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Homologous recombination

Yu Zhou, Chao Zhang, Qingwei Liu, Sitang Gong, Lanlan Geng, Zhong Huang
Coxsackievirus A10 (CVA10) has emerged worldwide as one of the main pathogens of hand, foot, and mouth disease (HFMD) in recent years. However, there is currently no commercial vaccine available to prevent CVA10 infection. Here we report the development of a recombinant virus-like particle (VLP) based candidate vaccine for CVA10. Co-expression of the capsid protein precursor P1 and the protease 3CD of CVA10 in Pichia pastoris resulted in cleavage of P1 into three capsid subunit proteins VP0, VP1, and VP3. These three subunit proteins co-assembled into CVA10 VLPs, which were visualized as spherical particles with a diameter of ∼30 nm under electron microscope...
February 19, 2018: Antiviral Research
Ming-Wei Lai, Ruey-Fen Liou
Plant cell walls are pivotal battlegrounds between microbial pathogens and their hosts. To penetrate the cell wall and thereby to facilitate infection, microbial pathogens are equipped with a wide array of cell wall-degrading enzymes to depolymerize the polysaccharides in the cell wall. However, many of these enzymes and their role in the pathogenesis of microbial pathogens are not characterized, especially those from Oomycetes. In this study, we analyzed the function of four putative endo-beta-1,4-xylanase-encoding genes (ppxyn1-ppxyn4) from Phytophthora parasitica, an oomycete plant pathogen known to cause severe disease in a wide variety of plant species...
February 22, 2018: Current Genetics
Veronika Ferencziová, Gábor M Harami, Julianna B Németh, Tibor Vellai, Mihály Kovács
Homologous recombination (HR) is crucial for the error-free repair of DNA double-strand breaks (DSBs) and the restart of stalled replication. However, imprecise HR can lead to genome instability, highlighting the importance of HR quality control. After DSB formation, HR proceeds via DNA end resection and recombinase loading, whereas helicase-catalyzed disruption of a subset of subsequently formed DNA invasions is thought to be essential for maintaining HR accuracy via inhibiting illegitimate (non-allelic) recombination...
2018: PloS One
Zheng Hu, Zhaoying Shi, Xiaogang Guo, Baishan Jiang, Guo Wang, Dixian Luo, Yonglong Chen, Yuan-Shan Zhu
Background: Precise genome editing is essential for both basic and translational research. The recently developed CRISPR/Cas9 system can specifically cleave a designated site of target gene to create a DNA double-strand break, which triggers cellular DNA repair mechanism of either inaccurate non-homologous end joining, or site-specific homologous recombination. Unfortunately, homology-directed repair (HDR) is challenging due to its very low efficiency. Herein, we focused on improving the efficiency of HDR using a combination of CRISPR/Cas9, eGFP, DNA ligase IV inhibitor SCR7, and single-stranded oligodeoxynucleotides (ssODN) in human cancer cells...
2018: Cell & Bioscience
Charlotte Pawlyn, Andrea Loehr, Cody Ashby, Ruslana Tytarenko, Shayu Deshpande, James Sun, Kyle Fedorchak, Tariq Mughal, Faith E Davies, Brian A Walker, Gareth J Morgan
PARP inhibitors can induce synthetic lethality in tumors characterized by homologous recombination deficiency (HRD), which can be detected by evaluating genome-wide loss of heterozygosity (LOH). Multiple myeloma (MM) is a genetically unstable tumor and we hypothesized that HRD-related LOH (HRD-LOH) could be detected in patient samples, supporting a potential role for PARP inhibition in MM. Using results from targeted next-generation sequencing studies (FoundationOne® Heme), we analyzed HRD-LOH in patients at all disease stages (MGUS (n = 7), smoldering MM (SMM, n = 30), newly diagnosed MM (NDMM, n = 71), treated MM (TRMM, n = 64), and relapsed MM (RLMM, n = 234)) using an algorithm to identify HRD-LOH segments...
