Single-Copy Gene Locus Chromatin Purification in <em>Saccharomyces cerevisiae</em>

Atiqa Sajid; Stephan Hamperl

doi:10.3791/65460

Journal

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Biochemistry

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Single-Copy Gene Locus Chromatin Purification in Saccharomyces cerevisiae

Single-Copy Gene Locus Chromatin Purification in <em>Saccharomyces cerevisiae</em>

JoVE Journal
Biochemistry

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JoVE Journal Biochemistry

Single-Copy Gene Locus Chromatin Purification in Saccharomyces cerevisiae

Single-Copy Gene Locus Chromatin Purification in Saccharomyces cerevisiae

908 Views

•

10:33 min

•

November 17, 2023

DOI:

10.3791/65460-v

DOI:

10.3791/65460-v

•

10:33 min

November 17, 2023

•

7 Views

Atiqa Sajid¹, Stephan Hamperl¹

¹Institute of Epigenetics and Stem Cells,Helmholtz Zentrum München

Transcript

A eukaryotic nucleus is very crowded with many biological processes occurring simultaneously. The scope of our research is to understand how all of these processes are coordinated on our genomes without major accidents that would otherwise lead to genomic instability and human diseases. This protocol allows us to purify a specific locus of the genome in its native state, allowing both the compositional and functional characterization of the isolated material, such as the measurements of protein DNA interactions.

This protocol enables an enormous level of enrichment of a targeted locus, which overcomes many current limitations of locus-specific chromatin isolation. The material has not been cross-linked. That also allows many downstream functional analysis, such as in vitro transcription or in vitro replication.

This protocol enabled us to excise and purify distinct replication origins from yeast chromosomes, allowing us to perform a proteomic analysis and identify novel origin interacting chromatin factors, and therefore, advance the research field of DNA replication. This protocol enables in vitro replication studies using the purified origin material to examine chromatin states, histone modifications, and perform structural analysis on the native nucleosomal templates from endogenous chromosomes, advancing chromatin research.

Summary

This protocol presents a locus-specific chromatin isolation method based on site-specific recombination to purify a single-copy gene locus of interest in its native chromatin context from budding yeast, Saccharomyces cerevisiae.