scholarly journals A Core Activity Associated with the N Terminus of the Yeast RAD52 Protein Is Revealed by RAD51 Overexpression Suppression of C-Terminal rad52 Truncation Alleles

Genetics ◽  
1999 ◽  
Vol 153 (2) ◽  
pp. 681-692 ◽  
Author(s):  
Erin N Asleson ◽  
Ron J Okagaki ◽  
Dennis M Livingston
Keyword(s):  
Amino Acids ◽  
Double Strand Breaks ◽  
Missense Mutations ◽  
Internal Deletion ◽  
Spore Viability ◽  
Strand Breaks ◽  
C Terminus ◽  
N Terminus ◽  
Rad52 Protein ◽  
Partial Activity

Abstract C-terminal rad52 truncation and internal deletion mutants were characterized for their ability to repair MMS-induced double-strand breaks and to produce viable spores during meiosis. The rad52-Δ251 allele, encoding the N-terminal 251 amino acids of the predicted 504-amino-acid polypeptide, supports partial activity for both functions. Furthermore, RAD51 overexpression completely suppresses the MMS sensitivity of a rad52-Δ251 mutant. The absence of the C terminus in the truncated protein makes it likely that suppression occurs by bypassing the C-terminal functions of Rad52p. RAD51 overexpression does not suppress the low level of spore viability that the rad52-Δ251 allele causes and only partially suppresses the defect in rad52 alleles encoding the N-terminal 292 or 327 amino acids. The results of this study also show that intragenic complementation between rad52 alleles is governed by a complex relationship that depends heavily on the two alleles involved and their relative dosage. In heteroallelic rad52 diploids, the rad52-Δ251 allele does not complement rad52 missense mutations altering residues 61 or 64 in the N terminus. However, complementation is achieved with each of these missense alleles when the rad52-Δ251 allele is overexpressed. Complementation also occurs between rad52-Δ327 and an internal deletion allele missing residues 210 through 327. We suggest that the first 251 amino acids of Rad52p constitute a core domain that provides critical RAD52 activities.

scholarly journals A conserved Ctp1/CtIP C-terminal peptide stimulates Mre11 endonuclease activity

2021 ◽  
Vol 118 (11) ◽  
pp. e2016287118
Author(s):  
Aleksandar Zdravković ◽  
James M. Daley ◽  
Arijit Dutta ◽  
Tatsuya Niwa ◽  
Yasuto Murayama ◽  
...  
Keyword(s):  
Amino Acids ◽  
Homologous Recombination ◽  
Schizosaccharomyces Pombe ◽  
Double Strand Breaks ◽  
Pivotal Role ◽  
Endonuclease Activity ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
C Terminus ◽  
Dna Strands

The Mre11-Rad50-Nbs1 complex (MRN) is important for repairing DNA double-strand breaks (DSBs) by homologous recombination (HR). The endonuclease activity of MRN is critical for resecting 5′-ended DNA strands at DSB ends, producing 3′-ended single-strand DNA, a prerequisite for HR. This endonuclease activity is stimulated by Ctp1, the Schizosaccharomyces pombe homolog of human CtIP. Here, with purified proteins, we show that Ctp1 phosphorylation stimulates MRN endonuclease activity by inducing the association of Ctp1 with Nbs1. The highly conserved extreme C terminus of Ctp1 is indispensable for MRN activation. Importantly, a polypeptide composed of the conserved 15 amino acids at the C terminus of Ctp1 (CT15) is sufficient to stimulate Mre11 endonuclease activity. Furthermore, the CT15 equivalent from CtIP can stimulate human MRE11 endonuclease activity, arguing for the generality of this stimulatory mechanism. Thus, we propose that Nbs1-mediated recruitment of CT15 plays a pivotal role in the activation of the Mre11 endonuclease by Ctp1/CtIP.

scholarly journals Structure of the human DNA-repair protein RAD52 containing surface mutations

2016 ◽  
Vol 72 (8) ◽  
pp. 598-603 ◽  
Author(s):  
Mika Saotome ◽  
Kengo Saito ◽  
Keiichi Onodera ◽  
Hitoshi Kurumizaka ◽  
Wataru Kagawa
Keyword(s):  
Ring Structure ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Homologous Recombinational Repair ◽  
Repair Protein ◽  
Rad52 Protein ◽  
Dna Repair Protein ◽  
Binding Groove ◽  
Partially Unfolded

