Protocol for studying topological DNA interactions by purified fission yeast condensin

TL;DR

This paper describes a protocol to study how the condensin protein complex interacts with DNA to shape chromosomes during cell division.

Contribution

A new protocol for purifying and studying fission yeast condensin's DNA entrapment activity is introduced.

Findings

Steps for purifying Schizosaccharomyces pombe condensin are detailed.

Bulk biochemical assays monitor DNA capture reactions by condensin.

The protocol is adaptable for studying other DNA-entrapping proteins.

Abstract

To understand the transition from interphase chromatin into well-shaped chromosomes during cell divisions, we need to understand the biochemical activities of the contributing proteins. Here, we present a protocol to investigate how the ring-shaped condensin complex sequentially and topologically entraps two DNA substrates. We describe the steps to prepare purified Schizosaccharomyces pombe condensin, as well as bulk biochemical assays to monitor the first and second DNA capture reactions. This protocol may facilitate further investigations of these essential genome organizers.

For complete details on the use and execution of this protocol, please refer to Tang et al.1

•Biochemically probing topological protein-DNA interactions by S. pombe condensin•Rapid screening for optimized subunit co-overexpression in S. cerevisiae•Purification steps to minimize contamination and denaturation•Adaptable to study other DNA-entrapping proteins

Biochemically probing topological protein-DNA interactions by S. pombe condensin

Rapid screening for optimized subunit co-overexpression in S. cerevisiae

Purification steps to minimize contamination and denaturation

Adaptable to study other DNA-entrapping proteins

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

To understand the transition from interphase chromatin into well-shaped chromosomes during cell divisions, we need to understand the biochemical activities of the contributing proteins. Here, we present a protocol to investigate how the ring-shaped condensin complex sequentially and topologically entraps two DNA substrates. We describe the steps to prepare purified Schizosaccharomyces pombe condensin, as well as bulk biochemical assays to monitor the first and second DNA capture reactions. This protocol may facilitate further investigations of these essential genome organizers.

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