scholarly journals Recruitment of Bacillus subtilis RecN to DNA Double-Strand Breaks in the Absence of DNA End Processing

2006 ◽  
Vol 188 (2) ◽  
pp. 353-360 ◽  
Author(s):  
Humberto Sanchez ◽  
Dawit Kidane ◽  
M. Castillo Cozar ◽  
Peter L. Graumann ◽  
Juan C. Alonso
Keyword(s):  
Bacillus Subtilis ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Double Strand Breaks ◽  
Live Cells ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Recombination Proteins ◽  
Killing Action ◽  
Green Fluorescent

ABSTRACT The recognition and processing of double-strand breaks (DSBs) to a 3′ single-stranded DNA (ssDNA) overhang structure in Bacillus subtilis is poorly understood. Mutations in addA and addB or null mutations in recJ (ΔrecJ), recQ (ΔrecQ), or recS (ΔrecS) genes, when present in otherwise-Rec+ cells, render cells moderately sensitive to the killing action of different DNA-damaging agents. Inactivation of a RecQ-like helicase (ΔrecQ or ΔrecS) in addAB cells showed an additive effect; however, when ΔrecJ was combined with addAB, a strong synergistic effect was observed with a survival rate similar to that of ΔrecA cells. RecF was nonepistatic with RecJ or AddAB. After induction of DSBs, RecN-yellow fluorescent protein (YFP) foci were formed in addAB ΔrecJ cells. AddAB and RecJ were required for the formation of a single RecN focus, because in their absence multiple RecN-YFP foci accumulated within the cells. Green fluorescent protein-RecA failed to form filamentous structures (termed threads) in addAB ΔrecJ cells. We propose that RecN is one of the first recombination proteins detected as a discrete focus in live cells in response to DSBs and that either AddAB or RecQ(S)-RecJ are required for the generation of a duplex with a 3′-ssDNA tail needed for filament formation of RecA.

scholarly journals Clp and Lon Proteases Occupy Distinct Subcellular Positions in Bacillus subtilis

2008 ◽  
Vol 190 (20) ◽  
pp. 6758-6768 ◽  
Author(s):  
Lyle A. Simmons ◽  
Alan D. Grossman ◽  
Graham C. Walker
Keyword(s):  
Bacillus Subtilis ◽  
Stress Responses ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Normal Growth ◽  
Temporal Organization ◽  
Hexameric Atpase ◽  
Fluorescent Moiety ◽  
Green Fluorescent

ABSTRACT Among other functions, ATP-dependent proteases degrade misfolded proteins and remove several key regulatory proteins necessary to activate stress responses. In Bacillus subtilis, ClpX, ClpE, and ClpC form homohexameric ATPases that couple to the ClpP peptidase. To understand where these peptidases and ATPases localize in living cells, each protein was fused to a fluorescent moiety. We found that ClpX-GFP (green fluorescent protein) and ClpP-GFP localized as focal assemblies in areas that were not occupied by the nucleoid. We found that the percentage of cells with ClpP-GFP foci increased following heat shock independently of protein synthesis. We determined that ClpE-YFP (yellow fluorescent protein) and ClpC-YFP formed foci coincident with nucleoid edges, usually near cell poles. Furthermore, we found that ClpQ-YFP (HslV) localized as small foci, usually positioned near the cell membrane. We found that ClpQ-YFP foci were dependent on the presence of the cognate hexameric ATPase ClpY (HslU). Moreover, we found that LonA-GFP is coincident with the nucleoid during normal growth and that LonA-GFP also localized to the forespore during development. We also investigated LonB-GFP and found that this protein localized to the forespore membrane early in development, followed by localization throughout the forespore later in development. Our comprehensive study has shown that in B. subtilis several ATP-fueled proteases occupy distinct subcellular locations. With these data, we suggest that substrate specificity could be determined, in part, by the spatial and temporal organization of proteases in vivo.

scholarly journals Replication Restart after Replication-Transcription Conflicts Requires RecA in Bacillus subtilis

