Abstract
The orientation of a DNA-binding protein bound on DNA is determinative in directing the assembly of other associated proteins in the complex for enzymatic action. As an example, in a replisome, the orientation of the DNA helicase at the replication fork directs the assembly of the other associated replisome proteins. We have recently determined the orientation of Saccharalobus solfataricus (Sso) Minichromosome maintenance (MCM) helicase at a DNA fork utilizing a site-specific DNA cleavage and mapping assay. Here, we describe a detailed protocol for site-specific DNA footprinting using 4-azidophenacyl bromide (APB). This method provides a straightforward, biochemical method to reveal the DNA binding orientation of SsoMCM helicase and can be applied to other DNA binding proteins.
Keywords: MCM helicase, DNA replication, Site-specific footprinting, Orientation, DNA translocation, DNA mapping
Background
DNA replication is the process in which the duplex genomic strands separate into two template strands, the leading and lagging strands. This function is executed by a ring-shaped hexameric helicase in all Domains of life. Like other ring-shaped hexameric helicases, MCM consists of two domains; an N-terminal domain (NTD) and a C-terminal domain (CTD). In theory, either of these domains could be oriented towards the replication fork during translocation and be consistent with the known 3′-5′ translocation directionality. The MCM helicase loads onto DNA origins as a double hexamer with NTDs facing each other. The orientation of the helicase during translocation determines whether the two hexamers dissociate away from each other or bypass one another during active unwinding. Our recent paper shows that the Saccharolobus solfataricus (SsoMCM) unwinds DNA with NTD leading the way (Perera and Trakselis, 2019).In order to directly determine the translocation orientation, we utilized a combination of site-specific DNA footprinting, single turnover unwinding, and translocation assays. Here, we provide detailed protocols of site-specific DNA footprinting assays with 4-azidophenacyl bromide (APB) to analyze the translocation orientation of SsoMCM (Pendergrast et al., 1992, Kassabov and Bartholomew, 2004, Nodelman et al., 2017).APB is a heterobifunctional photoactivatable crosslinking agent. Its bromide functional group reacts by S-alkylation with reduced thiols (i.e., cysteines) to form stable thioether products (Figure 1). After binding of the functionalized protein to DNA and then exposure to UV light, a reactive singlet nitrene forms that can crosslink to either protein or DNA (in close proximity) through multiple insertion or addition mechanisms (Figure 1). The resulting crosslinked protein-DNA complex can be cleaved at the crosslinked nucleotide(s) under induced alkali/heat treatment. The lengths of the resulting DNA fragments can be used to determine the orientation distribution of the SsoMCM helicase on DNA. This is a straightforward biochemical method that reveals unique positions of SsoMCM helicases on DNA. It has an important advantage over traditional footprinting in that it can determine protein binding orientation on DNA instead of just binding site size.Useful applications of this method can be employed at instances where the 3D structure of the protein is known or can be predicted, but the 3D structure of the protein-DNA complex is unknown (Pendergrast et al., 1992). It is especially useful in determining the orientation of proteins that translocate along DNA or for those that bind to specific DNA sequences. Alternatively, the orientation of DNA-binding proteins can also be determined by other biochemical methods that employ localized hydroxyl radical Fenton footprinting reactions utilizing 1-(p-Bromoacetamidobenzyl) ethylenediamine N,N,N (Fe-BABE), similarly (Owens et al., 1998).Figure 1. Conjugation of APB to free Cys on SsoMCM and UV induced crosslinking reaction mechanism to DNA
Materials and Reagents
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Data analysis
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Acknowledgments
This work was supported by the National Science Foundation Division of Molecular and Cellular Biosciences [NSF1613534 to M.A.T.] and supported by Baylor University. We thank Gregory Bowman for introducing us to the feasibility of this technique for orientation mapping (Nodelman et al., 2017).
Competing interests
The authors declare that they have no conflicts of interest with the contents of this article.
References
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