scholarly journals Znc2 module of RAG1 contributes towards structure-specific nuclease activity of RAGs

2020 ◽  
Vol 477 (18) ◽  
pp. 3567-3582
Author(s):  
Namrata M. Nilavar ◽  
Mayilaadumveettil Nishana ◽  
Amita M. Paranjape ◽  
Raghunandan Mahadeva ◽  
Rupa Kumari ◽  
...  
Keyword(s):  
Signal Sequence ◽  
Specific Binding ◽  
Dna Recombination ◽  
Point Mutations ◽  
Nuclease Activity ◽  
Heteroduplex Dna ◽  
Central Domain ◽  
Dna Structures ◽  
Recombination Activating Genes ◽  
Catalytic Motif

Recombination activating genes (RAGs), consisting of RAG1 and RAG2 have ability to perform spatially and temporally regulated DNA recombination in a sequence specific manner. Besides, RAGs also cleave at non-B DNA structures and are thought to contribute towards genomic rearrangements and cancer. The nonamer binding domain of RAG1 binds to the nonamer sequence of the signal sequence during V(D)J recombination. However, deletion of NBD did not affect RAG cleavage on non-B DNA structures. In the present study, we investigated the involvement of other RAG domains when RAGs act as a structure-specific nuclease. Studies using purified central domain (CD) and C-terminal domain (CTD) of the RAG1 showed that CD of RAG1 exhibited high affinity and specific binding to heteroduplex DNA, which was irrespective of the sequence of single-stranded DNA, unlike CTD which showed minimal binding. Furthermore, we show that ZnC2 of RAG1 is crucial for its binding to DNA structures as deletion and point mutations abrogated the binding of CD to heteroduplex DNA. Our results also provide evidence that unlike RAG cleavage on RSS, central domain of RAG1 is sufficient to cleave heteroduplex DNA harbouring pyrimidines, but not purines. Finally, we show that a point mutation in the DDE catalytic motif is sufficient to block the cleavage of CD on heteroduplex DNA. Therefore, in the present study we demonstrate that the while ZnC2 module in central domain of RAG1 is required for binding to non-B DNA structures, active site amino acids are important for RAGs to function as a structure-specific nuclease.

scholarly journals Determinants That Control the Specific Interactions between TAB1 and p38α

2006 ◽  
Vol 26 (10) ◽  
pp. 3824-3834 ◽  
Author(s):  
Huamin Zhou ◽  
Min Zheng ◽  
Jianming Chen ◽  
Changchuan Xie ◽  
Anand R. Kolatkar ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Conformational Changes ◽  
Transforming Growth Factor ◽  
Mutational Analysis ◽  
Specific Binding ◽  
Point Mutations ◽  
Transforming Growth Factor Β ◽  
Mitogen Activated Protein Kinase ◽  
Docking Site ◽  
C Terminus

ABSTRACT Previous studies have revealed that transforming growth factor-β-activated protein kinase 1 (TAB1) interacts with p38α and induces p38α autophosphorylation. Here, we examine the sequence requirements in TAB1 and p38α that drive their interaction. Deletion and point mutations in TAB1 reveal that a proline residue in the C terminus of TAB1 (Pro412) is necessary for its interaction with p38α. Furthermore, a cryptic D-domain-like docking site was identified adjacent to the N terminus of Pro412, putting Pro412 in the φB+3 position of the docking site. Through mutational analysis, we found that the previously identified hydrophobic docking groove in p38α is involved in this interaction, whereas the CD domain and ED domain are not. Furthermore, chimeric analysis with p38β (which does not bind to TAB1) revealed a previously unidentified locus of p38α comprising Thr218 and Ile275 that is essential for specific binding of p38α to TAB1. Converting either of these residues to the corresponding amino acid of p38β abolishes p38α interaction with TAB1. These p38α mutants still can be fully activated by p38α upstream activating kinase mitogen-activated protein kinase kinase 6, but their basal activity and activation in response to some extracellular stimuli are reduced. Adjacent to Thr218 and Ile275 is a site where large conformational changes occur in the presence of docking-site peptides derived from p38α substrates and activators. This suggests that TAB1-induced autophosphorylation of p38α results from conformational changes that are similar but unique to those seen in p38α interactions with its substrates and activating kinases.

