Probing hyper-negatively supercoiled mini-circles with nucleases and DNA binding proteins

Carole Saintomé,Emmanuelle Delagoutte

doi:10.1371/journal.pone.0202138

Carole Saintomé, Emmanuelle Delagoutte

Open Access

https://doi.org/10.1371/journal.pone.0202138

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Journal: PloS one	Publication Date: Aug 16, 2018
Citations: 1	License type: CC BY 4.0

Affiliation: Sorbonne Université, Université Sorbonne Nouvelle, Inserm

Abstract

It is well accepted that the introduction of negative supercoils locally unwinds the DNA double helix, influencing thus the activity of proteins. Despite the use of recent methods of molecular dynamics simulations to model the DNA supercoiling-induced DNA deformation, the precise extent and location of unpaired bases induced by the negative supercoiling have never been investigated at the nucleotide level. Our goals in this study were to use radiolabeled double-stranded DNA mini-circles (dsMCs) to locate the unpaired bases on dsMCs whose topology ranged from relaxed to hyper-negatively supercoiled states, and to characterize the binding of proteins involved in the DNA metabolism. Our results show that the Nuclease SI is nearly ten times more active on hyper-negatively supercoiled than relaxed DNA. The structural changes responsible for this stimulation of activity were mapped for the first time with a base pair resolution and shown to be subtle and distributed along the entire sequence. As divalent cations modify the DNA topology, our binding studies were conducted with or without magnesium. Without magnesium, the dsMCs topoisomers mostly differ by their twist. Under these conditions, the Escherichia coli topoisomerase I weakly binds relaxed dsMCs and exhibits a stronger binding on negatively and hyper-negatively supercoiled dsMCs than relaxed dsMCs, with no significant difference in the binding activity among the supercoiled topoisomers. For the human replication protein A (hRPA), the more negatively supercoiled is the DNA, the better the binding, illustrating the twist-dependent binding activity for this protein. The presence of magnesium permits the dsMCs to writhe upon introduction of negative supercoiling and greatly modifies the binding properties of the hRPA and Escherichia coli SSB on dsMCs, indicating a magnesium-dependent DNA binding behavior. Finally, our experiments that probe the topology of the DNA in the hRPA-dsMC complexes show that naked and hRPA-bound dsMCs have the same topology.

Highlights

The activity of proteins involved in the DNA metabolism such as specific DNA binding proteins, nucleases or RNA polymerases, is influenced by the topology of the DNA and by the degree of superhelicity [1,2,3,4,5,6,7,8,9,10,11,12]
To locate the nucleotides whose conformation changed in response to the introduction of negative and hyper-negative supercoiling, we first checked the sensitivity of the radiolabeled topoisomers of dsMCs with these two protein probes, Nuclease SI and DNAse I
Our goals in this study were first to locate at the nucleotide level, any structural changes induced by the hyper-negative supercoiling, by using two enzymatic probes, Nuclease SI and DNAse I, and second to characterize the effect of hyper-negative supercoiling on the binding of two proteins known to bind single-stranded DNA, the human replication protein A (hRPA) heterotrimer and Ec TopoI

Summary

Introduction

The activity of proteins involved in the DNA metabolism such as specific DNA binding proteins, nucleases or RNA polymerases, is influenced by the topology of the DNA and by the degree of superhelicity [1,2,3,4,5,6,7,8,9,10,11,12]. Wang monitored the transcriptional activity of the RNA polymerase on DNA samples containing varying numbers of superhelical turns and reported the influence of the level of supercoiling on transcription [1]. The inhibition of transcription at high negative supercoiling levels stems from the accumulation of hyper-negative supercoiled DNA behind the RNA polymerase, that hinders the progression of the transcription complex and may cause genome instability [14]. In this context, DNA topoisomerases of family IA perform an essential function in preventing the accumulation of hyper-negative supercoiled DNA, and their preferred substrate is often hyper-negatively supercoiled DNA. Structural data have suggested that even if Escherichia coli DNA topoisomerase I (Ec TopoI) can relax negatively supercoiled DNA, its preference for hypernegatively supercoiled DNA stems from the nature and the number of interactions between single-stranded DNA and its carboxy-terminal extremity that carries four cysteine zinc ribbon and zinc ribbon-like domains [16]

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