Single molecule MATAC-seq reveals key determinants of DNA replication origin efficiency.

Area of Science:

• Molecular Biology
• Genetics
• Epigenetics

Background:

• Cell-to-cell variability in genome replication patterns arises from stochastic origin activation.
• The molecular mechanisms underlying heterogeneity in DNA replication origin efficiency and timing remain incompletely understood.

Purpose of the Study:

• To develop a novel single-molecule assay for chromatin accessibility.
• To investigate the heterogeneity of chromatin states at specific DNA replication origins.
• To establish a mechanistic link between chromatin states and replication origin activation efficiency.

Main Methods:

• Methylation Accessibility of TArgeted Chromatin domain Sequencing (MATAC-Seq) was developed to assess single-molecule chromatin accessibility.
• MATAC-Seq utilizes methyltransferase modification of accessible DNA followed by Nanopore Sequencing for direct readout.
• The method was applied to early-efficient and late-inefficient yeast replication origins.

Main Results:

• MATAC-Seq revealed significant heterogeneity in chromatin states at individual replication origins.
• Disruption of INO80 or ISW2 chromatin remodeling complexes altered nucleosomal positions, correlating with replication efficiency changes.
• An accessible nucleosome-free region with specific +1 and +2 nucleosome positioning predicted efficient origin activation.

Conclusions:

• MATAC-Seq uncovers a spectrum of chromatin states at specific loci, resolving heterogeneity previously masked by population-based studies.
• The findings provide a mechanistic basis for heterogeneity in eukaryotic DNA replication origin activation.
• This single-molecule assay is applicable to studying heterogeneity in other fundamental biological processes like transcription and DNA repair.