Helicases are essential for DNA metabolism. Different helicases have different properties tailored to fulfill their specific tasks. RecQ-helicases are known to be important in DNA repair and DNA recombination. In higher organisms several RecQ homologues can be identified. For instance, seven RecQ homologues were identified in the model plant Arabidopsis thaliana. Specialization of those proteins can possibly be reflected by differences in their biochemical substrate spectrum. Moreover, a helicase of interest might be defined by its biochemical properties as a functional ortholog of a RecQ helicase in other organisms. In this chapter the initial steps that will provide the basis for a proper biochemical characterization are given. After the description of the expression of the helicase of interest in the heterologous host Escherichia coli, its purification with the help of two affinity tags and the preparation of a model DNA substrate for the strand displacement assay are described. Finally, it is shown how this model substrate can be used to ensure the purity of the enzymatic preparation of interest.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.1007/978-1-60327-355-8_14 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Mechanism of BRCA1-BARD1 function in DNA end resection and DNA protection.
Nature
October 2024
Institute for Research in Biomedicine, Università della Svizzera italiana (USI), Faculty of Biomedical Sciences, Bellinzona, Switzerland.
Article Synopsis
- DNA double-strand break (DSB) repair starts with DNA end resection, where the 5'-ended strands at break sites are carefully degraded, a process aided by the BRCA1-BARD1 protein complex.
- BRCA1-BARD1 not only promotes this resection process by activating specific nucleases (EXO1 and DNA2) but also works together with other proteins (like MRE11-RAD50-NBS1 and phosphorylated CtIP) to form the BRCA1-C complex, enhancing repair efficiency.
- Interestingly, while BRCA1-BARD1 supports resection, it also plays a protective role during DNA replication stress by working with the
Involvement of a BH3-only apoptosis sensitizer gene Blm-s in hippocampus-mediated mood control.
Transl Psychiatry
September 2022
Institute of Molecular Biology, Academia Sinica, 115, Taipei, Taiwan.
Mood disorders are an important public health issue and recent advances in genomic studies have indicated that molecules involved in neurodevelopment are causally related to mood disorders. BLM-s (BCL-2-like molecule, small transcript isoform), a BH3-only proapoptotic BCL-2 family member, mediates apoptosis of postmitotic immature neurons during embryonic cortical development, but its role in the adult brain is unknown. To better understand the physiological role of Blm-s gene in vivo, we generated a Blm-s-knockout (Blm-s) mouse.
View Article and Find Full Text PDFStable maintenance of the Mre11-Rad50-Nbs1 complex is sufficient to restore the DNA double-strand break response in cells lacking RecQL4 helicase activity.
J Biol Chem
October 2021
Interdisciplinary Graduate Program in Genetic Engineering, Seoul National University, Seoul, Korea; Department of Biology Education, Seoul National University, Seoul, Korea. Electronic address:
The proper cellular response to DNA double-strand breaks (DSBs) is critical for maintaining the integrity of the genome. RecQL4, a DNA helicase of which mutations are associated with Rothmund-Thomson syndrome (RTS), is required for the DNA DSB response. However, the mechanism by which RecQL4 performs these essential roles in the DSB response remains unknown.
View Article and Find Full Text PDFMapping yeast mitotic 5' resection at base resolution reveals the sequence and positional dependence of nucleases in vivo.
Nucleic Acids Res
December 2021
Department of Pathology, Ann Arbor, MI 48109, USA.
Resection of the 5'-terminated strand at DNA double-strand breaks (DSBs) is the critical regulated step in the transition to homologous recombination. Recent studies have described a multi-step model of DSB resection where endonucleolytic cleavage mediated by Mre11 and Sae2 leads to further degradation mediated by redundant pathways catalyzed by Exo1 and Sgs1/Dna2. These models have not been well tested at mitotic DSBs in vivo because most methods used to monitor resection cannot precisely map early cleavage events.
View Article and Find Full Text PDFSuper-resolution mapping of cellular double-strand break resection complexes during homologous recombination.
Proc Natl Acad Sci U S A
March 2021
Department of Biochemistry and Molecular Pharmacology, Perlmutter Cancer Center, New York University School of Medicine, New York, NY 10016
Homologous recombination (HR) is a major pathway for repair of DNA double-strand breaks (DSBs). The initial step that drives the HR process is resection of DNA at the DSB, during which a multitude of nucleases, mediators, and signaling proteins accumulates at the damage foci in a manner that remains elusive. Using single-molecule localization super-resolution (SR) imaging assays, we specifically visualize the spatiotemporal behavior of key mediator and nuclease proteins as they resect DNA at single-ended double-strand breaks (seDSBs) formed at collapsed replication forks.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!