall low level of homology, with 15% identity in the N-terminal core domains as well as important similarities with the Rad52 homolog Sak from phage ul36. Key conserved residues were chosen for mutagenesis and their impact on oligomer formation, ssDNA binding and annealing was probed. Two conserved regions were identified as sites important for binding ssDNA; a surface basic cluster and an intersubunit hydrophobic patch, consistent with findings for Rad52. Surprisingly, mutation of Redß residues in the basic cluster that in Rad52 are involved in ssDNA binding disrupted both oligomer formation and ssDNA binding. Mutations in the equivalent of the intersubunit hydrophobic patch in Rad52 did not affect Redß oligomerization but did impair DNA binding and annealing. We also identified a single amino acid substitution which had little effect on oligomerization and DNA binding but which inhibited DNA annealing, indicating that these two functions of Redß can be separated. Taken together, the results provide fresh insights into the structural basis for Redß function and the important role of quaternary structure.


Assuntos
Bacteriófago lambda/enzimologia , DNA de Cadeia Simples/química , DNA Viral/química , Recombinases/química , Proteínas Virais/química , Bacteriófago lambda/genética , DNA de Cadeia Simples/genética , DNA Viral/genética , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Recombinases/genética , Relação Estrutura-Atividade , Proteínas Virais/genética
4.
Mol Microbiol ; 89(6): 1240-58, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23888987

RESUMO

Viral and bacterial Holliday junction resolvases differ in specificity with the former typically being more promiscuous, acting on a variety of branched DNA substrates, while the latter exclusively targets Holliday junctions. We have determined the crystal structure of a RuvC resolvase from bacteriophage bIL67 to help identify features responsible for DNA branch discrimination. Comparisons between phage and bacterial RuvC structures revealed significant differences in the number and position of positively-charged residues in the outer sides of the junction binding cleft. Substitutions were generated in phage RuvC residues implicated in branch recognition and six were found to confer defects in Holliday junction and replication fork cleavage in vivo. Two mutants, R121A and R124A that flank the DNA binding site were purified and exhibited reduced in vitro binding to fork and linear duplex substrates relative to the wild-type, while retaining the ability to bind X junctions. Crucially, these two variants cleaved Holliday junctions with enhanced specificity and symmetry, a feature more akin to cellular RuvC resolvases. Thus, additional positive charges in the phage RuvC binding site apparently stabilize productive interactions with branched structures other than the canonical Holliday junction, a feature advantageous for viral DNA processing but deleterious for their cellular counterparts.


Assuntos
Bacteriófagos/enzimologia , DNA Cruciforme/metabolismo , Resolvases de Junção Holliday/genética , Resolvases de Junção Holliday/metabolismo , Substituição de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Resolvases de Junção Holliday/química , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/isolamento & purificação , Proteínas Mutantes/metabolismo , Ligação Proteica , Conformação Proteica , Especificidade por Substrato
5.
J Mol Recognit ; 24(2): 333-40, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21360615

RESUMO

Phage λ Orf substitutes for the activities of the Escherichia coli RecFOR proteins in vivo and is therefore implicated as a recombination mediator, encouraging the assembly of bacterial RecA onto single-stranded DNA (ssDNA) coated with SSB. Orf exists as a dimer in solution, associates with E. coli SSB and binds preferentially to ssDNA. To help identify interacting domains we analysed Orf and SSB proteins carrying mutations or truncations in the C-terminal region. A cluster of acidic residues at the carboxy-terminus of SSB is known to attract multiple protein partners to assist in DNA replication and repair. In this case an alternative domain must be utilized since Orf association with SSB was unaffected by an SSB113 point mutant (P176S) or removal of the last ten residues (ΔC10). Structurally the Orf C-terminus consists of a helix with a flexible tail that protrudes from each side of the dimer and could serve as a binding site for either SSB or DNA. Eliminating the six residue flexible tail (ΔC6) or the entire helix (ΔC19) had no significant impact on the Orf-SSB interaction. However, the OrfΔC6 protein exhibited reduced DNA binding, a feature shared by single amino acid substitutions within (W141F) or adjacent (R140A) to this region. The OrfΔC19 mutant bound poorly to DNA and secondary structure analysis in solution revealed that this truncation induces protein misfolding and aggregation. The results show that the carboxy-terminus of Orf is involved in nucleic acid recognition and also plays an unexpected role in maintaining structural integrity.


