Recombination Enzymes Research Articles

Computational structural and functional analysis of hypothetical proteins of Staphylococcus aureus.

Genome sequencing projects has led to an explosion of large amount of gene products in which many are of hypothetical proteins with unknown function. Analyzing and annotating the functions of hypothetical proteins is important in Staphylococcus aureus which is a pathogenic bacterium that cause multiple types of diseases by infecting various sites in humans and animals. In this study, ten hypothetical proteins of Staphylococcus aureus were retrieved from NCBI and analyzed for their structural and functional characteristics by using various bioinformatics tools and databases. The analysis revealed that some of them possessed functionally important domains and families and protein-protein interacting partners which were ABC transporter ATP-binding protein, Multiple Antibiotic Resistance (MAR) family, export proteins, Helix-Turn-helix domains, arsenate reductase, elongation factor, ribosomal proteins, Cysteine protease precursor, Type-I restriction endonuclease enzyme and plasmid recombination enzyme which might have the same functions in hypothetical proteins. The structural prediction of those proteins and binding sites prediction have been done which would be useful in docking studies for aiding in the drug discovery.

Molecular Organization of Small Plasmids Bearing bla TEM-1 and Conferring Resistance to β-Lactams in Haemophilus influenzae

TEM-1 is the dominant β-lactamase of Haemophilus influenzae and can be located on small plasmids. Three distinct plasmids with sizes from 4,304 to 5,646 nucleotides (nt) were characterized: pA1606, pA1209, and pPN223. In addition to TEM-1 and a replication enzyme of the Rep 3 superfamily, pA1606 carries a Tn3 resolvase gene and pA1606 and pA1209 carry an open reading frame (ORF) similar to a plasmid recombination enzyme gene described in Gram-positive bacteria. The plasmids transformed strain Rd to the ampicillin-resistant phenotype.

DNA breakage drives nuclear search

The search for a homologous template is a fundamental, yet largely uncharacterized, reaction in DNA double-strand break repair. Two reports now demonstrate that broken chromosomes increase their movement and explore large volumes of nuclear space searching for a homologous template. Break mobility requires resection and recombination enzymes, as well as damage-checkpoint components.

Genome-wide characterization of vibrio phage ϕpp2 with unique arrangements of the mob-like genes

BackgroundVibrio parahaemolyticus is associated with gastroenteritis, wound infections, and septicemia in human and animals. Phages can control the population of the pathogen. So far, the only one reported genome among giant vibriophages is KVP40: 244,835 bp with 26% coding regions that have T4 homologs. Putative homing endonucleases (HE) were found in Vibrio phage KVP40 bearing one segD and Vibrio cholerae phage ICP1 carrying one mobC/E and one segG.ResultsA newly isolated Vibrio phage ϕpp2, which was specific to the hosts of V. parahaemolyticus and V. alginolyticus, featured a long nonenveloped head of ~90 × 150 nm and tail of ~110 nm. The phage can survive at 50°C for more than one hour. The genome of the phage ϕpp2 was sequenced to be 246,421 bp, which is 1587 bp larger than KVP40. 383 protein-encoding genes (PEGs) and 30 tRNAs were found in the phage ϕpp2. Between the genomes of ϕpp2 and KVP40, 254 genes including 29 PEGs for viral structure were of high similarity, whereas 17 PEGs of KVP40 and 21 PEGs of ϕpp2 were unmatched. In both genomes, the capsid and tail genes have been identified, as well as the extensive representation of the DNA replication, recombination, and repair enzymes. In addition to the three giant indels of 1098, 1143 and 3330 nt, ϕpp2 possessed unique proteins involved in potassium channel, gp2 (DNA end protector), tRNA nucleotidyltransferase, and mob-type HEs, which were not reported in KVP40. The ϕpp2 PEG274, with strong promoters and translational initiation, was identified to be a mobE type, flanked by NrdA and NrdB/C homologs. Coincidently, several pairs of HE-flanking homologs with empty center were found in the phages of Vibrio phages ϕpp2 and KVP40, as well as in Aeromonas phages (Aeh1 and Ae65), and cyanophage P-SSM2.ConclusionsVibrio phage ϕpp2 was characterized by morphology, growth, and genomics with three giant indels and different types of HEs. The gene analysis on the required elements for transcription and translation suggested that the ϕpp2 PEG274 was an active mobE gene. The phage was signified to be a new species of T4-related, differing from KVP40.

