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Article ; Online: High‐throughput, microscope‐based sorting to dissect cellular heterogeneity

Nicholas Hasle / Anthony Cooke / Sanjay Srivatsan / Heather Huang / Jason J Stephany / Zachary Krieger / Dana Jackson / Weiliang Tang / Sriram Pendyala / Raymond J Monnat Jr. / Cole Trapnell / Emily M Hatch / Douglas M Fowler

Molecular Systems Biology, Vol 16, Iss 6, Pp n/a-n/a (2020)

2020

Abstract: Abstract Microscopy is a powerful tool for characterizing complex cellular phenotypes, but linking these phenotypes to genotype or RNA expression at scale remains challenging. Here, we present Visual Cell Sorting, a method that physically separates ... ...

Abstract	Abstract Microscopy is a powerful tool for characterizing complex cellular phenotypes, but linking these phenotypes to genotype or RNA expression at scale remains challenging. Here, we present Visual Cell Sorting, a method that physically separates hundreds of thousands of live cells based on their visual phenotype. Automated imaging and phenotypic analysis directs selective illumination of Dendra2, a photoconvertible fluorescent protein expressed in live cells; these photoactivated cells are then isolated using fluorescence‐activated cell sorting. First, we use Visual Cell Sorting to assess hundreds of nuclear localization sequence variants in a pooled format, identifying variants that improve nuclear localization and enabling annotation of nuclear localization sequences in thousands of human proteins. Second, we recover cells that retain normal nuclear morphologies after paclitaxel treatment, and then derive their single‐cell transcriptomes to identify pathways associated with paclitaxel resistance in cancers. Unlike alternative methods, Visual Cell Sorting depends on inexpensive reagents and commercially available hardware. As such, it can be readily deployed to uncover the relationships between visual cellular phenotypes and internal states, including genotypes and gene expression programs.
Keywords	genetic screening ; microscopy ; pharmacology ; subcellular localization ; transcriptomics ; Biology (General) ; QH301-705.5 ; Medicine (General) ; R5-920
Subject code	612
Language	English
Publishing date	2020-06-01T00:00:00Z
Publisher	Wiley
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Fanconi anemia-isogenic head and neck cancer cell line pairs: A basic and translational science resource.

International journal of cancer

2023 Volume 153, Issue 1, Page(s) 183–196

Abstract: Fanconi anemia (FA) is a heritable malformation, bone marrow failure and cancer predisposition syndrome that confers an exceptionally high risk of squamous carcinomas. These carcinomas originate in epithelia lining the mouth, proximal esophagus, vulva ... ...

Abstract	Fanconi anemia (FA) is a heritable malformation, bone marrow failure and cancer predisposition syndrome that confers an exceptionally high risk of squamous carcinomas. These carcinomas originate in epithelia lining the mouth, proximal esophagus, vulva and anus: their origins are not understood, and no effective ways have been identified to prevent or delay their appearance. Many FA-associated carcinomas are also therapeutically challenging: they may be multi-focal and stage-advanced at diagnosis, and most individuals with FA cannot tolerate standard-of-care systemic therapies such as DNA cross-linking drugs or ionizing radiation due to constitutional DNA damage hypersensitivity. We developed the Fanconi Anemia Cancer Cell Line Resource (FA-CCLR) to foster new work on the origins, treatment and prevention of FA-associated carcinomas. The FA-CCLR consists of Fanconi-isogenic head and neck squamous cell carcinoma (HNSCC) cell line pairs generated from five individuals with FA-associated HNSCC, and five individuals with sporadic HNSCC. Sporadic, isogenic HNSCC cell line pairs were generated in parallel with FA patient-derived isogenic cell line pairs to provide comparable experimental material to use to identify cell and molecular phenotypes driven by germline or somatic loss of Fanconi pathway function, and the subset of these FA-dependent phenotypes that can be modified, complemented or suppressed. All 10 FANC-isogenic cell line pairs are available to academic, non-profit and industry investigators via the "Fanconi Anemia Research Materials" Resource and Repository at Oregon Health & Sciences University, Portland OR.
MeSH term(s)	Female ; Humans ; Squamous Cell Carcinoma of Head and Neck ; Fanconi Anemia/genetics ; Fanconi Anemia/complications ; Fanconi Anemia/pathology ; Translational Science, Biomedical ; Head and Neck Neoplasms/genetics ; Carcinoma, Squamous Cell/genetics ; Cell Line, Tumor
Language	English
Publishing date	2023-03-30
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	218257-9
ISSN	1097-0215 ; 0020-7136
ISSN (online)	1097-0215
ISSN	0020-7136
DOI	10.1002/ijc.34506
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Article: Comprehensive computational design of mCreI homing endonuclease cleavage specificity for genome engineering

Ulge, Umut Y / Baker, David A / Monnat, Raymond J. Jr

Nucleic acids research. 2011 May, v. 39, no. 10

2011

Abstract: Homing endonucleases (HEs) cleave long (~20 bp) DNA target sites with high site specificity to catalyze the lateral transfer of parasitic DNA elements. In order to determine whether comprehensive computational design could be used as a general strategy ... ...