February 2, 2018: Leukemia: Official Journal of the Leukemia Society of America, Leukemia Research Fund, U.K
George E Ronson, Ann Liza Piberger, Martin R Higgs, Anna L Olsen, Grant S Stewart, Peter J McHugh, Eva Petermann, Nicholas D Lakin
PARP1 regulates the repair of DNA single-strand breaks generated directly, or during base excision repair (BER). However, the role of PARP2 in these and other repair mechanisms is unknown. Here, we report a requirement for PARP2 in stabilising replication forks that encounter BER intermediates through Fbh1-dependent regulation of Rad51. Whereas PARP2 is dispensable for tolerance of cells to SSBs or homologous recombination dysfunction, it is redundant with PARP1 in BER. Therefore, combined disruption of PARP1 and PARP2 leads to defective BER, resulting in elevated levels of replication-associated DNA damage owing to an inability to stabilise Rad51 at damaged replication forks and prevent uncontrolled DNA resection...
February 21, 2018: Nature Communications
Yanbin Liu, Chong Mei John Koh, Sihui Amy Yap, Minge Du, Mya Myintzu Hlaing, Lianghui Ji
BACKGROUND: Rhodotorula toruloides is an outstanding producer of lipids and carotenoids. Currently, information on the key metabolic pathways and their molecular basis of regulation remains scarce, severely limiting efforts to engineer it as an industrial host. RESULTS: We have adapted Agrobacterium tumefaciens-mediated transformation (ATMT) as a gene-tagging tool for the identification of novel genes in R. toruloides. Multiple factors affecting transformation efficiency in several species in the Pucciniomycotina subphylum were optimized...
February 21, 2018: BMC Microbiology
Xi Wang, Cheng Chen, Nan Zhang, Jiang Li, Fei Deng, Hualin Wang, Just M Vlak, Zhihong Hu, Manli Wang
Autographa californica nucleopolyhedrovirus (AcMNPV) orf5 (ac5) is a group I alphabaculovirus-specific gene of unknown function, although the protein (AC5) was previously reported to be associated with the per os infectivity factor (PIF) complex. The purpose of this study was to study the dynamics of AC5 during AcMNPV infection and to verify whether it is indeed a component of the PIF complex. Transcription and expression analyses suggested that ac5 is a late viral gene. An ac5-deleted recombinant AcMNPV was generated by homologous recombination...
February 21, 2018: Journal of General Virology
Hao Wang, Xiao-Meng Zhang, Go Tomiyoshi, Rika Nakamura, Natsuko Shinmen, Hideyuki Kuroda, Risa Kimura, Seiichiro Mine, Ikuo Kamitsukasa, Takeshi Wada, Akiyo Aotsuka, Yoichi Yoshida, Eiichi Kobayashi, Tomoo Matsutani, Yasuo Iwadate, Kazuo Sugimoto, Masahiro Mori, Akiyuki Uzawa, Mayumi Muto, Satoshi Kuwabara, Minoru Takemoto, Kazuki Kobayashi, Harukiyo Kawamura, Ryoichi Ishibashi, Koutaro Yokote, Mikiko Ohno, Po-Min Chen, Eiichiro Nishi, Koh Ono, Takeshi Kimura, Toshio Machida, Hirotaka Takizawa, Koichi Kashiwado, Hideaki Shimada, Masaaki Ito, Ken-Ichiro Goto, Katsuro Iwase, Hiromi Ashino, Akiko Taira, Emiko Arita, Masaki Takiguchi, Takaki Hiwasa
Transient ischemic attack (TIA) is a predictor for cerebral infarction (CI), and early diagnosis of TIA is extremely important for the prevention of CI. We set out to identify novel antibody biomarkers for TIA and CI, and detected matrix metalloproteinase 1 (MMP1), chromobox homolog 1 (CBX1), and chromobox homolog 5 (CBX5) as candidate antigens using serological identification of antigens by recombinant cDNA expression cloning (SEREX) and Western blotting to confirm the presence of serum antibodies against the antigens...