2015 ◽  
Vol 197 (14) ◽  
pp. 2374-2382 ◽  
Author(s):  
Samuel Million-Weaver ◽  
Ariana Nakta Samadpour ◽  
Houra Merrikh
Keyword(s):  
Bacillus Subtilis ◽  
Double Strand Breaks ◽  
Holliday Junction ◽  
Replication Forks ◽  
Dna Breaks ◽  
Site Specific ◽  
Content Type ◽  
Strand Breaks ◽  
Recombination Proteins ◽  
Replication Restart

ABSTRACTEfficient duplication of genomes depends on reactivation of replication forks outside the origin. Replication restart can be facilitated by recombination proteins, especially if single- or double-strand breaks form in the DNA. Each type of DNA break is processed by a distinct pathway, though both depend on the RecA protein. One common obstacle that can stall forks, potentially leading to breaks in the DNA, is transcription. Though replication stalling by transcription is prevalent, the nature of DNA breaks and the prerequisites for replication restart in response to these encounters remain unknown. Here, we used an engineered site-specific replication-transcription conflict to identify and dissect the pathways required for the resolution and restart of replication forks stalled by transcription inBacillus subtilis. We found that RecA, its loader proteins RecO and AddAB, and the Holliday junction resolvase RecU are required for efficient survival and replication restart after conflicts with transcription. Genetic analyses showed that RecO and AddAB act in parallel to facilitate RecA loading at the site of the conflict but that they can each partially compensate for the other's absence. Finally, we found that RecA and either RecO or AddAB are required for the replication restart and helicase loader protein, DnaD, to associate with the engineered conflict region. These results suggest that conflicts can lead to both single-strand gaps and double-strand breaks in the DNA and that RecA loading and Holliday junction resolution are required for replication restart at regions of replication-transcription conflicts.IMPORTANCEHead-on conflicts between replication and transcription occur when a gene is expressed from the lagging strand. These encounters stall the replisome and potentially break the DNA. We investigated the necessary mechanisms forBacillus subtiliscells to overcome a site-specific engineered conflict with transcription of a protein-coding gene. We found that the recombination proteins RecO and AddAB both load RecA onto the DNA in response to the head-on conflict. Additionally, RecA loading by one of the two pathways was required for both replication restart and efficient survival of the collision. Our findings suggest that both single-strand gaps and double-strand DNA breaks occur at head-on conflict regions and demonstrate a requirement for recombination to restart replication after collisions with transcription.

scholarly journals Retention but Not Recruitment of Crb2 at Double-Strand Breaks Requires Rad1 and Rad3 Complexes

2003 ◽  
Vol 23 (17) ◽  
pp. 6150-6158 ◽  
Author(s):  
Li-Lin Du ◽  
Toru M. Nakamura ◽  
Bettina A. Moser ◽  
Paul Russell
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Double Strand Breaks ◽  
Live Cells ◽  
Adapter Protein ◽  
Wild Type ◽  
Dna Double Strand Breaks ◽  
Checkpoint Signaling ◽  
Strand Breaks ◽  
Checkpoint Protein

ABSTRACT The fission yeast checkpoint protein Crb2, related to budding yeast Rad9 and human 53BP1 and BRCA1, has been suggested to act as an adapter protein facilitating the phosphorylation of specific substrates by Rad3-Rad26 kinase. To further understand its role in checkpoint signaling, we examined its localization in live cells by using fluorescence microscopy. In response to DNA damage, Crb2 localizes to distinct nuclear foci, which represent sites of DNA double-strand breaks (DSBs). Crb2 colocalizes with Rad22 at persistent foci, suggesting that Crb2 is retained at sites of DNA damage during repair. Damage-induced Crb2 foci still form in cells defective in Rad1, Rad3, and Rad17 complexes, but these foci do not persist as long as in wild-type cells. Our results suggest that Crb2 functions at the sites of DNA damage, and its regulated persistent localization at damage sites may be involved in facilitating DNA repair and/or maintaining the checkpoint arrest while DNA repair is under way.

scholarly journals Mouse TEX15 is essential for DNA double-strand break repair and chromosomal synapsis during male meiosis

2008 ◽  
Vol 180 (4) ◽  
pp. 673-679 ◽  
Author(s):  
Fang Yang ◽  
Sigrid Eckardt ◽  
N. Adrian Leu ◽  
K. John McLaughlin ◽  
Peijing Jeremy Wang
Keyword(s):  
Meiotic Recombination ◽  
Strand Break ◽  
Male Meiosis ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Homologous Chromosomes ◽  
Recombination Proteins ◽  
Meiotic Chromosomes ◽  
Dna Double Strand Break