Abstract	Homing endonucleases (HEs) cleave long (~20 bp) DNA target sites with high site specificity to catalyze the lateral transfer of parasitic DNA elements. In order to determine whether comprehensive computational design could be used as a general strategy to engineer new HE target site specificities, we used RosettaDesign (RD) to generate 3200 different variants of the mCreI LAGLIDADG HE towards 16 different base pair positions in the 22 bp mCreI target site. Experimental verification of a range of these designs demonstrated that over 2/3 (24 of 35 designs, 69%) had the intended new site specificity, and that 14 of the 15 attempted specificity shifts (93%) were achieved. These results demonstrate the feasibility of using structure-based computational design to engineer HE variants with novel target site specificities to facilitate genome engineering.
Keywords	DNA ; bioinformatics ; genetic engineering ; genetic variation ; site-specific recombination
Language	English
Dates of publication	2011-05
Size	p. 4330-4339.
Document type	Article
ZDB-ID	186809-3
ISSN	0301-5610 ; 0305-1048
ISSN	0301-5610 ; 0305-1048
DOI	10.1093/nar/gkr022
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Human RECQ helicases

Sidorova JM / Monnat Jr RJ

Advances in Genomics and Genetics, Vol 2015, Iss default, Pp 19-

roles in cancer, aging, and inherited disease

2014 Volume 33

Abstract: Julia M Sidorova,1,* Raymond J Monnat Jr,1,2,* 1Department of Pathology, 2Department of Genome ...

Abstract	Julia M Sidorova,1,* Raymond J Monnat Jr,1,2,* 1Department of Pathology, 2Department of Genome Sciences, University of Washington, Seattle, WA, USA *The authors contributed equally to this review Abstract: DNA helicases use the energy of ATP hydrolysis to disrupt DNA base pairing and displace proteins from DNA in order to facilitate replication, recombination, transcription, and repair. This article focuses on the human RECQ helicases, five DNA-dependent helicases that play key roles in cellular physiology and disease. Loss of function of three RECQ helicases causes the cancer predisposition syndromes Bloom syndrome, Werner syndrome, and Rothmund–Thomson and related syndromes. We summarize recent work on these syndromes and proteins and discuss disease pathogenesis in light of RECQ helicase biochemical activities and in vivo functions. Keywords: ATP-dependent DNA helicase, Bloom syndrome, Werner syndrome, Rothmund–Thomson syndrome, DNA replication, DNA repair, genetic instability, cancer predisposition syndrome
Keywords	Genetics ; QH426-470 ; Biology (General) ; QH301-705.5 ; Science ; Q
Subject code	612
Language	English
Publishing date	2014-12-01T00:00:00Z
Publisher	Dove Medical Press
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article: Generation of single-chain LAGLIDADG homing endonucleases from native homodimeric precursor proteins

Li, Hui / Pellenz, Stefan / Ulge, Umut / Stoddard, Barry L / Monnat, Raymond J. Jr

Nucleic acids research. 2009 Apr., v. 37, no. 5

2009

Abstract: Homing endonucleases (HEs) cut long DNA target sites with high specificity to initiate and target the lateral transfer of mobile introns or inteins. This high site specificity of HEs makes them attractive reagents for gene targeting to promote DNA ... ...