Timing of molecular events in meiosis in Saccharomyces cerevisiae: stable heteroduplex DNA is formed late in meiotic prophase

1993 ◽  
Vol 13 (1) ◽  
pp. 373-382 ◽  
Author(s):  
C Goyon ◽  
M Lichten
Keyword(s):  
Meiotic Prophase ◽  
Double Strand Breaks ◽  
Dna Breaks ◽  
Base Pairs ◽  
Heteroduplex Dna ◽  
Strand Breaks ◽  
Dna Structures ◽  
Double Strand Dna Breaks ◽  
Molecular Events ◽  
The Times

To better understand the means by which chromosomes pair and recombine during meiosis, we have determined the time of appearance of heteroduplex DNA relative to the times of appearance of double-strand DNA breaks and of mature recombined molecules. Site-specific double-strand breaks appeared early in meiosis and were formed and repaired with a timing consistent with a role for breaks as initiators of recombination. Heteroduplex-containing molecules appeared about 1 h after double-strand breaks and were followed shortly by crossover products and the first meiotic nuclear division. We conclude that parental chromosomes are stably joined in heteroduplex-containing structures late in meiotic prophase and that these structures are rapidly resolved to yield mature crossover products. If the chromosome pairing and synapsis observed earlier in meiotic prophase is mediated by formation of biparental DNA structures, these structures most likely either contain regions of non-Watson-Crick base pairs or contain regions of heteroduplex DNA that either are very short or dissociate during DNA purification. Two loci were examined in this study: the normal ARG4 locus, and an artificial locus consisting of an arg4-containing plasmid inserted at MAT. Remarkably, sequences in the ARG4 promoter that suffered double-strand cleavage at the normal ARG4 locus were not cut at significant levels when present at MAT::arg4. These results indicate that the formation of double-strand breaks during meiosis does not simply involve the specific recognition and cleavage of a short nucleotide sequence.

scholarly journals Uncoupling of the apyrimidinic/apurinic endonucleolytic and 3′→5′ exonucleolytic activities of the Nfo protein of Mycoplasma pneumoniae through mutation of specific amino acid residues

Microbiology ◽  
2014 ◽  
Vol 160 (6) ◽  
pp. 1087-1100 ◽  
Author(s):  
Silvia Estevão ◽  
Pieternella E. van der Spek ◽  
Annemarie M. C. van Rossum ◽  
Cornelis Vink
Keyword(s):  
Mycoplasma Pneumoniae ◽  
Divalent Cations ◽  
Single Point ◽  
Dna Recombination ◽  
Point Mutations ◽  
Mycoplasma Genitalium ◽  
Amino Acid Residues ◽  
Specific Amino Acid ◽  
Cleavage Activity ◽  
Ap Sites

The DNA recombination and repair machineries of Mycoplasma pneumoniae and Mycoplasma genitalium were predicted to consist of a set of ~11 proteins. The function of one of these proteins was inferred from its homology with proteins belonging to the Endo IV enzyme family. The members of this family function in the repair of apyrimidinic/apurinic (AP) sites in DNA. As such activity may be crucial in the mycoplasmal life cycle, we set out to study the Endo IV-like proteins encoded by M. pneumoniae and M. genitalium. Both proteins, termed Nfo Mpn and Nfo Mge , respectively, were assessed for their ability to interact with damaged and undamaged DNA. In the absence of divalent cations, both proteins exhibited specific cleavage of AP sites. Surprisingly, the proteins also recognized and cleaved cholesteryl-bound deoxyribose moieties in DNA, showing that these Nfo proteins may also function in repair of large DNA adducts. In the presence of Mg2+, Nfo Mpn and Nfo Mge also showed 3′→5′ exonucleolytic activity. By introduction of 13 single point mutations at highly conserved positions within Nfo Mpn , two major types of mutants could be distinguished: (i) mutants that showed no, or limited, AP cleavage activity in the presence of EDTA, but displayed significant levels of AP cleavage activity in the presence of Mg2+; these mutants displayed no, or very low, exonucleolytic activity; and (ii) mutants that only demonstrated marginal levels of AP site cleavage activity in the presence of Mg2+ and did not show exonucleolytic activity. Together, these results indicated that the AP endonucleolytic activity of the Nfo Mpn protein can be uncoupled from its 3′→5′ exonucleolytic activity.

