Single-copy locus proteomics of early- and late-firing DNA replication origins identifies a role of Ask1/DASH complex in replication timing control

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

“…We focused on four selected replication origins on yeast chromosome III, which display previously characterized major differences in their replication timing and efficiency properties ( 43 , 44 ) (Figure 1A ). We analyzed their replication timing properties in wildtype and CRE mutant strains using a quantitative PCR (qPCR)-based DNA copy number assay (Figure 1B , and ( 45 )). Briefly, cells are arrested in G1-phase using α-factor and then synchronously released into S-phase.…”

“…MATAC-Seq is built on the conceptual foundations of NOMe-Seq and SMAC-Seq ( 50–54 ) that rely on the preferential modification of accessible DNA by DNA methyltransferases combined with Nanopore sequencing for direct readout of the methylated DNA bases. In MATAC-Seq, we additionally take advantage of our previously established single-copy locus chromatin purification approach ( 38 , 45 , 55 , 56 ). This strategy allows strong enrichment for our targeted replication origins prior to methylation footprinting and Nanopore sequencing analysis, thereby overcoming current limitations of DNA sequencing coverage of similar genome-wide approaches ( 53 ).…”

“…One complexity in interpreting our MATAC-Seq data is the fact that besides nucleosome core particles also other protein complexes bound to the native chromatin domains can contribute to the differential accessibilities and observed heterogeneity. Indeed, our published mass spectrometry study of the four origins showed a clear enrichment of all subunits of the MCM2-7 pre-RC complex on the purified chromatin domains ( 45 ), suggesting that loaded MCM2-7 double hexamers may likely contribute to the observed accessibilities. Our initial attempts to identify a characteristic ∼50–60 bp MCM2-7 double hexamer footprint were unsuccessful (data not shown), which can be explained by several technical and/or biological reasons.…”

“…As the process of transcription is mostly unidirectional, it is tempting to speculate that the observed bias in reduced nucleosome occupancy on one side of the origin could be functionally related to the occurrence of cryptic transcription processes. To monitor the enrichment of transcription-associated factors, an interesting perspective would be to perform proteomic analysis of the purified chromatin domains in the CRE mutant strains, as we have recently established in the wildtype condition ( 45 ).…”