Abstract	Homing endonucleases (HEs) cut long DNA target sites with high specificity to initiate and target the lateral transfer of mobile introns or inteins. This high site specificity of HEs makes them attractive reagents for gene targeting to promote DNA modification or repair. We have generated several hundred catalytically active, monomerized versions of the well-characterized homodimeric I-CreI and I-MsoI LAGLIDADG family homing endonuclease (LHE) proteins. Representative monomerized I-CreI and I-MsoI proteins (collectively termed mCreIs or mMsoIs) were characterized in detail by using a combination of biochemical, biophysical and structural approaches. We also demonstrated that both mCreI and mMsoI proteins can promote cleavage-dependent recombination in human cells. The use of single chain LHEs should simplify gene modification and targeting by requiring the expression of a single small protein in cells, rather than the coordinate expression of two separate protein coding genes as is required when using engineered heterodimeric zinc finger or homing endonuclease proteins.
Keywords	DNA ; gene targeting ; genes ; humans ; introns ; proteins ; zinc finger motif
Language	English
Dates of publication	2009-04
Size	p. 1650-1662.
Document type	Article
ZDB-ID	186809-3
ISSN	0301-5610 ; 0305-1048
ISSN	0301-5610 ; 0305-1048
DOI	10.1093/nar/gkp004
Database	NAL-Catalogue (AGRICOLA)

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Article: Altered target site specificity variants of the I-PpoI His-Cys box homing endonuclease

Eklund, Jennifer L / Ulge, Umut Y / Eastberg, Jennifer / Monnat, Raymond J. Jr

Nucleic acids research. 2007 Sept., v. 35, no. 17

2007

Abstract: We used a yeast one-hybrid assay to isolate and characterize variants of the eukaryotic homing endonuclease I-PpoI that were able to bind a mutant, cleavage-resistant I-PpoI target or 'homing' site DNA in vivo. Native I-PpoI recognizes and cleaves a semi- ...

Abstract	We used a yeast one-hybrid assay to isolate and characterize variants of the eukaryotic homing endonuclease I-PpoI that were able to bind a mutant, cleavage-resistant I-PpoI target or 'homing' site DNA in vivo. Native I-PpoI recognizes and cleaves a semi-palindromic 15-bp target site with high specificity in vivo and in vitro. This target site is present in the 28S or equivalent large subunit rDNA genes of all eukaryotes. I-PpoI variants able to bind mutant target site DNA had from 1 to 8 amino acid substitutions in the DNA-protein interface. Biochemical characterization of these proteins revealed a wide range of site-binding affinities and site discrimination. One-third of variants were able to cleave target site DNA, but there was no systematic relationship between site-binding affinity and site cleavage. Computational modeling of several variants provided mechanistic insight into how amino acid substitutions that contact, or are adjacent to, specific target site DNA base pairs determine I-PpoI site-binding affinity and site discrimination, and may affect cleavage efficiency.
Keywords	amino acid substitution ; eukaryotic cells ; genes ; models ; mutants ; proteins ; ribosomal DNA ; yeasts
Language	English
Dates of publication	2007-09
Size	p. 5839-5850.
Document type	Article
ZDB-ID	186809-3
ISSN	0301-5610 ; 0305-1048
ISSN	0301-5610 ; 0305-1048
DOI	10.1093/nar/gkm624
Database	NAL-Catalogue (AGRICOLA)

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Article: Generation of a nicking enzyme that stimulates site-specific gene conversion from the I-AniI LAGLIDADG homing endonuclease

McConnell Smith, Audrey / Takeuchi, Ryo / Pellenz, Stefan / Davis, Luther / Maizels, Nancy / Monnat, Raymond J. Jr / Stoddard, Barry L

Proceedings of the National Academy of Sciences of the United States of America. 2009 Mar. 31, v. 106, no. 13

2009

Abstract: Homing endonucleases stimulate gene conversion by generating site-specific DNA double-strand breaks that are repaired by homologous recombination. These enzymes are potentially valuable tools for targeted gene correction and genome engineering. We have ... ...

Abstract	Homing endonucleases stimulate gene conversion by generating site-specific DNA double-strand breaks that are repaired by homologous recombination. These enzymes are potentially valuable tools for targeted gene correction and genome engineering. We have engineered a variant of the I-AniI homing endonuclease that nicks its cognate target site. This variant contains a mutation of a basic residue essential for proton transfer and solvent activation in one active site. The cleavage mechanism, DNA-binding affinity, and substrate specificity profile of the nickase are similar to the wild-type enzyme. I-AniI nickase stimulates targeted gene correction in human cells, in cis and in trans, at [almost equal to]1/4 the efficiency of the wild-type enzyme. The development of sequence-specific nicking enzymes like the I-AniI nickase will facilitate comparative analyses of DNA repair and mutagenesis induced by single- or double-strand breaks.
Keywords	DNA damage ; DNA repair ; active sites ; enzymes ; gene conversion ; genes ; genetic engineering ; homologous recombination ; humans ; mutagenesis ; solvents ; substrate specificity
Language	English
Dates of publication	2009-0331
Size	p. 5099-5104.
Publishing place	National Academy of Sciences
Document type	Article
ZDB-ID	209104-5
ISSN	1091-6490 ; 0027-8424
ISSN (online)	1091-6490
ISSN	0027-8424
DOI	10.1073/pnas.0810588106
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Genomic and Molecular Landscape of DNA Damage Repair Deficiency across The Cancer Genome Atlas