Molecular Investigation of Virulence Determinants between a Virulent Clinical Strain and an Attenuated Strain of Burkholderia pseudomallei

Burkholderia pseudomallei is the causative agent of melioidosis. We initiated this investigation with a virulent and an attenuated strain of B. pseudomallei. Pulsed-field gel electrophoresis was carried out initially for macrogenomic comparison of both strains of B. pseudomallei. However, the pulsotypes obtained were identical and therefore we applied a subtractive hybridization technique to distinguish and determine the possible differences between the two strains. Six virulence strain-specific DNA fragments were obtained and the encoding homolog proteins were identified as a xenobiotic-responsive element family of transcriptional regulator, a hypothetical protein, an unknown protein, a plasmid recombination enzyme, a regulatory protein and a putative hemolysin activator protein. A combination of at least three of these determinants was identified in 45 clinical isolates when screening was carried out with self-designed multiplex PCR targeting the six putative virulent determinants. Our data demonstrated that different combinations of the six putative virulence genes were present in the clinical isolates indicating their probable role in the pathogenesis of B. pseudomallei infections.

Toll-like receptor function in primary B cell defects

Primary immunodeficiency diseases include more than 150 different genetic defects, classified on the basis of the mutations or physiological defects involved. The first immune defects to be well recognized were those of adaptive immunity affecting B cell function and resulting in hypogammaglobulinemia and defects of specific antibody production; more recently, novel defects of innate immunity have been described, some involving Toll-like receptors (TLRs) and their signaling pathways. Furthermore, it is increasingly evident that the innate and adaptive pathways intersect and reinforce each other. B cells express a number of TLRs, which when activated lead to cell activation, up-regulation of co-stimulatory molecules, secretion of cytokines, up-regulation of recombination enzymes, isotype switch and immune globulin production. TLR activation of antigen presenting cells leads to heightened cytokine production, providing additional stimuli for B cell development and maturation. Recent studies have demonstrated that patients with common variable immunodeficiency (CVID) and X-linked agammaglobulinemia (XLA) have altered TLR responsiveness. We review TLR defects in these disorders of B cell development, and discuss how B cell gene defects may modulate TLR signaling.

BRCA1 requirement for the fidelity of plasmid DNA double‐strand break repair in cultured breast epithelial cells

The tumor suppressor breast cancer susceptibility protein 1 (BRCA1) protects our cells from genomic instability in part by facilitating the efficient repair of DNA double-strand breaks (DSBs). BRCA1 promotes the error-free repair of DSBs through homologous recombination and is also implicated in the regulation of nonhomologous end joining (NHEJ) repair fidelity. Here, we investigate the role of BRCA1 in NHEJ repair mutagenesis following a DSB. We examined the frequency of microhomology-mediated end joining (MMEJ) and the fidelity of DSB repair relative to BRCA1 protein levels in both control and tumorigenic breast epithelial cells. In addition to altered BRCA1 protein levels, we tested the effects of cellular exposure to mirin, an inhibitor of meiotic recombination enzyme 11 (Mre11) 3'-5'-exonuclease activity. Knockdown or loss of BRCA1 protein resulted in an increased frequency of overall plasmid DNA mutagenesis and MMEJ following a DSB. Inhibition of Mre11-exonuclease activity with mirin significantly decreased the occurrence of MMEJ, but did not considerably affect the overall mutagenic frequency of plasmid DSB repair. The results suggest that BRCA1 protects DNA from mutagenesis during nonhomologous DSB repair in plasmid-based assays. The increased frequency of DSB mutagenesis and MMEJ repair in the absence of BRCA1 suggests a potential mechanism for carcinogenesis.

Linker histone H1 stimulates DNA strand exchange between short oligonucleotides retaining high sensitivity to heterology

The interaction of human linker histone H1(0) with short oligonucleotides was characterized. The capability of the histone to promote DNA strand exchange in this system has been demonstrated. The reaction is reversible at saturating amounts of H1 corresponding to complete binding of the oligonucleotide substrates with the histone. In our conditions the complete saturation of DNA with the histone occurs at a ratio of one protein molecule per about 60 nucleotides irrespectively of DNA strandedness. In contrast to the DNA strand exchange promoted by RecA-like enzymes of homologous recombination the H1 promoted reaction exhibits low tolerance to interruptions of homology between oligonucleotide substrates comparable to those for the case of spontaneous strand exchange between free DNA molecules at elevated temperatures and the exchange promoted by some synthetic polycations.