The Rad52 protein is a eukaryotic single-strand DNA-annealing protein that is involved in the homologous recombinational repair of DNA double-strand breaks. The isolated N-terminal half of the human RAD52 protein (RAD521–212) forms an undecameric ring structure with a surface that is mostly positively charged. In the present study, it was found that RAD521–212containing alanine mutations of the charged surface residues (Lys102, Lys133 and Glu202) is highly amenable to crystallization. The structure of the mutant RAD521–212was solved at 2.4 Å resolution. The structure revealed an association between the symmetry-related RAD521–212rings, in which a partially unfolded, C-terminal region of RAD52 extended into the DNA-binding groove of the neighbouring ring in the crystal. The alanine mutations probably reduced the surface entropy of the RAD521–212ring and stabilized the ring–ring association observed in the crystal.

scholarly journals The Extracellular Transport Signal of the Vibrio cholerae Endochitinase (ChiA) Is a Structural Motif Located between Amino Acids 75 and 555

2002 ◽  
Vol 184 (8) ◽  
pp. 2225-2234 ◽  
Author(s):  
Jason P. Folster ◽  
Terry D. Connell
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Vibrio Cholerae ◽  
Structural Information ◽  
Amino Acid Sequences ◽  
Structural Motif ◽  
Terminal Branch ◽  
C Terminus ◽  
N Terminus ◽  
Extracellular Transport

ABSTRACT ChiA, an 88-kDa endochitinase encoded by the chiA gene of the gram-negative enteropathogen Vibrio cholerae, is secreted via the eps-encoded main terminal branch of the general secretory pathway (GSP), a mechanism which also transports cholera toxin. To localize the extracellular transport signal of ChiA that initiates transport of the protein through the GSP, a chimera comprised of ChiA fused at the N terminus with the maltose-binding protein (MalE) of Escherichia coli and fused at the C terminus with a 13-amino-acid epitope tag (E-tag) was expressed in strain 569B(chiA::Kanr), a chiA-deficient but secretion-competent mutant of V. cholerae. Fractionation studies revealed that blockage of the natural N terminus and C terminus of ChiA did not prevent secretion of the MalE-ChiA-E-tag chimera. To locate the amino acid sequences which encoded the transport signal, a series of truncations of ChiA were engineered. Secretion of the mutant polypeptides was curtailed only when ChiA was deleted from the N terminus beyond amino acid position 75 or from the C terminus beyond amino acid 555. A mutant ChiA comprised of only those amino acids was secreted by wild-type V. cholerae but not by an epsD mutant, establishing that amino acids 75 to 555 independently harbored sufficient structural information to promote secretion by the GSP of V. cholerae. Cys77 and Cys537, two cysteines located just within the termini of ChiA(75-555), were not required for secretion, indicating that those residues were not essential for maintaining the functional activity of the ChiA extracellular transport signal.

scholarly journals CTCF cooperates with CtIP to drive homologous recombination repair of double-strand breaks

2019 ◽  
Vol 47 (17) ◽  
pp. 9160-9179 ◽  
Author(s):  
Soon Young Hwang ◽  
Mi Ae Kang ◽  
Chul Joon Baik ◽  
Yejin Lee ◽  
Ngo Thanh Hang ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Critical Role ◽  
Control Point ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
C Terminus ◽  
Dna End Resection ◽  
End Resection

Abstract The pleiotropic CCCTC-binding factor (CTCF) plays a role in homologous recombination (HR) repair of DNA double-strand breaks (DSBs). However, the precise mechanistic role of CTCF in HR remains largely unclear. Here, we show that CTCF engages in DNA end resection, which is the initial, crucial step in HR, through its interactions with MRE11 and CtIP. Depletion of CTCF profoundly impairs HR and attenuates CtIP recruitment at DSBs. CTCF physically interacts with MRE11 and CtIP and promotes CtIP recruitment to sites of DNA damage. Subsequently, CTCF facilitates DNA end resection to allow HR, in conjunction with MRE11–CtIP. Notably, the zinc finger domain of CTCF binds to both MRE11 and CtIP and enables proficient CtIP recruitment, DNA end resection and HR. The N-terminus of CTCF is able to bind to only MRE11 and its C-terminus is incapable of binding to MRE11 and CtIP, thereby resulting in compromised CtIP recruitment, DSB resection and HR. Overall, this suggests an important function of CTCF in DNA end resection through the recruitment of CtIP at DSBs. Collectively, our findings identify a critical role of CTCF at the first control point in selecting the HR repair pathway.

scholarly journals Insight from TonB Hybrid Proteins into the Mechanism of Iron Transport through the Outer Membrane