Theo A. Knijnenburg / Linghua Wang / Michael T. Zimmermann / Nyasha Chambwe / Galen F. Gao / Andrew D. Cherniack / Huihui Fan / Hui Shen / Gregory P. Way / Casey S. Greene / Yuexin Liu / Rehan Akbani / Bin Feng / Lawrence A. Donehower / Chase Miller / Yang Shen / Mostafa Karimi / Haoran Chen / Pora Kim /

Peilin Jia / Eve Shinbrot / Shaojun Zhang / Jianfang Liu / Hai Hu / Matthew H. Bailey / Christina Yau / Denise Wolf / Zhongming Zhao / John N. Weinstein / Lei Li / Li Ding / Gordon B. Mills / Peter W. Laird / David A. Wheeler / Ilya Shmulevich / Raymond J. Monnat, Jr. / Yonghong Xiao / Chen Wang / Samantha J. Caesar-Johnson / John A. Demchok / Ina Felau / Melpomeni Kasapi / Martin L. Ferguson / Carolyn M. Hutter / Heidi J. Sofia / Roy Tarnuzzer / Zhining Wang / Liming Yang / Jean C. Zenklusen / Jiashan (Julia) Zhang

Cell Reports, Vol 23, Iss 1, Pp 239-254.e

2018 Volume 6

Abstract: Summary: DNA damage repair (DDR) pathways modulate cancer risk, progression, and therapeutic response. We systematically analyzed somatic alterations to provide a comprehensive view of DDR deficiency across 33 cancer types. Mutations with accompanying ... ...

Abstract	Summary: DNA damage repair (DDR) pathways modulate cancer risk, progression, and therapeutic response. We systematically analyzed somatic alterations to provide a comprehensive view of DDR deficiency across 33 cancer types. Mutations with accompanying loss of heterozygosity were observed in over 1/3 of DDR genes, including TP53 and BRCA1/2. Other prevalent alterations included epigenetic silencing of the direct repair genes EXO5, MGMT, and ALKBH3 in ∼20% of samples. Homologous recombination deficiency (HRD) was present at varying frequency in many cancer types, most notably ovarian cancer. However, in contrast to ovarian cancer, HRD was associated with worse outcomes in several other cancers. Protein structure-based analyses allowed us to predict functional consequences of rare, recurrent DDR mutations. A new machine-learning-based classifier developed from gene expression data allowed us to identify alterations that phenocopy deleterious TP53 mutations. These frequent DDR gene alterations in many human cancers have functional consequences that may determine cancer progression and guide therapy. : Knijnenburg et al. present The Cancer Genome Atlas (TCGA) Pan-Cancer analysis of DNA damage repair (DDR) deficiency in cancer. They use integrative genomic and molecular analyses to identify frequent DDR alterations across 33 cancer types, correlate gene- and pathway-level alterations with genome-wide measures of genome instability and impaired function, and demonstrate the prognostic utility of DDR deficiency scores. Keywords: The Cancer Genome Atlas PanCanAtlas project, DNA damage repair, somatic mutations, somatic copy-number alterations, epigenetic silencing, DNA damage footprints, mutational signatures, integrative statistical analysis, protein structure analysis
Keywords	Biology (General) ; QH301-705.5
Subject code	616
Language	English
Publishing date	2018-04-01T00:00:00Z
Publisher	Elsevier
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article: Flow cytometric analysis of DNA binding and cleavage by cell surface-displayed homing endonucleases

VolnÃ¡, Petra / Jarjour, Jordan / Baxter, Sarah / Roffler, Steve R / Monnat, Raymond J. Jr / Stoddard, Barry L / Scharenberg, Andrew M

Nucleic acids research. 2007 Apr., v. 35, no. 8

2007

Abstract: LAGLIDADG homing endonucleases (LHEs) cleave 18-24 bp DNA sequences and are promising enzymes for applications requiring sequence-specific DNA cleavage amongst genome-sized DNA backgrounds. Here, we report a method for cell surface display of LHEs, which ...