Genome of low-temperature T4-related bacteriophage vB_EcoM-VR7

The complete genome sequence of the T4-related low-temperature Escherichia coli bacteriophage vB_EcoM-VR7 was determined. The genome sequence is 169,285bp long, with a G+C content of 40.3%. Overall, 95% of the phage genome is coding. It encodes 293 putative protein-encoding open reading frames (ORFs) and tRNA(Met). More than half (59%) of the genomic DNA lacks significant homology with the DNA of T4, but once translated, 72% of the vB_EcoM-VR7 genome (211 ORFs) encodes protein homologues of T4 genes. Overall, 46 vB_EcoM-VR7 ORFs have no homologues in T4 but are derived from other T4-related phages, nine ORFs show similarities to bacterial or non-T4-related phage genes, and 27 ORFs are unique to vB_EcoM-VR7. This phage lacks several T4 enzymes involved in host DNA degradation; however, there is extensive representation of the DNA replication, recombination and repair enzymes as well as the viral capsid and tail structural genes.

A Versatile Set of Ligation-Independent Cloning Vectors for Functional Studies in Plants

With plant molecular biology entering the omics era, there is a need for simple cloning strategies that allow high throughput to systematically study the expression and function of large numbers of genes. Such strategies would facilitate the analysis of gene (sub)families and/or sets of coexpressed genes identified by transcriptomics. Here, we provide a set of 34 ligation-independent cloning (LIC) binary vectors for expression analysis, protein localization studies, and misexpression that will be made freely available. This set of plant LIC vectors offers a fast alternative to standard cloning strategies involving ligase or recombination enzyme technology. We demonstrate the use of this strategy and our new vectors by analyzing the expression domains of genes belonging to two subclades of the basic helix-loop-helix transcription factor family. We show that neither the closest homologs of TARGET OF MONOPTEROS7 (TMO7/ATBS1) nor the members of the ATBS1 INTERACTING FACTOR subclade of putative TMO7 interactors are expressed in the embryo and that there is very limited coexpression in the primary root meristem. This suggests that these basic helix-loop-helix transcription factors are most likely not involved in TMO7-dependent root meristem initiation.

RecA-dependent replication in the nrdA101(Ts) mutant of Escherichia coli under restrictive conditions.

Cells carrying the thermosensitive nrdA101 allele are able to replicate entire chromosomes at 42°C when new DNA initiation events are inhibited. We investigated the role of the recombination enzymes on the progression of the DNA replication forks in the nrdA101 mutant at 42°C in the presence of rifampin. Using pulsed-field gel electrophoresis (PFGE), we demonstrated that the replication forks stalled and reversed during the replication progression under this restrictive condition. DNA labeling and flow cytometry experiments supported this finding as the deleterious effects found in the RecB-deficient background were suppressed specifically by the absence of RuvABC; however, this did not occur in a RecG-deficient background. Furthermore, we show that the RecA protein is absolutely required for DNA replication in the nrdA101 mutant at restrictive temperature when the replication forks are reversed. The detrimental effect of the recA deletion is not related to the chromosomal degradation caused by the absence of RecA. The inhibition of DNA replication observed in the nrdA101 recA mutant at 42°C in the presence of rifampin was reverted by the presence of the wild-type RecA protein expressed ectopically but only partially suppressed by the RecA protein with an S25P mutation [RecA(S25P)], deficient in the rescue of the stalled replication forks. We propose that RecA is required to maintain the integrity of the reversed forks in the nrdA101 mutant under certain restrictive conditions, supporting the relationship between DNA replication and recombination enzymes through the stabilization and repair of the stalled replication forks.

Cancer-Specific Nuclear Positioning of Translocation Prone Gene Loci In Non-Malignant B-Cells From Patients with Multiple Myeloma