scholarly journals Effects of Adeno-Associated Virus DNA Hairpin Structure on Recombination

2005 ◽  
Vol 79 (11) ◽  
pp. 6801-6807 ◽  
Author(s):  
Vivian W. Choi ◽  
R. Jude Samulski ◽  
Douglas M. McCarty
Keyword(s):  
Dna Recombination ◽  
Dna Hairpin ◽  
Reporter System ◽  
Controlled System ◽  
Adeno Associated Virus ◽  
Dna Structures ◽  
Evolutionarily Conserved ◽  
Intramolecular Recombination ◽  
Hairpin Structures ◽  
Dna Ends

ABSTRACT Hairpin DNA ends are evolutionarily conserved intermediates in DNA recombination. The hairpin structures present on the ends of the adeno-associated virus (AAV) genome are substrates for recombination that give rise to persistent circular and concatemeric DNA episomes through intramolecular and intermolecular recombination, respectively. We have developed circularization-dependent and orientation-specific self-complementary AAV (scAAV) vectors as a reporter system to examine recombination events involving distinct hairpin structures, i.e., closed versus open hairpins. The results suggest that intramolecular recombination (circularization) is far more efficient than intermolecular recombination (concatemerization). Among all possible combinations of terminal repeats (TRs) involved in intermolecular recombination, the closed-closed TR structures are twice as efficient as the open-open TR substrates for recombination. In addition, both intramolecular recombination and intermolecular recombination exhibit the common dependency on specific DNA polymerases and topoisomerases. The circularization-dependent and orientation-specific scAAV vectors can serve as an efficient and controlled system for the delivery of DNA structures that mimic mammalian recombination intermediates and should be useful in assaying recombination in different experimental settings as well as elucidating the molecular mechanism of recombinant AAV genome persistence.

scholarly journals A yeast ARS-binding protein activates transcription synergistically in combination with other weak activating factors.

1990 ◽  
Vol 10 (3) ◽  
pp. 887-897 ◽  
Author(s):  
A R Buchman ◽  
R D Kornberg
Keyword(s):  
Dna Sequences ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Essential Gene ◽  
Specific Binding ◽  
Binding Protein ◽  
Point Mutations ◽  
Yeast Genes

ABFI (ARS-binding protein I) is a yeast protein that binds specific DNA sequences associated with several autonomously replicating sequences (ARSs). ABFI also binds sequences located in promoter regions of some yeast genes, including DED1, an essential gene of unknown function that is transcribed constitutively at a high level. ABFI was purified by specific binding to the DED1 upstream activating sequence (UAS) and was found to recognize related sequences at several other promoters, at an ARS (ARS1), and at a transcriptional silencer (HMR E). All ABFI-binding sites, regardless of origin, provided weak UAS function in vivo when examined in test plasmids. UAS function was abolished by point mutations that reduced ABFI binding in vitro. Analysis of the DED1 promoter showed that two ABFI-binding sites combine synergistically with an adjacent T-rich sequence to form a strong constitutive activator. The DED1 T-rich element acted synergistically with all other ABFI-binding sites and with binding sites for other multifunctional yeast activators. An examination of the properties of sequences surrounding ARS1 left open the possibility that ABFI enhances the initiation of DNA replication at ARS1 by transcriptional activation.

scholarly journals Comparative Genomics of Streptococcus thermophilus Phage Species Supports a Modular Evolution Theory

1999 ◽  
Vol 73 (10) ◽  
pp. 8647-8656 ◽  
Author(s):  
Sacha Lucchini ◽  
Frank Desiere ◽  
Harald Brüssow
Keyword(s):  
Dna Recombination ◽  
Point Mutations ◽  
Streptococcus Thermophilus ◽  
Tail Fiber ◽  
Dna Repeats ◽  
Modular Evolution ◽  
Fiber Protein ◽  
Variable Domains ◽  
Replication Region ◽  
Fiber Gene