Abstract	LAGLIDADG homing endonucleases (LHEs) cleave 18-24 bp DNA sequences and are promising enzymes for applications requiring sequence-specific DNA cleavage amongst genome-sized DNA backgrounds. Here, we report a method for cell surface display of LHEs, which facilitates analysis of their DNA binding and cleavage properties by flow cytometry. Cells expressing surface LHEs can be stained with fluorescently conjugated double-stranded oligonucleotides (dsOligos) containing their respective target sequences. The signal is absolutely sequence specific and undetectable with dsOligos carrying single base-pair substitutions. LHE-dsOligo interactions facilitate rapid enrichment and viable recovery of rare LHE expressing cells by both fluorescence-activated cell sorting (FACS) and magnetic cell sorting (MACS). Additionally, dsOligos conjugated with unique fluorophores at opposite termini can be tethered to the cell surface and used to detect DNA cleavage. Recapitulation of DNA binding and cleavage by surface-displayed LHEs provides a high-throughput approach to library screening that should facilitate rapid identification and analysis of enzymes with novel sequence specificities.
Keywords	DNA ; DNA damage ; enzymes ; flow cytometry ; fluorescent dyes ; nucleotide sequences ; oligonucleotides ; screening
Language	English
Dates of publication	2007-04
Size	p. 2748-2758.
Document type	Article
ZDB-ID	186809-3
ISSN	0301-5610 ; 0305-1048
ISSN	0301-5610 ; 0305-1048
DOI	10.1093/nar/gkm182
Database	NAL-Catalogue (AGRICOLA)

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Article: The Werner Syndrome Helicase Is a Cofactor for HIV-1 Long Terminal Repeat Transactivation and Retroviral Replication

Sharma, Anima / Awasthi, Soumya / Harrod, Carolyn K / Matlock, Elizabeth F / Khan, Saiqa / Xu, Louisa / Chan, Stephanie / Yang, Helen / Thammavaram, Charu K / Rasor, Randall A / Burns, Dennis K / Skiest, Daniel J / Van Lint, Carine / Girard, Anne-Marie / McGee, Monnie / Monnat, Raymond J. Jr / Harrod, Robert

Journal of biological chemistry. 2007 Apr. 20, v. 282, no. 16

2007

Abstract: The Werner syndrome helicase (WRN) participates in DNA replication, double strand break repair, telomere maintenance, and p53 activation. Mutations of wrn cause Werner syndrome (WS), an autosomal recessive premature aging disorder associated with cancer ... ...

Abstract	The Werner syndrome helicase (WRN) participates in DNA replication, double strand break repair, telomere maintenance, and p53 activation. Mutations of wrn cause Werner syndrome (WS), an autosomal recessive premature aging disorder associated with cancer predisposition, atherosclerosis, and other aging related symptoms. Here, we report that WRN is a novel cofactor for HIV-1 replication. Immortalized human WRN⁻/⁻ WS fibroblasts, lacking a functional wrn gene, are impaired for basal and Tat-activated HIV-1 transcription. Overexpression of wild-type WRN transactivates the HIV-1 long terminal repeat (LTR) in the absence of Tat, and WRN cooperates with Tat to promote high-level LTR transactivation. Ectopic WRN induces HIV-1 p24Gag production and retroviral replication in HIV-1-infected H9HIV₋₁IIIB lymphocytes. A dominant-negative helicase-minus mutant, WRNK₅₇₇M, inhibits LTR transactivation and HIV-1 replication. Inhibition of endogenous WRN, through co-expression of WRNK₅₇₇M, diminishes recruitment of p300/CREB-binding protein-associated factor (PCAF) and positive transcription elongation factor b (P-TEFb) to Tat/transactivation response-RNA complexes, and immortalized WRN⁻/⁻ WS fibroblasts exhibit comparable defects in recruitment of PCAF and P-TEFb to the HIV-1 LTR. Our results demonstrate that WRN is a novel cellular cofactor for HIV-1 replication and suggest that the WRN helicase participates in the recruitment of PCAF/P-TEFb-containing transcription complexes. WRN may be a plausible target for antiretroviral therapy.
Language	English
Dates of publication	2007-0420
Size	p. 12048-12057.
Publishing place	American Society for Biochemistry and Molecular Biology
Document type	Article
ZDB-ID	2997-x
ISSN	1083-351X ; 0021-9258
ISSN (online)	1083-351X
ISSN	0021-9258
Database	NAL-Catalogue (AGRICOLA)

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