AbstractAbstract 783Malignant plasma cells from patients with multiple myeloma (MM) display recurrent chromosome translocations involving the immunoglobulin heavy chain locus (IGH) and CCND1 [t(11;14)] or FGFR [t(4;14)]. It is poorly understood why recombination events between chromosomes recur at specific breakpoints in the human genome, but spatial proximity of translocation-prone gene loci (TPGL) is influential. Utilizing 3D fluorescent in situ hybridization and 3D analysis techniques, we have measured the spatial and radial positioning of translocation-prone and control loci in the nuclei of non-malignant cells to determine a propensity to translocate. Purified CD34+ progenitors and CD19+ B cells were analyzed; none had translocations. Utilizing these cell subsets (n=900 cells) from MM patients having recurrent IGH translocations in autologous plasma cells, and comparable subsets (n=300 cells) from healthy donors, we show that spatial genome organization contributes to the formation of recurrent translocations. We show that IGH, CCND1, and FGFR3 are preferentially positioned in close proximity relative to each other in presumptively normal CD34+ progenitors and CD19+ B-cells from these MM patients, and that the clinical frequency of IGH translocations correlates with relative positioning (p=0.002 for CD34+ cells; p=0.017 for CD19+ cells), and with radial positioning (p=0.05 for CD34+ cells; p=0.043 for CD19+ cells). We also show that non-malignant CD19+ B-cells from MM patients display cancer-specific radial positioning of TPGL in the nucleus (p≤0.008), suggesting a predisposition to translocate. In addition to preferred positions within the nucleus, genes also localize to favoured locations within their respective chromosome territory (CT). Genes have been shown to position outside of their respective chromosome territory. Although proximity of potential translocation partners is necessary for translocation events to occur, others have shown that translocation events between adjacent chromosomes take place at CT boundaries, or in the space of intermingling between adjacent CTs. Active transcription ‘factories’ are also present within the area of intermingling. We observed that at least one of the two alleles of CCND1 and FGFR3, is positioned outside of its chromosome territory in 59% of CD19+ cells from MM patients, whereas a control locus TGFBR2, is positioned outside its chromosome territory in only 25% of the same cells. We find myeloma-specific positioning of TPGL in a subset of B-cells that lack translocations and are predominantly polyclonal, defining them as non-malignant. It seems likely that the original parent B-cell that gave rise to MM also harboured spatially proximal TPGL prior to the formation of physically translocated loci. Together, these observations support the likelihood that translocations occur only in specific sites within the nucleus. Recent reports indicate that ongoing transcription is necessary for functional activity of IGH recombination enzymes, and that these same enzymes have off-target effects on proximal genes. IGH and its translocation partners FGFR3 and CCND1 may come together briefly to a transcription ‘factory’ outside of their respective CTs and be acted upon by IGH recombination enzymes. We speculate that cancer-specific positioning of active TPGL in B-cells from MM patients promotes the formation of clinically important IGH translocations, perhaps through co-localization of TPGL to nuclear transcription ‘factories’ outside of CTs. The strong correlation between locus positioning and the clinical frequency of recurrent IGH translocations involving these loci is consistent with the idea that spatial proximity in the nucleus is an important contributor to the high frequency of recurrent translocations. CCND1 and FGFR3 are positioned outside their CT and in close proximity to IGH as compared to TGFBR2 and c-MAF, both of which are more peripheral in the nucleus and position out of their respective CTs at lower frequency. This is consistent with the fact that c-MAF/IGH translocations are relatively infrequent and TGFBR2/IGH translocations have not been reported. Our results suggest that the formation of recurrent IGH translocations in MM is determined not only by spatial proximity of gene loci and radial positioning in the cell nucleus, but also by positioning of loci outside of their chromosomal territories. Disclosures:No relevant conflicts of interest to declare.

Structural mechanisms of ssDNA/SSB‐processing enzymes

Unwinding genomic DNA to form single‐stranded (ss) DNA is an obligatory step in many DNA replication, recombination, and repair pathways. However, because ssDNA is sensitive to chemical and nucleolytic attack and can self‐associate to form structures that impede genome maintenance, DNA unwinding also presents a potential threat to genomic integrity. To help mediate this risk, cells have evolved ssDNA‐binding proteins (SSBs) that bind and protect ssDNA from biochemical attacks and maintain its single stranded structure. SSB/ssDNA nucleoprotein complexes thus form a common substrate utilized in genome maintenance reactions. Defining how DNA replication, recombination, and repair enzymes recognize and process SSB/ssDNA structures is key to understanding cellular genome maintenance mechanisms.Bacterial SSBs facilitate DNA replication, recombination, and repair processes in part by recruiting diverse genome maintenance enzymes to ssDNA. This function utilizes the C‐terminus of SSB (SSB‐Ct) as a common binding site for SSB's protein partners. The SSB‐Ct is a highly conserved, amphipathic sequence, comprising acidic and hydrophobic elements. I will present our most recent studies that examine the structural mechanisms that support protein interaction with the SSB‐Ct element, focusing on the roles of these interactions in DNA replication.

Yeast Exonuclease 5 Is Essential for Mitochondrial Genome Maintenance

Yeast exonuclease 5 is encoded by the YBR163w (DEM1) gene, and this gene has been renamed EXO5. It is distantly related to the Escherichia coli RecB exonuclease class. Exo5 is localized to the mitochondria, and EXO5 deletions or nuclease-defective EXO5 mutants invariably yield petites, amplifying either the ori3 or ori5 region of the mitochondrial genome. These petites remain unstable and undergo continuous rearrangement. The mitochondrial phenotype of exo5Delta strains suggests an essential role for the enzyme in DNA replication and recombination. No nuclear phenotype associated with EXO5 deletions has been detected. Exo5 is a monomeric 5' exonuclease that releases dinucleotides as products. It is specific for single-stranded DNA and does not hydrolyze RNA. However, Exo5 has the capacity to slide across 5' double-stranded DNA or 5' RNA sequences and resumes cutting two nucleotides downstream of the double-stranded-to-single-stranded junction or RNA-to-DNA junction, respectively.

