Elucidation of the molecular mechanism of the breakage-fusion-bridge (BFB) cycle using a CRISPR-dCas9 cellular model.

Chromosome instability (CIN) is frequently observed in many tumors. The breakage-fusion-bridge (BFB) cycle has been proposed to be one of the main drivers of CIN during tumorigenesis and tumor evolution. However, the detailed mechanisms for the individual steps of the BFB cycle warrants further investigation.

A local ATR-dependent checkpoint pathway is activated by a site-specific replication fork block in human cells.

When replication forks encounter DNA lesions that cause polymerase stalling, a checkpoint pathway is activated. The ATR-dependent intra-S checkpoint pathway mediates detection and processing of sites of replication fork stalling to maintain genomic integrity. Several factors involved in the global checkpoint pathway have been identified, but the response to a single replication fork barrier (RFB) is poorly understood.

A local ATR-dependent checkpoint pathway is activated by a site-specific replication fork block in human cells.

When replication forks encounter DNA lesions that cause polymerase stalling a checkpoint pathway is activated. The ATR-dependent intra-S checkpoint pathway mediates detection and processing of sites of replication fork stalling to maintain genomic integrity. Several factors involved in the global checkpoint pathway have been identified, but the response to a single replication fork barrier (RFB) is poorly understood.

Translesion polymerase eta both facilitates DNA replication and promotes increased human genetic variation at common fragile sites.

Common fragile sites (CFSs) are difficult-to-replicate genomic regions that form gaps and breaks on metaphase chromosomes under replication stress. They are hotspots for chromosomal instability in cancer. Repetitive sequences located at CFS loci are inefficiently copied by replicative DNA polymerase (Pol) delta.

TRF2 Mediates Replication Initiation within Human Telomeres to Prevent Telomere Dysfunction.

The telomeric shelterin protein telomeric repeat-binding factor 2 (TRF2) recruits origin recognition complex (ORC) proteins, the foundational building blocks of DNA replication origins, to telomeres. We seek to determine whether TRF2-recruited ORC proteins give rise to functional origins in telomere repeat tracts. We find that reduction of telomeric recruitment of ORC2 by expression of an ORC interaction-defective TRF2 mutant significantly reduces telomeric initiation events in human cells.

FANCD2 Facilitates Replication through Common Fragile Sites.

Common fragile sites (CFSs) are genomic regions that are unstable under conditions of replicative stress. Although the characteristics of CFSs that render them vulnerable to stress are associated mainly with replication, the cellular pathways that protect CFSs during replication remain unclear. Here, we identify and describe a role for FANCD2 as a trans-acting facilitator of CFS replication, in the absence of exogenous replicative stress.

BLM helicase facilitates telomere replication during leading strand synthesis of telomeres.

Based on its in vitro unwinding activity on G-quadruplex (G4) DNA, the Bloom syndrome-associated helicase BLM is proposed to participate in telomere replication by aiding fork progression through G-rich telomeric DNA. Single molecule analysis of replicated DNA (SMARD) was used to determine the contribution of BLM helicase to telomere replication. In BLM-deficient cells, replication forks initiating from origins within the telomere, which copy the G-rich strand by leading strand synthesis, moved slower through the telomere compared with the adjacent subtelomere.

Single molecule analysis of Trypanosoma brucei DNA replication dynamics.

Eukaryotic genome duplication relies on origins of replication, distributed over multiple chromosomes, to initiate DNA replication. A recent genome-wide analysis of Trypanosoma brucei, the etiological agent of sleeping sickness, localized its replication origins to the boundaries of multigenic transcription units. To better understand genomic replication in this organism, we examined replication by single molecule analysis of replicated DNA.

Regulation of DNA replication within the immunoglobulin heavy-chain locus during B cell commitment.

The temporal order of replication of mammalian chromosomes appears to be linked to their functional organization, but the process that establishes and modifies this order during cell differentiation remains largely unknown. Here, we studied how the replication of the Igh locus initiates, progresses, and terminates in bone marrow pro-B cells undergoing B cell commitment. We show that many aspects of DNA replication can be quantitatively explained by a mechanism involving the stochastic firing of origins (across the S phase and the Igh locus) and extensive variations in their firing rate (along the locus).

Human telomeres replicate using chromosome-specific, rather than universal, replication programs.

Telomeric and adjacent subtelomeric heterochromatin pose significant challenges to the DNA replication machinery. Little is known about how replication progresses through these regions in human cells. Using single molecule analysis of replicated DNA (SMARD), we delineate the replication programs-i.

Single molecule analysis of replicated DNA reveals the usage of multiple KSHV genome regions for latent replication.

Kaposi's sarcoma associated herpesvirus (KSHV), an etiologic agent of Kaposi's sarcoma, Body Cavity Based Lymphoma and Multicentric Castleman's Disease, establishes lifelong latency in infected cells. The KSHV genome tethers to the host chromosome with the help of a latency associated nuclear antigen (LANA). Additionally, LANA supports replication of the latent origins within the terminal repeats by recruiting cellular factors.

Single-molecule analysis reveals changes in the DNA replication program for the POU5F1 locus upon human embryonic stem cell differentiation.

Human embryonic stem cells (hESCs), due to their pluripotent nature, represent a particularly relevant model system to study the relationship between the replication program and differentiation state. Here, we define the basic properties of the replication program in hESCs and compare them to the programs of hESC-derived multipotent cells (neural rosette cells) and primary differentiated cells (microvascular endothelial cells [MECs]). We characterized three genomic loci: two pluripotency regulatory genes, POU5F1 (OCT4) and NANOG, and the IGH locus, a locus that is transcriptionally active specifically in B-lineage cells.

Decreased replication origin activity in temporal transition regions.

In the mammalian genome, early- and late-replicating domains are often separated by temporal transition regions (TTRs) with novel properties and unknown functions. We identified a TTR in the mouse immunoglobulin heavy chain (Igh) locus, which contains replication origins that are silent in embryonic stem cells but activated during B cell development. To investigate which factors contribute to origin activation during B cell development, we systematically modified the genetic and epigenetic status of the endogenous Igh TTR and used a single-molecule approach to analyze DNA replication.

Mammalian telomeres resemble fragile sites and require TRF1 for efficient replication.

Telomeres protect chromosome ends through the interaction of telomeric repeats with shelterin, a protein complex that represses DNA damage signaling and DNA repair reactions. The telomeric repeats are maintained by telomerase, which solves the end replication problem. We report that the TTAGGG repeat arrays of mammalian telomeres pose a challenge to the DNA replication machinery, giving rise to replication-dependent defects that resemble those of aphidicolin-induced common fragile sites.

Progressive activation of DNA replication initiation in large domains of the immunoglobulin heavy chain locus during B cell development.

In mammalian cells, the replication of tissue-specific gene loci is believed to be under developmental control. Here, we provide direct evidence of the existence of developmentally regulated origins of replication in both cell lines and primary cells. By using single-molecule analysis of replicated DNA (SMARD), we identified various groups of coregulated origins that are activated within the Igh locus.