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We review transient kinetic methods developed to study the mechanism of translocation of nucleic acid motor proteins. One useful stopped-flow fluorescence method monitors arrival of the translocase at the end of a fluorescently labeled nucleic acid. When conducted under single-round conditions the time courses can be analyzed quantitatively using n-step sequential models to determine the kinetic parameters...
In eukaryotes, homologous recombination (HR) provides an important means to eliminate DNA double-stranded breaks and other chromosomal lesions. Accordingly, failure in HR leads to genomic instability and a predisposition to various cancer types. While HR is clearly beneficial for genome maintenance, inappropriate or untimely events can be harmful. For this reason, HR must be tightly regulated. Several...
Methods are described to show how different fluorescent labeling strategies can be used to probe various aspects of the helicase mechanism. Fluorophores on the adenine nucleotide, the DNA or the helicase can modify the activity of the system to a greater or lesser extent. Reagentless biosensors, binding proteins that are labeled with a fluorophore, target products of the helicase reaction, namely...
The importance of helicases in nucleic acid metabolism and human disease has raised the bar for understanding how these unique enzymes function to perform their biological roles at the molecular level. Here we will describe experimental procedures and strategies to investigate the functions of helicases. These functional assays have been used to study DNA helicases important for the maintenance of...
Many quantitative approaches for analysis of helicase–nucleic acid interactions require a robust and specific signal, which reports on the presence of the helicase and its position on a nucleic acid lattice. Since 2006, iron–sulfur (FeS) clusters have been found in a number of helicases. They serve as endogenous quenchers of Cy3 and Cy5 fluorescence which can be exploited to characterize FeS cluster...
Dbf4–Cdc7 phosphorylation of the Mcm2–7 complex is required for the activation of the replication fork helicase in budding yeast cells. There is a genetic interaction between Dbf4–Cdc7 and Mcm2, and Dbf4–Cdc7 phosphorylates Mcm2 in vitro and in vivo. We initiated a focused study of how Dbf4–Cdc7 phosphorylates Mcm2 in budding yeast, and we also investigated the in vivo implications of this kinase...
In order to elucidate molecular mechanism of helicases, we have developed two new rapid and sensitive fluorescence assays to measure helicase-mediated DNA unwinding. The fluorescence anisotropy (FA) assay takes the advantage of the substantial change in fluorescence polarization upon helicase binding to DNA and DNA unwinding. The extent of depolarization depends on the rate of tumbling of the fluorescently...
Most processes involving an organism’s genetic material, including replication, repair and recombination, require access to single-stranded DNA as a template or reaction intermediate. To disrupt the hydrogen bonds between the two strands in double-stranded DNA, organisms utilize proteins called DNA helicases. DNA helicases use duplex DNA as a substrate to create single-stranded DNA in a reaction that...
Magnetic tweezers is a versatile and easy to implement single-molecule technique that has become increasingly prevalent in the study of nucleic acid based molecular motors. Here, we provide a description of the magnetic tweezers instrument and guidelines for measuring and analyzing DNA helicase activity. Along with experimental methods, we describe a robust method of single-molecule trajectory analysis...
Single-molecule picometer resolution nanopore tweezers (SPRNT) is a new tool for analyzing the motion of nucleic acids through molecular motors. With SPRNT, individual enzymatic motions along DNA as small as 40pm can be resolved on sub-millisecond time scales. Additionally, SPRNT reveals an enzyme’s exact location with respect to a DNA strand’s nucleotide sequence, enabling identification of sequence-specific...
The growing number of DNA helicases implicated in hereditary disorders and cancer indicates that this particular class of enzymes plays key roles in genomic stability and cellular homeostasis. Indeed, a large body of work has provided molecular and cellular evidence that helicases act upon a variety of nucleic acid substrates and interact with numerous proteins to enact their functions in replication,...
Faithful duplication of genetic material during every cell division is essential to ensure accurate transmission of genetic information to daughter cells. DNA helicases play a crucial role in promoting this process by facilitating almost all transactions occurring on DNA, including DNA replication and repair. They are responsible not only for DNA double helix unwinding ahead of progressing replication...
Helicases and translocases are nucleic acid (NA)-based molecular motors that use the free energy liberated during the nucleoside triphosphate (NTP, usually ATP) hydrolysis cycle for unidirectional translocation along their NA (DNA, RNA or heteroduplex) substrates. Determination of the kinetic and thermodynamic parameters of their mechanoenzymatic cycle serves as a basis for the exploration of their...
Gene expression changes in the functional absence of a specific RecQ protein, and how that relates to disease outcomes including cancer predisposition and premature aging in RecQ helicase associated syndromes, are poorly understood. Here we describe detailed experimental strategy for identification of RECQ1-regulated transcriptome that led us to uncover a novel association of RECQ1 in regulation of...
Magnetic tweezers provide a versatile toolkit supporting the mechanistic investigation of helicases. In the present article, we show that custom magnetic tweezers setups are straightforward to construct and can easily be extended to provide adaptable platforms, capable of addressing a multitude of enquiries regarding the functions of these fascinating molecular machines. We first address the fundamental...
Helicases are proposed to unwind dsDNA primarily by translocating on one strand to sterically exclude and separate the two strands. Hexameric helicases in particular have been shown to encircle one strand while physically excluding the other strand. In this article, we will detail experimental methods used to validate specific interactions with the excluded strand on the exterior surface of hexameric...
FANCJ is a superfamily 2 DNA helicase, which also belongs to the iron-sulfur domain containing helicases that include XPD, ChlR1 (DDX11), and RTEL1. Mutations in FANCJ are genetically linked to Fanconi anemia (FA), breast cancer, and ovarian cancer. FANCJ plays a critical role in genome stability and participates in DNA interstrand crosslink and double-strand break repair. Enormous sequence alterations...
Helicases are enzymes involved in nucleic acid metabolism, playing major roles in replication, transcription, and repair. Defining helicases oligomerization state and transient and persistent protein interactions is essential for understanding of their function. In this article we review current methods for the protein-protein interaction analysis, and discuss examples of its application to the study...
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