DNA strand exchange catalyzed by molecular crowding in PEG solutions

DNA strand exchange is catalyzed by molecular crowding and hydrophobic interactions in concentrated aqueous solutions of polyethylene glycol, a discovery of relevance for understanding the function of recombination enzymes and with potential applications to DNA nanotechnology.

Insights from the structure of a smallpox virus topoisomerase-DNA transition state mimic.

Poxviruses encode their own type IB topoisomerases (TopIBs), which release superhelical tension generated by replication and transcription of their genomes. To investigate the reaction catalyzed by viral TopIBs, we have determined the structure of a variola virus topoisomerase-DNA complex trapped as a vanadate transition state mimic. The structure reveals how the viral TopIB enzymes are likely to position the DNA duplex for ligation following relaxation of supercoils and identifies the sources of friction observed in single-molecule experiments that argue against free rotation. The structure also identifies a conformational change in the leaving group sugar that must occur prior to cleavage and reveals a mechanism for promoting ligation following relaxation of supercoils that involves an Asp-minor groove interaction. Overall, the new structural data support a common catalytic mechanism for the TopIB superfamily but indicate distinct methods for controlling duplex rotation in the small versus large enzyme subfamilies.

Reverse Transcribing the Code for Chromosome Stability

The linearity of eukaryotic chromosomes presents challenges to cells, as the presence of DNA "ends" poses problems for the DNA replication machinery and the cell's damage response systems. This year's Nobel Prize in Physiology or Medicine recognized groundbreaking studies establishing the telomere field as a crucial area of biomedical research.

An insight into the biological functions of family X-DNA polymerase in DNA replication and repair of plant genome

Recently we have reported the characterization of a novel single subunit 62-kDa polypeptide with ddNTP-sensitive DNA polymerase activity from the developing seeds of mungbean (Vigna radiata). The protein showed higher expression and activity level during nuclear endoreduplication stages of mungbean seeds and similarity with mammalian DNA polymerase in many physicochemical properties.1 The enzyme was found to specifically interact with PCNA (proliferating cell nuclear antigen),2 and expressed in both meristematic and meiotic tissues. Functional assays have demonstrated binding of the enzyme to normal and mismatched DNA substrates and with fidelity DNA synthesis in moderately processive mode, suggesting probable involvement of the enzyme in both replication and recombination.3 Here we have discussed the position of mungbean DNA polymerase as a homologue of DNA Pol λone of the newly identified member of family-X DNA polymerase in plants and illustrated the functional relevance of this enzyme in maintaining the coordination between DNA replication and repair in plant genome.

New yeast recombineering tools for bacteria

Recombineering with Saccharomyces cerevisiae is a powerful methodology that can be used to clone multiple unmarked pieces of DNA to generate complex constructs with high efficiency. Here, we introduce two new tools that utilize the native recombination enzymes of S. cerevisiae to facilitate the manipulation of DNA. First, yeast recombineering was used to make directed nested deletions in a bacteria–yeast shuttle plasmid using only one or two single stranded oligomers, thus obviating the need for a PCR step. Second, we have generated several new shuttle vectors for yeast recombineering capable of replication in a wide variety of bacterial genera. As a demonstration of utility, some of the approaches and vectors generated in this study were used to make a pigP deletion mutation in the opportunistic pathogen Serratia marcescens.

Oligomeric Structure Diversity within the GIY-YIG Nuclease Family

The GIY-YIG nuclease domain has been identified in homing endonucleases, DNA repair and recombination enzymes, and restriction endonucleases. The Type II restriction enzyme Eco29kI belongs to the GIY-YIG nuclease superfamily and, like most of other family members, including the homing endonuclease I-TevI, is a monomer. It recognizes the palindromic sequence 5'-CCGC/GG-3' ("/" marks the cleavage position) and cuts it to generate 3'-staggered ends. The Eco29kI monomer, which contains a single active site, either has to nick sequentially individual DNA strands or has to form dimers or even higher-order oligomers upon DNA binding to make a double-strand break at its target site. Here, we provide experimental evidence that Eco29kI monomers dimerize on a single cognate DNA molecule forming the catalytically active complex. The mechanism described here for Eco29kI differs from that of Cfr42I isoschisomer, which also belongs to the GIY-YIG family but is functional as a tetramer. This novel mechanism may have implications for the function of homing endonucleases and other enzymes of the GIY-YIG family.