January 19, 2018: Oncotarget
Sylvia de Pater, Bart J P M Klemann, Paul J J Hooykaas
In recent years, several tools have become available for improved gene-targeting (GT) in plants. DNA breaks at specific sites activate local DNA repair and recombination, including recombination with ectopic sequences leading to GT. Large-scale transformation with the repair template can be avoided by pre-insertion of the repair template in the genome and liberation by sequence-specific nucleases (in planta GT procedure). Here, we tested whether release of the repair template was required for GT. Plants were transformed with constructs encoding a CRISPR/Cas nuclease with a recognition site in the endogenous PPO gene and a repair template harboring a 5' truncated PPO gene with two amino acid substitutions rendering the enzyme insensitive to the herbicide butafenacil...
February 20, 2018: Scientific Reports
Ye Hong, Remi Sonneville, Bin Wang, Viktor Scheidt, Bettina Meier, Alexander Woglar, Sarah Demetriou, Karim Labib, Verena Jantsch, Anton Gartner
Faithful chromosome segregation and genome maintenance requires the removal of all DNA bridges that physically link chromosomes before cells divide. Using C. elegans embryos we show that the LEM-3/Ankle1 nuclease defines a previously undescribed genome integrity mechanism by processing DNA bridges right before cells divide. LEM-3 acts at the midbody, the structure where abscission occurs at the end of cytokinesis. LEM-3 localization depends on factors needed for midbody assembly, and LEM-3 accumulation is increased and prolonged when chromatin bridges are trapped at the cleavage plane...
February 20, 2018: Nature Communications
Heïdi Serra, Christophe Lambing, Catherine H Griffin, Stephanie D Topp, Divyashree C Nageswaran, Charles J Underwood, Piotr A Ziolkowski, Mathilde Séguéla-Arnaud, Joiselle B Fernandes, Raphaël Mercier, Ian R Henderson
During meiosis, homologous chromosomes undergo reciprocal crossovers, which generate genetic diversity and underpin classical crop improvement. Meiotic recombination initiates from DNA double-strand breaks (DSBs), which are processed into single-stranded DNA that can invade a homologous chromosome. The resulting joint molecules can ultimately be resolved as crossovers. In Arabidopsis , competing pathways balance the repair of ∼100-200 meiotic DSBs into ∼10 crossovers per meiosis, with the excess DSBs repaired as noncrossovers...
February 20, 2018: Proceedings of the National Academy of Sciences of the United States of America
Kazi Mokim Ahmed, Raj K Pandita, Dharmendra Kumar Singh, Clayton R Hunt, Tej K Pandita
The molecular mechanisms underlying resistance to radiotherapy in breast cancer cells remain elusive. Previously, we reported that elevated β1-integrin is associated with enhanced breast cancer cell survival post-irradiation, but how β1-integrin conferred radioresistance was unclear. Ionizing radiation (IR) induced cell killing correlates with the efficiency of DNA DSB repair and we found that non-malignant breast epithelial (S1) cells with low β1-integrin expression have a higher frequency of S-phase specific IR-induced chromosomal aberrations as compared to the derivative malignant breast (T4-2) cells with high β1-integrin expression...
February 20, 2018: Molecular and Cellular Biology
Paula M De Angelis, Aasa R Schjølberg, Juliana B Hughes, Henrik S Huitfeldt, Solveig Norheim Andersen, Anne Carine Østvold
Background: The colon and rectum are continuously exposed to oxidative stress that generates reactive oxygen species, which are a major cause of DNA double-strand breaks (DSB). Furthermore, chronic inflammatory diseases such as ulcerative colitis (UC) are characterized by an excess of reactive nitrogen species that can also lead to DNA double-strand breakage and genomic instability. We investigated the expression of the nuclear casein kinase and cyclin-dependent kinase substrate 1 (NUCKS1) protein in UC and sporadic colorectal cancer (CRC) due to its involvement in both DNA double-strand break repair and inflammatory signaling...