During meiosis, homologous chromosomes undergo synapsis and recombination. We identify TEX15 as a novel protein that is required for chromosomal synapsis and meiotic recombination. Loss of TEX15 function in mice causes early meiotic arrest in males but not in females. Specifically, TEX15-deficient spermatocytes exhibit a failure in chromosomal synapsis. In mutant spermatocytes, DNA double-strand breaks (DSBs) are formed, but localization of the recombination proteins RAD51 and DMC1 to meiotic chromosomes is severely impaired. Based on these data, we propose that TEX15 regulates the loading of DNA repair proteins onto sites of DSBs and, thus, its absence causes a failure in meiotic recombination.

scholarly journals Targeted Gene Knockout Reveals a Role in Meiotic Recombination for ZHP-3, a Zip3-Related Protein in Caenorhabditis elegans

2004 ◽  
Vol 24 (18) ◽  
pp. 7998-8006 ◽  
Author(s):  
Verena Jantsch ◽  
Pawel Pasierbek ◽  
Michael M. Mueller ◽  
Dieter Schweizer ◽  
Michael Jantsch ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Meiotic Recombination ◽  
Fluorescent Protein ◽  
Gene Knockout ◽  
Chiasma Formation ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
C Elegans ◽  
Reciprocal Recombination

ABSTRACT The meiotically expressed Zip3 protein is found conserved from Saccharomyces cerevisiae to humans. In baker's yeast, Zip3p has been implicated in synaptonemal complex (SC) formation, while little is known about the protein's function in multicellular organisms. We report here the successful targeted gene disruption of zhp-3 (K02B12.8), the ZIP3 homolog in the nematode Caenorhabditis elegans. Homozygous zhp-3 knockout worms show normal homologue pairing and SC formation. Also, the timing of appearance and the nuclear localization of the recombination protein Rad-51 seem normal in these animals, suggesting proper initiation of meiotic recombination by DNA double-strand breaks. However, the occurrence of univalents during diplotene indicates that C. elegans ZHP-3 protein is essential for reciprocal recombination between homologous chromosomes and thus chiasma formation. In the absence of ZHP-3, reciprocal recombination is abolished and double-strand breaks seem to be repaired via alternative pathways, leading to achiasmatic chromosomes and the occurrence of univalents during meiosis I. Green fluorescent protein-tagged C. elegans ZHP-3 forms lines between synapsed chromosomes and requires the SC for its proper localization.

scholarly journals Proteins pinpoint double strand breaks

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Michael M Cox
Keyword(s):  
Dna Damage ◽  
Green Fluorescent Protein ◽  
High Efficiency ◽  
Fluorescent Protein ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
Green Fluorescent ◽  
Important Form ◽  
Living Bacteria

Combining green fluorescent protein with a protein that only binds to double strand breaks in DNA allows these breaks—which are an important form of DNA damage—to be detected with high efficiency in living bacteria.

scholarly journals A Zip3-like protein plays a role in crossover formation in the SC-less meiosis of the protist Tetrahymena

2017 ◽  
Vol 28 (6) ◽  
pp. 825-833 ◽  
Author(s):  
Anura Shodhan ◽  
Kensuke Kataoka ◽  
Kazufumi Mochizuki ◽  
Maria Novatchkova ◽  
Josef Loidl
Keyword(s):  
Ring Finger ◽  
Specific Protein ◽  
Double Strand Breaks ◽  
Recombinational Repair ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Recombination Proteins ◽  
Evolutionarily Conserved ◽  
Ring Finger Proteins ◽  
Segregation Of Chromosomes