ABSTRACT The comparative analysis of five completely sequencedStreptococcus thermophilus bacteriophage genomes demonstrated that their diversification was achieved by a combination of DNA recombination events and an accumulation of point mutations. The five phages included lytic and temperate phages, both pacsite and cos site, from three distinct geographical areas. The units of genetic exchange were either large, comprising the entire morphogenesis gene cluster, excluding the putative tail fiber genes, or small, consisting of one or maximally two genes or even segments of a gene. Many indels were flanked by DNA repeats. Differences in a single putative tail fiber gene correlated with the host ranges of the phages. The predicted tail fiber protein consisted of highly conserved domains containing conspicuous glycine repeats interspersed with highly variable domains. As in the T-even coliphage adhesins, the glycine-containing domains were recombinational hot spots. Downstream of a highly conserved DNA replication region, all lytic phages showed a short duplication; in three isolates the origin of replication was repeated. The lytic phages could conceivably be derived from the temperate phages by deletion and multiple rearrangement events in the lysogeny module, giving rise to occasional selfish phages that defy the superinfection control systems of the corresponding temperate phages.

cis- and trans-acting regulatory elements of the yeast URA3 promoter

1990 ◽  
Vol 10 (10) ◽  
pp. 5257-5270
Author(s):  
A Roy ◽  
F Exinger ◽  
R Losson
Keyword(s):  
Specific Binding ◽  
Point Mutations ◽  
Regulatory Elements ◽  
Cis Acting ◽  
Cell Extracts ◽  
Dnase I Footprinting ◽  
Pair Sequence ◽  
Dyad Symmetry

Expression of the yeast pyrimidine biosynthetic gene, URA3, is induced three- to fivefold in response to uracil starvation, and this regulation is mediated by the transcriptional activator PPR1 (pyrimidine pathway regulator 1). In this study, we have analyzed the regulatory elements of the URA3 promoter by DNase I footprinting, using partially purified yeast cell extracts, by deletion mutagenesis, and by 5'-end mapping of RNA transcripts. Two DNA-binding activities have been detected, and at least four distinct cis-acting regions have been identified. A region rich in poly(dA-dT) serves as an upstream promoter element necessary for the basal level of URA3 expression. A 16-base-pair sequence with dyad symmetry acts acts as a uracil-controlled upstream activating site (UASURA) and shows a specific binding only with cell extracts from strains overproducing PPR1. This in vitro binding does not require dihydroorotic acid, the physiological inducer of URA3. The TATA region appears to be composed of two functionally distinct (constitutive and regulatory) elements. Two G + A-rich regions surrounding this TATA box bind an unidentified factor called GA-binding factor. The 5' copy, GA1, is involved in PPR1 induction and overlaps the constitutive TATA region. The 3' region, GA2, is necessary for maximal expression. Neither of these GA sequences acts as a UAS in a CYC1-lacZ context. The promoters of the unlinked but coordinately regulated URA1 and URA4 genes contain highly conserved copies of the UASURA sequence, which prompted us to investigate the effects of many point mutations within this UASURA sequence on PPR1-dependent binding. In this way, we have identified the most important residues of this binding site and found that a nonsymmetrical change of these bases is sufficient to prevent the specific binding and to suppress the UASURA activity in vivo. In addition, we showed that UASURA contains a constitutive activating element which can stimulate transcription from a heterologous promoter independently of dihydroorotic acid and PPR1.

scholarly journals Efficient production of active chicken avidin using a bacterial signal peptide in Escherichia coli

2004 ◽  
Vol 384 (2) ◽  
pp. 385-390 ◽  
Author(s):  
Vesa P. HYTÖNEN ◽  
Olli H. LAITINEN ◽  
Tomi T. AIRENNE ◽  
Heidi KIDRON ◽  
Niko J. MELTOLA ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Signal Sequence ◽  
Specific Binding ◽  
Affinity Purification ◽  
Active Form ◽  
Efficient Production ◽  
E Coli ◽  
Secretion Signal ◽  
Technology Applications ◽  
Biotin Binding

Chicken avidin is a highly popular tool with countless applications in the life sciences. In the present study, an efficient method for producing avidin protein in the periplasmic space of Escherichia coli in the active form is described. Avidin was produced by replacing the native signal sequence of the protein with a bacterial OmpA secretion signal. The yield after a single 2-iminobiotin–agarose affinity purification step was approx. 10 mg/l of virtually pure avidin. Purified avidin had 3.7 free biotin-binding sites per tetramer and showed the same biotin-binding affinity and thermal stability as egg-white avidin. Avidin crystallized under various conditions, which will enable X-ray crystallographic studies. Avidin produced in E. coli lacks the carbohydrate chains of chicken avidin and the absence of glycosylation should decrease the non-specific binding that avidin exhibits towards many materials [Rosebrough and Hartley (1996) J. Nucl. Med. 37, 1380–1384]. The present method provides a feasible and inexpensive alternative for the production of recombinant avidin, avidin mutants and avidin fusion proteins for novel avidin–biotin technology applications.

