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.

TERRA G-quadruplex RNA interaction with TRF2 GAR domain is required for telomere integrity.

Telomere dysfunction causes chromosomal instability which is associated with many cancers and age-related diseases. The non-coding telomeric repeat-containing RNA (TERRA) forms a structural and regulatory component of the telomere that is implicated in telomere maintenance and chromosomal end protection. The basic N-terminal Gly/Arg-rich (GAR) domain of telomeric repeat-binding factor 2 (TRF2) can bind TERRA but the structural basis and significance of this interaction remains poorly understood.

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.

Dynamic Assembly and Disassembly of the Human DNA Polymerase δ Holoenzyme on the Genome In Vivo.

Human DNA polymerase delta (Pol δ) forms a holoenzyme complex with the DNA sliding clamp proliferating cell nuclear antigen (PCNA) to perform its essential roles in genome replication. Here, we utilize live-cell single-molecule tracking to monitor Pol δ holoenzyme interaction with the genome in real time. We find holoenzyme assembly and disassembly in vivo are highly dynamic and ordered.

FANCM, BRCA1, and BLM cooperatively resolve the replication stress at the ALT telomeres.

In the mammalian genome, certain genomic loci/regions pose greater challenges to the DNA replication machinery (i.e., the replisome) than others.

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.

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.

Cohesin-SA1 deficiency drives aneuploidy and tumourigenesis in mice due to impaired replication of telomeres.

Cohesin is a protein complex originally identified for its role in sister chromatid cohesion, although increasing evidence portrays it also as a major organizer of interphase chromatin. Vertebrate cohesin consists of Smc1, Smc3, Rad21/Scc1 and either stromal antigen 1 (SA1) or SA2. To explore the functional specificity of these two versions of cohesin and their relevance for embryonic development and cancer, we generated a mouse model deficient for SA1.

The telomerase-specific T motif is a restrictive determinant of repetitive reverse transcription by human telomerase.

The central hallmark of telomerases is repetitive copying of a short, defined sequence within its integral RNA subunit. We sought to identify structural determinants of this unique activity in the catalytic protein subunit telomerase reverse transcriptase (TERT) of telomerase. Residues within the highly conserved telomerase-specific T motif of human TERT were mutationally probed, leading to variant telomerases with increased repeat extension rates and wild-type processivity.

The active site residue Valine 867 in human telomerase reverse transcriptase influences nucleotide incorporation and fidelity.

Human telomerase reverse transcriptase (hTERT), the catalytic subunit of human telomerase, contains conserved motifs common to retroviral reverse transcriptases and telomerases. Within the C motif of hTERT is the Leu866-Val867-Asp868-Asp869 tetrapeptide that includes a catalytically essential aspartate dyad. Site-directed mutagenesis of Tyr183 and Met184 residues in HIV-1 RT, residues analogous to Leu866 and Val867, revealed that they are key determinants of nucleotide binding, processivity and fidelity.

Human telomerase RNA template sequence is a determinant of telomere repeat extension rate.

Human telomerase is a specialized reverse transcriptase that utilizes an integral RNA subunit to template the synthesis of telomeres. In the present study, we demonstrate that the human telomerase template sequence not only determines the composition, but also the rate of synthesis, of telomere repeats. Mutagenesis of the template sequence identified variants that reconstitute enzymes with repeat extension rates that were either faster or slower than wild type template.