An automated image analysis pipeline to quantify the coordination and overlap of transcription and replication activity in mammalian genomes.

Transcription-replication conflicts (TRCs) represent a potent endogenous source of replication stress. Besides the spatial and temporal coordination of replication and transcription programs, cells employ many additional mechanisms to resolve TRCs in a timely manner, thereby avoiding replication fork stalling and genomic instability. Proximity ligation assays (PLA) using antibodies against actively elongating RNA Polymerase II (RNAPIIpS2) and PCNA to detect proximity (<40nm) between transcribing RNA polymerases and replication forks can be used to assess and quantify TRC levels in cells.

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

Stochastic origin activation gives rise to significant cell-to-cell variability in the pattern of genome replication. The molecular basis for heterogeneity in efficiency and timing of individual origins is a long-standing question. Here, we developed Methylation Accessibility of TArgeted Chromatin domain Sequencing (MATAC-Seq) to determine single-molecule chromatin accessibility of four specific genomic loci.

Emerging roles of the CIP2A-TopBP1 complex in genome integrity.

CIP2A is an inhibitor of the tumour suppressor protein phosphatase 2A. Recently, CIP2A was identified as a synthetic lethal interactor of BRCA1 and BRCA2 and a driver of basal-like breast cancers. In addition, a joint role of TopBP1 (topoisomerase IIβ-binding protein 1) and CIP2A for maintaining genome integrity during mitosis was discovered.

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

The chromatin environment at origins of replication is thought to influence DNA replication initiation in eukaryotic genomes. However, it remains unclear how and which chromatin features control the firing of early-efficient (EE) or late-inefficient (LI) origins. Here, we use site-specific recombination and single-locus chromatin isolation to purify EE and LI replication origins in Saccharomyces cerevisiae.

Proximity Labeling Techniques to Study Chromatin.

Mammals contain over 200 different cell types, yet nearly all have the same genomic DNA sequence. It is a key question in biology how the genetic instructions in DNA are selectively interpreted by cells to specify various transcriptional programs and therefore cellular identity. The structural and functional organization of chromatin governs the transcriptional state of individual genes.