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A G-quadruplex DNA-affinity Approach for Purification of Enzymatically Active G4 Resolvase1.
Routh, Eric D; Creacy, Steven D; Beerbower, Peter E; Akman, Steven A; Vaughn, James P; Smaldino, Philip J.
Afiliação
  • Routh ED; Department of Cancer Biology, Wake Forest School of Medicine.
  • Creacy SD; YX Genomics.
  • Beerbower PE; Department of Biology, Ball State University.
  • Akman SA; Department of Hematology and Oncology, Roper St. Francis Hospital.
  • Vaughn JP; Department of Cancer Biology, Wake Forest School of Medicine; jpvaughn@nanomedica.com.
  • Smaldino PJ; Department of Biology, Ball State University; pjsmaldino@bsu.edu.
J Vis Exp ; (121)2017 03 18.
Article em En | MEDLINE | ID: mdl-28362374
Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly thermally stable. There are >375,000 putative G4-forming sequences in the human genome, and they are enriched in promoter regions, untranslated regions (UTRs), and within the telomeric repeat. Due to the potential for these structures to affect cellular processes, such as replication and transcription, the cell has evolved enzymes to manage them. One such enzyme is G4 Resolvase 1 (G4R1), which was biochemically co-characterized by our laboratory and Nagamine et al. and found to bind extremely tightly to both G4-DNA and G4-RNA (Kd in the low-pM range). G4R1 is the source of the majority of G4-resolving activity in HeLa cell lysates and has since been implicated to play a role in telomere metabolism, lymph development, gene transcription, hematopoiesis, and immune surveillance. The ability to efficiently express and purify catalytically active G4R1 is of importance for laboratories interested in gaining further insight into the kinetic interaction of G4 structures and G4-resolving enzymes. Here, we describe a detailed method for the purification of recombinant G4R1 (rG4R1). The described procedure incorporates the traditional affinity-based purification of a C-terminal histidine-tagged enzyme expressed in human codon-optimized bacteria with the utilization of the ability of rG4R1 to bind and unwind G4-DNA to purify highly active enzyme in an ATP-dependent elution step. The protocol also includes a quality-control step where the enzymatic activity of rG4R1 is measured by examining the ability of the purified enzyme to unwind G4-DNA. A method is also described that allows for the quantification of purified rG4R1. Alternative adaptations of this protocol are discussed.
Assuntos

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: DNA / Cromatografia de Afinidade / RNA Helicases DEAD-box / Quadruplex G Limite: Humans Idioma: En Ano de publicação: 2017 Tipo de documento: Article

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: DNA / Cromatografia de Afinidade / RNA Helicases DEAD-box / Quadruplex G Limite: Humans Idioma: En Ano de publicação: 2017 Tipo de documento: Article