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.

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.

Consequences and Resolution of Transcription-Replication Conflicts.

Transcription-replication conflicts occur when the two critical cellular machineries responsible for gene expression and genome duplication collide with each other on the same genomic location. Although both prokaryotic and eukaryotic cells have evolved multiple mechanisms to coordinate these processes on individual chromosomes, it is now clear that conflicts can arise due to aberrant transcription regulation and premature proliferation, leading to DNA replication stress and genomic instability. As both are considered hallmarks of aging and human diseases such as cancer, understanding the cellular consequences of conflicts is of paramount importance.

TERRA, a Multifaceted Regulator of Telomerase Activity at Telomeres.

In eukaryotes, telomeres are repetitive sequences at the end of chromosomes, which are maintained in a constitutive heterochromatin state. It is now known that telomeres can be actively transcribed, leading to the production of a telomeric repeat-containing noncoding RNA called TERRA. Due to its sequence complementarity to the telomerase template, it was suggested early on that TERRA could be an inhibitor of telomerase.

Induction and relocalization of telomeric repeat-containing RNAs during diauxic shift in budding yeast.

Telomeres are maintained in a heterochromatic state that represses transcription of subtelomeric genes, a phenomenon known as telomere position effect. Nevertheless, telomeric DNA is actively transcribed, leading to the synthesis of telomeric repeat-containing noncoding RNA or TERRA. This nuclear noncoding RNA has been proposed to play important roles at telomeres, regulating their silencing, capping, repair and elongation by telomerase.

Telomerase RNA Imaging in Budding Yeast and Human Cells by Fluorescent In Situ Hybridization.

Telomerase, the enzyme that elongates telomeres in most eukaryotes, is a ribonucleoprotein complex composed of a reverse transcriptase catalytic subunit (TERT in human, Est2 in the budding yeast S. cerevisiae), regulatory factors and a noncoding RNA called hTERC (in human) or TLC1 (in budding yeast). Telomerase trafficking is a major process in the biogenesis and regulation of telomerase action at telomeres.

Cell cycle-dependent spatial segregation of telomerase from sites of DNA damage.

Telomerase can generate a novel telomere at DNA double-strand breaks (DSBs), an event called de novo telomere addition. How this activity is suppressed remains unclear. Combining single-molecule imaging and deep sequencing, we show that the budding yeast telomerase RNA ( RNA) is spatially segregated to the nucleolus and excluded from sites of DNA repair in a cell cycle-dependent manner.

Smc5/6 Is a Telomere-Associated Complex that Regulates Sir4 Binding and TPE.

SMC proteins constitute the core members of the Smc5/6, cohesin and condensin complexes. We demonstrate that Smc5/6 is present at telomeres throughout the cell cycle and its association with chromosome ends is dependent on Nse3, a subcomponent of the complex. Cells harboring a temperature sensitive mutant, nse3-1, are defective in Smc5/6 localization to telomeres and have slightly shorter telomeres.

Live-cell imaging of budding yeast telomerase RNA and TERRA.

In most eukaryotes, the ribonucleoprotein complex telomerase is responsible for maintaining telomere length. In recent years, single-cell microscopy techniques such as fluorescent in situ hybridization and live-cell imaging have been developed to image the RNA subunit of the telomerase holoenzyme. These techniques are now becoming important tools for the study of telomerase biogenesis, its association with telomeres and its regulation.