2008 ◽  
Vol 190 (11) ◽  
pp. 4001-4016 ◽  
Author(s):  
Wallace A. Kaserer ◽  
Xiaoxu Jiang ◽  
Qiaobin Xiao ◽  
Daniel C. Scott ◽  
Matthew Bauler ◽  
...  
Keyword(s):  
Amino Acids ◽  
Outer Membrane ◽  
Cell Envelope ◽  
Outer Membrane Proteins ◽  
Binding Motif ◽  
Inner Membrane ◽  
E Coli ◽  
Hybrid Proteins ◽  
C Terminus ◽  
N Terminus

ABSTRACT We created hybrid proteins to study the functions of TonB. We first fused the portion of Escherichia coli tonB that encodes the C-terminal 69 amino acids (amino acids 170 to 239) of TonB downstream from E. coli malE (MalE-TonB69C). Production of MalE-TonB69C in tonB + bacteria inhibited siderophore transport. After overexpression and purification of the fusion protein on an amylose column, we proteolytically released the TonB C terminus and characterized it. Fluorescence spectra positioned its sole tryptophan (W213) in a weakly polar site in the protein interior, shielded from quenchers. Affinity chromatography showed the binding of the TonB C-domain to other proteins: immobilized TonB-dependent (FepA and colicin B) and TonB-independent (FepAΔ3-17, OmpA, and lysozyme) proteins adsorbed MalE-TonB69C, revealing a general affinity of the C terminus for other proteins. Additional constructions fused full-length TonB upstream or downstream of green fluorescent protein (GFP). TonB-GFP constructs had partial functionality but no fluorescence; GFP-TonB fusion proteins were functional and fluorescent. The activity of the latter constructs, which localized GFP in the cytoplasm and TonB in the cell envelope, indicate that the TonB N terminus remains in the inner membrane during its biological function. Finally, sequence analyses revealed homology in the TonB C terminus to E. coli YcfS, a proline-rich protein that contains the lysin (LysM) peptidoglycan-binding motif. LysM structural mimicry occurs in two positions of the dimeric TonB C-domain, and experiments confirmed that it physically binds to the murein sacculus. Together, these findings infer that the TonB N terminus remains associated with the inner membrane, while the downstream region bridges the cell envelope from the affinity of the C terminus for peptidoglycan. This architecture suggests a membrane surveillance model of action, in which TonB finds occupied receptor proteins by surveying the underside of peptidoglycan-associated outer membrane proteins.

scholarly journals The BRCA2-MEILB2-BRME1 complex governs meiotic recombination and impairs the mitotic BRCA2-RAD51 function in cancer cells

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Jingjing Zhang ◽  
Manickam Gurusaran ◽  
Yasuhiro Fujiwara ◽  
Kexin Zhang ◽  
Meriem Echbarthi ◽  
...  
Keyword(s):  
Cancer Susceptibility ◽  
Susceptibility Gene ◽  
Breast Cancer Susceptibility ◽  
Double Strand Breaks ◽  
Breast Cancer Susceptibility Gene ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
C Terminus ◽  
Self Association ◽  
Cancer Susceptibility Gene

AbstractBreast cancer susceptibility gene II (BRCA2) is central in homologous recombination (HR). In meiosis, BRCA2 binds to MEILB2 to localize to DNA double-strand breaks (DSBs). Here, we identify BRCA2 and MEILB2-associating protein 1 (BRME1), which functions as a stabilizer of MEILB2 by binding to an α-helical N-terminus of MEILB2 and preventing MEILB2 self-association. BRCA2 binds to the C-terminus of MEILB2, resulting in the formation of the BRCA2-MEILB2-BRME1 ternary complex. In Brme1 knockout (Brme1−/−) mice, the BRCA2-MEILB2 complex is destabilized, leading to defects in DSB repair, homolog synapsis, and crossover formation. Persistent DSBs in Brme1−/− reactivate the somatic-like DNA-damage response, which repairs DSBs but cannot complement the crossover formation defects. Further, MEILB2-BRME1 is activated in many human cancers, and somatically expressed MEILB2-BRME1 impairs mitotic HR. Thus, the meiotic BRCA2 complex is central in meiotic HR, and its misregulation is implicated in cancer development.

scholarly journals Localization of Functional Domains of the Mitogenic Toxin of Pasteurella multocida