February 15, 2018: Inflammatory Bowel Diseases
D V Grishin, D D Zhdanov, Ju A Gladilina, V S Pokrovsky, O V Podobed, M V Pokrovskaya, S S Aleksandrova, A L Milyushkina, M A Vigovskiy, N N Sokolov
In the work a recombinant chemotaxis protein CheW from Thermotoga petrophila RKU-1 (TpeCheW) and its mutant homolog (TpeCheW-mut) were created. It was shown that, despite the low homology with CheW prototypes from intestinal bacteria, these proteins didn't cause metabolic overload and were well expressed by cells of E. coli laboratory strains. We have discovered a broad spectrum of industrial valuable properties of the TpeCheW-mut protein such as stability in a wide range of temperatures and pH, high expression level, solubility and possibility of the application of a simple low-stage purification methodology with the use of preliminary heat treatment...
January 2018: Biomedit︠s︡inskai︠a︡ Khimii︠a︡
Laura J Eccles, Andrew C Bell, Simon N Powell
When Fanconi Anemia (FA) proteins were depleted in human U2OS cells with integrated DNA repair reporters, we observed decreases in homologous recombination (HR), decreases in mutagenic non-homologous end joining (m-NHEJ) and increases in canonical NHEJ, which was independently confirmed by measuring V(D)J recombination. Furthermore, depletion of FA proteins resulted in reduced HR protein foci and increased NHEJ protein recruitment to replication-associated DSBs, consistent with our observation that the use of canonical NHEJ increases after depletion of FA proteins in cycling cells...
February 10, 2018: DNA Repair
Cosimo Pinto, Roopesh Anand, Petr Cejka
DNA end resection initiates the largely accurate repair of DNA double-strand breaks (DSBs) by homologous recombination. Specifically, recombination requires the formation of 3' overhangs at DSB sites, which is carried out by nucleases that specifically degrade 5'-terminated DNA. In most cases, DNA end resection is a two-step process, comprising of initial short-range followed by more processive long-range resection. In this chapter, we describe selected assays that reconstitute both the short- and long-range pathways...
2018: Methods in Enzymology
Qingwen Zhou, William K Holloman, Milorad Kojic
Primary components of the homologous recombination pathway in eukaryotes include Rad51 whose function is to search for DNA sequence homology and promote strand exchange, its mediator BRCA2, and Dss1, a key regulator of BRCA2. We seek to understand the role of BRCA2 in governing the activity of Rad51 and to learn how BRCA2 function is regulated by Dss1. We use the microbe Ustilago maydis as a model system for experimentation because it has a well-conserved BRCA2-homolog, Brh2, and is amenable to biochemical and molecular genetic manipulations and analysis...
2018: Methods in Enzymology
Catharina von Nicolai, Åsa Ehlén, Juan S Martinez, Aura Carreira
Homologous recombination (HR) is an essential pathway to restart stalled replication forks, repair spontaneous DNA double-strand breaks, and generate genetic diversity. Together with genetic studies in model organisms, the development of purification protocols and biochemical assays has allowed investigators to begin to understand how the complex machinery of HR functions. At the core of the HR process is the recombination enzyme RecA in bacteria or RAD51 and DMC1 in eukaryotes. The main steps of HR can be reconstituted in vitro and involve: (1) The formation of a ssDNA-RAD51 complex into a helical structure termed the nucleoprotein filament after one DNA strand has been resected at the site of the break...
2018: Methods in Enzymology
Fletcher E Bain, Laura A Fischer, Ran Chen, Marc S Wold
Replication protein A (RPA) is a highly conserved, eukaryotic ssDNA-binding protein essential for genome stability. RPA interacts with ssDNA and with protein partners to coordinate DNA replication, repair, and recombination. Single-molecule analysis of RPA-DNA interactions is leading to a better understanding of the molecular interactions and dynamics responsible for RPA function in cells. Here, we first describe how to express, purify, and label RPA. We then describe how to prepare materials and carry out single-molecule experiments examining RPA-DNA interactions using total internal reflection fluorescence microscopy (TIRFM)...
2018: Methods in Enzymology
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