When programmed meiotic DNA double-strand breaks (DSBs) undergo recombinational repair, genetic crossovers (COs) may be formed. A certain level of this is required for the faithful segregation of chromosomes, but the majority of DSBs are processed toward a safer alternative, namely noncrossovers (NCOs), via nonreciprocal DNA exchange. At the crossroads between these two DSB fates is the Msh4-Msh5 (MutSγ) complex, which stabilizes CO-destined recombination intermediates and members of the Zip3/RNF212 family of RING finger proteins, which in turn stabilize MutSγ. These proteins function in the context of the synaptonemal complex (SC) and mainly act on SC-dependent COs. Here we show that in the SC-less ciliate Tetrahymena, Zhp3 (a protein distantly related to Zip3/RNF212), together with MutSγ, is responsible for the majority of COs. This activity of Zhp3 suggests an evolutionarily conserved SC-independent strategy for balancing CO:NCO ratios. Moreover, we report a novel meiosis-specific protein, Sa15, as an interacting partner of Zhp3. Sa15 forms linear structures in meiotic prophase nuclei to which Zhp3 localizes. Sa15 is required for a wild-type level of CO formation. Its linear organization suggests the existence of an underlying chromosomal axis that serves as a scaffold for Zhp3 and other recombination proteins.

scholarly journals Construction and Application of Epitope- and Green Fluorescent Protein-Tagging Integration Vectors for Bacillus subtilis

2002 ◽  
Vol 68 (5) ◽  
pp. 2624-2628 ◽  
Author(s):  
Marcus Kaltwasser ◽  
Thomas Wiegert ◽  
Wolfgang Schumann
Keyword(s):  
Bacillus Subtilis ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Pcr Amplification ◽  
Epitope Tag ◽  
Inner Membrane Protein ◽  
Green Fluorescent Protein Tagging ◽  
Green Fluorescent ◽  
Protein Tagging

ABSTRACT Here we describe the construction and application of six new tagging vectors allowing the fusion of two different types of tagging sequences, epitope and localization tags, to any Bacillus subtilis protein. These vectors are based on the backbone of pMUTIN2 and replace the lacZ gene with tagging sequences. Fusion of the tagging sequences occurs by PCR amplification of the 3′ terminal part of the gene of interest (about 300 bp), insertion into the tagging vector in such a way that a fusion protein will be synthesized upon integration of the whole vector via homologous recombination with the chromosomal gene. Three of these tagging sequences (FLAG, hemagglutinin, and c-Myc) allow the covalent addition of a short epitope tag and thereby detection of the fusion proteins in immunoblots, while three other tags (green fluorescent protein+, yellow fluorescent protein, and cyan fluorescent protein) are helpful in assigning proteins within one of the compartments of the cell. The versatility of these vectors was demonstrated by fusing these tags to the cytoplasmically located HtpG and the inner membrane protein FtsH.

Repair pathway choice for double-strand breaks

2020 ◽  
Vol 64 (5) ◽  
pp. 765-777 ◽  
Author(s):  
Yixi Xu ◽  
Dongyi Xu
Keyword(s):  
Cell Cycle ◽  
Double Strand Breaks ◽  
Homologous Region ◽  
Gene Repair ◽  
Repair Pathway ◽  
Dna Double Strand Breaks ◽  
Environmental Radiation ◽  
Strand Breaks ◽  
Dna End Resection ◽  
End Resection

Abstract Deoxyribonucleic acid (DNA) is at a constant risk of damage from endogenous substances, environmental radiation, and chemical stressors. DNA double-strand breaks (DSBs) pose a significant threat to genomic integrity and cell survival. There are two major pathways for DSB repair: nonhomologous end-joining (NHEJ) and homologous recombination (HR). The extent of DNA end resection, which determines the length of the 3′ single-stranded DNA (ssDNA) overhang, is the primary factor that determines whether repair is carried out via NHEJ or HR. NHEJ, which does not require a 3′ ssDNA tail, occurs throughout the cell cycle. 53BP1 and the cofactors PTIP or RIF1-shieldin protect the broken DNA end, inhibit long-range end resection and thus promote NHEJ. In contrast, HR mainly occurs during the S/G2 phase and requires DNA end processing to create a 3′ tail that can invade a homologous region, ensuring faithful gene repair. BRCA1 and the cofactors CtIP, EXO1, BLM/DNA2, and the MRE11–RAD50–NBS1 (MRN) complex promote DNA end resection and thus HR. DNA resection is influenced by the cell cycle, the chromatin environment, and the complexity of the DNA end break. Herein, we summarize the key factors involved in repair pathway selection for DSBs and discuss recent related publications.

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