2001 ◽  
Vol 69 (12) ◽  
pp. 7839-7850 ◽  
Author(s):  
Gillian D. Pullinger ◽  
R. Sowdhamini ◽  
Alistair J. Lax
Keyword(s):  
Amino Acids ◽  
Fusion Proteins ◽  
Pasteurella Multocida ◽  
Transmembrane Domain ◽  
Polyclonal Antibodies ◽  
Full Length ◽  
Cell Binding ◽  
Terminal Fragment ◽  
C Terminus ◽  
N Terminus

ABSTRACT The locations of the catalytic and receptor-binding domains of thePasteurella multocida toxin (PMT) were investigated. N- and C-terminal fragments of PMT were cloned and expressed as fusion proteins with affinity tags. Purified fusion proteins were assessed in suitable assays for catalytic activity and cell-binding ability. A C-terminal fragment (amino acids 681 to 1285) was catalytically active. When microinjected into quiescent Swiss 3T3 cells, it induced changes in cell morphology typical of toxin-treated cells and stimulated DNA synthesis. An N-terminal fragment with a His tag at the C terminus (amino acids 1 to 506) competed with full-length toxin for binding to surface receptors and therefore contains the cell-binding domain. The inactive mutant containing a mutation near the C terminus (C1165S) also bound to cells in this assay. Polyclonal antibodies raised to the N-terminal PMT region bound efficiently to full-length native toxin, suggesting that the N terminus is surface located. Antibodies to the C terminus of PMT were microinjected into cells and inhibited the activity of toxin added subsequently to the medium, confirming that the C terminus contains the active site. Analysis of the PMT sequence predicted a putative transmembrane domain with predicted hydrophobic and amphipathic helices near the N terminus over the region of homology to the cytotoxic necrotizing factors. The C-terminal end of PMT was predicted to be a mixed α/β domain, a structure commonly found in catalytic domains. Homology to proteins of known structure and threading calculations supported these assignments.

scholarly journals Schizosaccharomyces pombeRdh54 (TID1) Acts with Rhp54 (RAD54) to Repair Meiotic Double-Strand Breaks

2003 ◽  
Vol 14 (11) ◽  
pp. 4707-4720 ◽  
Author(s):  
Michael G. Catlett ◽  
Susan L. Forsburg
Keyword(s):  
Meiotic Recombination ◽  
Double Strand Breaks ◽  
Cell Analysis ◽  
Yeast Two Hybrid ◽  
Spore Viability ◽  
Strand Breaks ◽  
Functional Homology ◽  
Damage Repair ◽  
Two Hybrid

We report the characterization of rdh54+, the second fission yeast Schizosaccharomyces pombe Rad54 homolog. rdh54+shares sequence and functional homology to budding yeast RDH54/TID1. Rdh54p is present during meiosis with appropriate timing for a meiotic recombination factor. It interacts with Rhp51 and the meiotic Rhp51 homolog Dmc1 in yeast two-hybrid assays. Deletion of rdh54+has no effect on DNA damage repair during the haploid vegetative cell cycle. In meiosis, however, rdh54Δ shows decreased spore viability and homologous recombination with a concomitant increase in sister chromatid exchange. The rdh54Δ single mutant repairs meiotic breaks with similar timing to wild type, suggesting redundancy of meiotic recombination factors. Consistent with this, the rdh54Δ rhp54Δ double mutant fails to repair meiotic double strand breaks. Live cell analysis shows that rdh54Δ rhp54Δ asci do not arrest, but undergo both meiotic divisions with near normal timing, suggesting that failure to repair double strand breaks in S. pombe meiosis does not result in checkpoint arrest.

scholarly journals The unique tropism ofMycobacterium lepraeto the nasal epithelial cells can be explained by the mammalian cell entry protein 1A

2018 ◽  
Author(s):  
Viesta Beby Fadlitha ◽  
Fuki Yamamoto ◽  
Irfan Idris ◽  
Haslindah Dahlan ◽  
Naoya Sato ◽  
...  
Keyword(s):  
Amino Acids ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Recombinant Proteins ◽  
Cell Entry ◽  
Important Region ◽  
C Terminus ◽  
Protein Encoding ◽  
N Terminus ◽  
Encoding Genes