A non-B DNA can replace heptamer of V(D)J recombination when present along with a nonamer: implications in chromosomal translocations and cancer

2012 ◽  
Vol 448 (1) ◽  
pp. 115-125 ◽  
Author(s):  
Mayilaadumveettil Nishana ◽  
Sathees C. Raghavan
Keyword(s):  
Dna Cleavage ◽  
Signal Sequence ◽  
B Cell Lymphoma ◽  
Chromosomal Translocations ◽  
Heteroduplex Dna ◽  
Spacer Length ◽  
Lymphoid Malignancies ◽  
Recombination Activating Gene ◽  
Dependent Mechanism

The RAG (recombination-activating gene) complex is responsible for the generation of antigen receptor diversity by acting as a sequence-specific nuclease. Recent studies have shown that it also acts as a structure-specific nuclease. However, little is known about the factors regulating this activity at the genomic level. We show in the present study that the proximity of a V(D)J nonamer to heteroduplex DNA significantly increases RAG cleavage and binding efficiencies at physiological concentrations of MgCl2. The position of the nonamer with respect to heteroduplex DNA was important, but not orientation. A spacer length of 18 bp between the nonamer and mismatch was optimal for RAG-mediated DNA cleavage. Mutations to the sequence of the nonamer and deletion of the nonamer-binding domain of RAG1 reinforced the role of the nonamer in the enhancement in RAG cleavage. Interestingly, partial mutation of the nonamer did not significantly reduce RAG cleavage on heteroduplex DNA, suggesting that even cryptic nonamers were sufficient to enhance RAG cleavage. More importantly, we show that the fragile region involved in chromosomal translocations associated with BCL2 (B-cell lymphoma 2) can be cleaved by RAGs following a nonamer-dependent mechanism. Hence our results from the present study suggest that a non-B DNA can replace the heptamer of RSS (recombination signal sequence) when present adjacent to nonamers, explaining the generation of certain chromosomal translocations in lymphoid malignancies.

scholarly journals Cleavage of Stalled Forks by Fission Yeast Mus81/Eme1 in Absence of DNA Replication Checkpoint

2008 ◽  
Vol 19 (2) ◽  
pp. 445-456 ◽  
Author(s):  
Benoît Froget ◽  
Joël Blaisonneau ◽  
Sarah Lambert ◽  
Giuseppe Baldacci
Keyword(s):  
Dna Replication ◽  
Fission Yeast ◽  
Nuclease Activity ◽  
S Phase ◽  
Dna Breaks ◽  
Replication Checkpoint ◽  
Dna Structures ◽  
Replication Arrest ◽  
Dna Replication Checkpoint ◽  
Stalled Fork

During replication arrest, the DNA replication checkpoint plays a crucial role in the stabilization of the replisome at stalled forks, thus preventing the collapse of active forks and the formation of aberrant DNA structures. How this checkpoint acts to preserve the integrity of replication structures at stalled fork is poorly understood. In Schizosaccharomyces pombe, the DNA replication checkpoint kinase Cds1 negatively regulates the structure-specific endonuclease Mus81/Eme1 to preserve genomic integrity when replication is perturbed. Here, we report that, in response to hydroxyurea (HU) treatment, the replication checkpoint prevents S-phase–specific DNA breakage resulting from Mus81 nuclease activity. However, loss of Mus81 regulation by Cds1 is not sufficient to produce HU-induced DNA breaks. Our results suggest that unscheduled cleavage of stalled forks by Mus81 is permitted when the replisome is not stabilized by the replication checkpoint. We also show that HU-induced DNA breaks are partially dependent on the Rqh1 helicase, the fission yeast homologue of BLM, but are independent of its helicase activity. This suggests that efficient cleavage of stalled forks by Mus81 requires Rqh1. Finally, we identified an interplay between Mus81 activity at stalled forks and the Chk1-dependent DNA damage checkpoint during S-phase when replication forks have collapsed.

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