Assuntos
Bacteriófago lambda/enzimologia , DNA/metabolismo , Recombinases/química , Recombinases/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Cromatografia em Gel , Dicroísmo Circular , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Cinética , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação/genética , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Deleção de Sequência , Soluções , Relação Estrutura-Atividade
6.
Bioorg Med Chem ; 14(14): 4731-9, 2006 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-16581254

RESUMO

Holliday junctions (HJs) are formed as transient DNA intermediates during site-specific and homologous recombination. Both of these genetic exchange pathways are critical for normal DNA metabolism and repair. Trapping HJs leads to bacterial cell death by preventing proper segregation of the resulting interlinked chromosomes. Macrocyclic peptides designed to target this intermediate were synthesized with the goal of identifying compounds with specificity for this unique molecular target. We discovered ten macrocycles, both hexameric and octameric peptides, capable of trapping HJs in vitro. Those macrocycles containing tyrosine residues proved most effective. These data demonstrate that C-2 symmetrical macrocycles offer excellent synthetic targets for the development of novel antibiotic agents. Furthermore, the active compounds identified provide valuable tools for probing different pathways of recombinational exchange.


Assuntos
Antibacterianos/síntese química , Antibacterianos/farmacologia , DNA Cruciforme/efeitos dos fármacos , DNA Cruciforme/metabolismo , Endodesoxirribonucleases/metabolismo , Proteínas de Escherichia coli/metabolismo , Oligopeptídeos/síntese química , Oligopeptídeos/farmacologia , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/farmacologia , Antibacterianos/química , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Técnicas In Vitro , Substâncias Macromoleculares , Modelos Moleculares , Estrutura Molecular , Conformação de Ácido Nucleico , Oligopeptídeos/química , Peptídeos Cíclicos/química , Conformação Proteica , Staphylococcus epidermidis/efeitos dos fármacos
7.
Proc Natl Acad Sci U S A ; 102(32): 11260-5, 2005 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-16076958

RESUMO

Genetic recombination in bacteriophage lambda relies on DNA end processing by Exo to expose 3'-tailed strands for annealing and exchange by beta protein. Phage lambda encodes an additional recombinase, Orf, which participates in the early stages of recombination by supplying a function equivalent to the Escherichia coli RecFOR complex. These host enzymes assist loading of the RecA strand exchange protein onto ssDNA coated with ssDNA-binding protein. In this study, we purified the Orf protein, analyzed its biochemical properties, and determined its crystal structure at 2.5 angstroms. The homodimeric Orf protein is arranged as a toroid with a shallow U-shaped cleft, lined with basic residues, running perpendicular to the central cavity. Orf binds DNA, favoring single-stranded over duplex and with no obvious preference for gapped, 3'-tailed, or 5'-tailed substrates. An interaction between Orf and ssDNA-binding protein was indicated by far Western analysis. The functional similarities between Orf and RecFOR are discussed in relation to the early steps of recombinational exchange and the interplay between phage and bacterial recombinases.


Assuntos
Bacteriófago lambda/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Modelos Moleculares , Recombinação Genética/genética , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Western Blotting , Clonagem Molecular , Cristalografia , Proteínas de Ligação a DNA/genética , Proteínas de Escherichia coli/genética , Dados de Sequência Molecular , Análise de Sequência de DNA , Proteínas Virais/genética
8.
Genetics ; 171(3): 873-83, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16020779

RESUMO

In models of Escherichia coli recombination and DNA repair, the RuvABC complex directs the branch migration and resolution of Holliday junction DNA. To probe the validity of the E. coli paradigm, we examined the impact of mutations in DeltaruvAB and DeltarecU (a ruvC functional analog) on DNA repair. Under standard transformation conditions we failed to construct DeltaruvAB DeltarecG, DeltarecU DeltaruvAB, DeltarecU DeltarecG, or DeltarecU DeltarecJ strains. However, DeltaruvAB could be combined with addAB (recBCD), recF, recH, DeltarecS, DeltarecQ, and DeltarecJ mutations. The DeltaruvAB and DeltarecU mutations rendered cells extremely sensitive to DNA-damaging agents, although less sensitive than a DeltarecA strain. When damaged cells were analyzed, we found that RecU was recruited to defined double-stranded DNA breaks (DSBs) and colocalized with RecN. RecU localized to these centers at a later time point during DSB repair, and formation was dependent on RuvAB. In addition, expression of RecU in an E. coli ruvC mutant restored full resistance to UV light only when the ruvAB genes were present. The results demonstrate that, as with E. coli RuvABC, RuvAB targets RecU to recombination intermediates and that all three proteins are required for repair of DSBs arising from lesions in chromosomal DNA.


Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/fisiologia , Dano ao DNA/fisiologia , DNA Helicases/fisiologia , Reparo do DNA/fisiologia , DNA Bacteriano/metabolismo , Proteínas de Escherichia coli/fisiologia , Resolvases de Junção Holliday/fisiologia , Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Enzimas de Restrição do DNA/metabolismo , DNA Bacteriano/efeitos da radiação , DNA Cruciforme/fisiologia , DNA Cruciforme/efeitos da radiação , Endodesoxirribonucleases/genética , Escherichia coli/genética , Escherichia coli/efeitos da radiação , Proteínas de Escherichia coli/genética , Genes Reporter , Mutação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Raios Ultravioleta
9.
Mol Microbiol ; 55(5): 1332-45, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15720544

RESUMO

Resolution of Holliday junction recombination intermediates in most Gram-negative bacteria is accomplished by the RuvC endonuclease acting in concert with the RuvAB branch migration machinery. Gram-positive species, however, lack RuvC, with the exception of distantly related orthologues from bacteriophages infecting Lactococci and Streptococci. We have purified one of these proteins, 67RuvC, from Lactococcus lactis phage bIL67 and demonstrated that it functions as a Holliday structure resolvase. Differences in the sequence selectivity of resolution between 67RuvC and Escherichia coli RuvC were noted, although both enzymes prefer to cleave 3' of thymidine residues. However, unlike its cellular counterpart, 67RuvC readily binds and cleaves a variety of branched DNA substrates in addition to Holliday junctions. Plasmids expressing 67RuvC induce chromosomal breaks, probably as a consequence of replication fork cleavage, and cannot be recovered from recombination-defective E. coli strains. Despite these deleterious effects, 67RuvC constructs suppress the UV light sensitivity of ruvA, ruvAB and ruvABC mutant strains confirming that the phage protein mediates Holliday junction resolution in vivo. The characterization of 67RuvC offers a unique insight into how a Holliday junction-specific resolvase can evolve into a debranching endonuclease tailored to the requirements of phage recombination.


Assuntos
Bacteriófagos/enzimologia , DNA Helicases/metabolismo , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Lactococcus lactis/virologia , Reparo do DNA , DNA Bacteriano/química , DNA Bacteriano/genética , Proteínas de Ligação a DNA/química , Endodesoxirribonucleases/química , Escherichia coli/genética , Proteínas de Escherichia coli , Evolução Molecular , Lactococcus lactis/genética
10.
J Mol Biol ; 340(4): 739-51, 2004 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-15223317

RESUMO

Rap endonuclease targets recombinant joint molecules arising from phage lambda Red-mediated genetic exchange. Previous studies revealed that Rap nicks DNA at the branch point of synthetic Holliday junctions and other DNA structures with a branched component. However, on X junctions incorporating a three base-pair core of homology or with a fixed crossover, Rap failed to make the bilateral strand cleavages characteristic of a Holliday junction resolvase. Here, we demonstrate that Rap can mediate symmetrical resolution of 50 bp and chi Holliday structures containing larger homologous cores. On two different mobile 50 bp junctions Rap displays a weak preference for cleaving the phosphodiester backbone between 5'-GC dinucleotides. The products of resolution on both large and small DNA substrates can be sealed by T4 DNA ligase, confirming the formation of nicked duplexes. Rap protein was also assessed for its capacity to influence the global conformation of junctions in the presence or absence of magnesium ions. Unlike the known Holliday junction binding proteins, Rap does not affect the angle of duplex arms, implying an unorthodox mode of junction binding. The results demonstrate that Rap can function as a Holliday junction resolvase in addition to eliminating other branched structures that may arise during phage recombination.


Assuntos
Bacteriófago lambda/enzimologia , Bacteriófago lambda/metabolismo , Endodesoxirribonucleases/metabolismo , Resolvases de Junção Holliday/metabolismo , Bacteriófago lambda/genética , Sequência de Bases , Sítios de Ligação , Eletroforese em Gel de Ágar , Resolvases de Junção Holliday/química , Magnésio/farmacologia , Manganês/farmacologia , Recombinação Genética , Relação Estrutura-Atividade , Especificidade por Substrato
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