AbstractLeprosy is a chronic infection where the skin and peripheral nervous system is invaded byMycobacterium leprae. The infection mechanism remains unknown in part because culture methods have not been established yet forM.leprae.Mce1A protein (442 aa) is coded by mce1A (1326 bp) ofM.leprae. The mce1A homolog inMycobacterium tuberculosisis known to be associated withM.tuberculosisepithelial cell entry, and survival and multiplication within macrophages. Studies using recombinant proteins have indicated that mce1A ofM.lepraeis also associated with epithelial cell entry. This study is aimed at identifying particular sequences within mce1A associated withM.lepraeepithelial cell entry.Recombinant proteins having N-terminus and C-terminus truncations of the mce1A region ofM.lepraewere created inEschericia coli.Entry activity of latex beads, coated with these truncated proteins (r-lep37kDa and r-lep27kDa), into HeLa cells was observed by electron microscopy. The entry activity was preserved even when 315 bp (105 aa) and 922 bp (308 aa) was truncated from the N-terminus and C-terminus, respectively. This 316 – 921 bp region was divided into three sub-regions: 316 – 531 bp (InvX), 532 – 753 bp (InvY), and 754 – 921 bp (InvZ). Each sub-region was cloned into an AIDA vector and expressed on the surface ofE.coli.Entry of theseE.coliinto monolayer-cultured HeLa and RPMI2650 cells was observed by electron microscopy. OnlyE.coliharboring the InvX sub-region exhibited cell entry. InvX was further divided into 4 domains, InvXa - InvXd, containing sequences 1 – 24 aa, 25 – 46 aa, 47 – 57 aa, and 58 – 72 aa, respectively.RecombinantE.coli, expressing each of InvXa - InvXd on the surface, were treated with antibodies against these domains, then added to monolayer cultured RPMI cells. The effectiveness of these antibodies in preventing cell entry was studied by colony counting. Entry activity was suppressed by antibodies against InvXa, InvXb, and InvXd. This suggests that these three InvX domains of mce1A are important forM.lepraeinvasion into nasal epithelial cells.Author SummaryMce1A protein is encoded by the mce1A region of mce1 locus ofM.tuberculosisandM.leprae, and is involved in the bacteria’s invasion into epithelial cells. The present study revealed that the active sequence ofM.lepraeinvolved in the invasion into nasal mucosa epithelial cells is present in the 316-531 bp region of mce1A.The most important region of mce1A protein involved in the invasion ofM.tuberculosisinto human epithelial cells is called the InvIII region, which is located between amino acids at position 130 to 152. The InvIII region ofM.tuberculosiscorresponds to InvXb ofM.leprae. The sequences of these regions are identical between amino acids at positions 10 to position 22 as counted from the N terminus, except that amino acids at positions 1 to 3, 5, 8, 9, 13 are different betweenM.lepraeandM.tuberculosis. Suppression test results also indicated that the most important region of mce1A protein ofM.lepraeinvolved in the invasion into human epithelial cells is different from thatM.tuberculosis. WhileM.tuberculosishas 3,959 protein-encoding genes and only 6 pseudogenes,M.lepraehas only 1,604 protein-encoding genes but has 1,116 pseudogenes indicating that inM.leprae, far more proteins are inactivated as compared toM.tuberculosis. The present study also revealed that, as inM.tuberculosis,the mce1A protein is expressed on the surface of bacteria as a native protein. In light of these data, the mce1A protein is considered to be one of the most important proteins involved in the invasion ofM.lepraeinto nasal mucosa epithelial cells.

scholarly journals Treacle controls the nucleolar response to rDNA breaks via TOPBP1 recruitment and ATR activation

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Clémence Mooser ◽  
Ioanna-Eleni Symeonidou ◽  
Pia-Amata Leimbacher ◽  
Alison Ribeiro ◽  
Ann-Marie K. Shorrocks ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Large Scale ◽  
Rna Synthesis ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Transcriptional Inhibition ◽  
C Terminus ◽  
Atr Activation ◽  
Nucleolar Segregation

AbstractInduction of DNA double-strand breaks (DSBs) in ribosomal DNA (rDNA) repeats is associated with ATM-dependent repression of ribosomal RNA synthesis and large-scale reorganization of nucleolar architecture, but the signaling events that regulate these responses are largely elusive. Here we show that the nucleolar response to rDNA breaks is dependent on both ATM and ATR activity. We further demonstrate that ATM- and NBS1-dependent recruitment of TOPBP1 in the nucleoli is required for inhibition of ribosomal RNA synthesis and nucleolar segregation in response to rDNA breaks. Mechanistically, TOPBP1 recruitment is mediated by phosphorylation-dependent interactions between three of its BRCT domains and conserved phosphorylated Ser/Thr residues at the C-terminus of the nucleolar phosphoprotein Treacle. Our data thus reveal an important cooperation between TOPBP1 and Treacle in the signaling cascade that triggers transcriptional inhibition and nucleolar segregation in response to rDNA breaks.

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