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. However, translesion synthesis Pol eta has been shown to efficiently polymerize CFS-associated repetitive sequences in vitro and facilitate CFS stability by a mechanism that is not fully understood. Here, by locus-specific, single-molecule replication analysis, we identified a crucial role for Pol eta (encoded by the gene POLH) in the in vivo replication of CFSs, even without exogenous stress. We find that Pol eta deficiency induces replication pausing, increases initiation events, and alters the direction of replication-fork progression at CFS-FRA16D in both lymphoblasts and fibroblasts. Furthermore, certain replication pause sites at CFS-FRA16D were associated with the presence of non-B DNA-forming motifs, implying that non-B DNA structures could increase replication hindrance in the absence of Pol eta. Further, in Pol eta-deficient fibroblasts, there was an increase in fork pausing at fibroblast-specific CFSs. Importantly, while not all pause sites were associated with non-B DNA structures, they were embedded within regions of increased genetic variation in the healthy human population, with mutational spectra consistent with Pol eta activity. From these findings, we propose that Pol eta replicating through CFSs may result in genetic variations found in the human population at these sites.

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. Here, we show that TRF2 GAR recognizes G-quadruplex features of TERRA. We show that small molecules that disrupt the TERRA-TRF2 GAR complex, such as N-methyl mesoporphyrin IX (NMM) or genetic deletion of TRF2 GAR domain, result in the loss of TERRA, and the induction of γH2AX-associated telomeric DNA damage associated with decreased telomere length, and increased telomere aberrations, including telomere fragility. Taken together, our data indicates that the G-quadruplex structure of TERRA is an important recognition element for TRF2 GAR domain and this interaction between TRF2 GAR and TERRA is essential to maintain telomere stability.

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. This reduction in initiation events is accompanied by telomere repeat loss, telomere aberrations and dysfunction. We demonstrate that telomeric origins are activated by induced replication stress to provide a key rescue mechanism for completing compromised telomere replication. Importantly, our studies also indicate that the chromatin remodeler SNF2H promotes telomeric initiation events by providing access for ORC2. Collectively, our findings reveal that active recruitment of ORC by TRF2 leads to formation of functional origins, providing an important mechanism for avoiding telomere dysfunction and rescuing challenged telomere replication.

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. PCNA generally loads onto the genome before Pol δ. Once assembled, the holoenzyme has a relatively short lifetime on the genome, implying multiple Pol δ binding events may be needed to synthesize an Okazaki fragment. During disassembly, Pol δ dissociation generally precedes PCNA unloading. We also find that Pol δ p125, the catalytic subunit of the holoenzyme, is maintained at a constant cellular level, indicating an active mechanism for control of Pol δ levels in vivo. Collectively, our studies reveal that Pol δ holoenzyme assembly and disassembly follow a predominant pathway in vivo; however, alternate pathways are observed.

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. Such known genomic loci/regions include centromeres, common fragile sites, subtelomeres, and telomeres. However, the detailed mechanism of how mammalian cells cope with the replication stress at these loci/regions is largely unknown. Here we show that depletion of FANCM, or of one of its obligatory binding partners, FAAP24, MHF1, and MHF2, induces replication stress primarily at the telomeres of cells that use the alternative lengthening of telomeres (ALT) pathway as their telomere maintenance mechanism. Using the telomere-specific single-molecule analysis of replicated DNA technique, we found that depletion of FANCM dramatically reduces the replication efficiency at ALT telomeres. We further show that FANCM, BRCA1, and BLM are actively recruited to the ALT telomeres that are experiencing replication stress and that the recruitment of BRCA1 and BLM to these damaged telomeres is interdependent and is regulated by both ATR and Chk1. Mechanistically, we demonstrated that, in FANCM-depleted ALT cells, BRCA1 and BLM help to resolve the telomeric replication stress by stimulating DNA end resection and homologous recombination (HR). Consistent with their roles in resolving the replication stress induced by FANCM deficiency, simultaneous depletion of BLM and FANCM, or of BRCA1 and FANCM, leads to increased micronuclei formation and synthetic lethality in ALT cells. We propose that these synthetic lethal interactions can be explored for targeting the ALT cancers.

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. Fork progression through the telomere was further slowed in the presence of a G4 stabilizer. Using a G4-specific antibody, we found that deficiency of BLM, or another G4-unwinding helicase, the Werner syndrome-associated helicase WRN, resulted in increased G4 structures in cells. Importantly, deficiency of either helicase led to greater increases in G4 DNA detected in the telomere compared with G4 seen genome-wide. Collectively, our findings are consistent with BLM helicase facilitating telomere replication by resolving G4 structures formed during copying of the G-rich strand by leading strand synthesis.

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. We determined the average speed of replication forks of procyclic and bloodstream form cells and we found that T. brucei DNA replication rate is similar to rates seen in other eukaryotes. We also analyzed the replication dynamics of a central region of chromosome 1 in procyclic forms. We present evidence for replication terminating within the central part of the chromosome and thus emanating from both sides, suggesting a previously unmapped origin toward the 5' extremity of chromosome 1. Also, termination is not at a fixed location in chromosome 1, but is rather variable. Importantly, we found a replication origin located near an ORC1/CDC6 binding site that is detected after replicative stress induced by hydroxyurea treatment, suggesting it may be a dormant origin activated in response to replicative stress. Collectively, our findings support the existence of more replication origins in T. brucei than previously appreciated.

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.e., origin distribution, termination site location, and fork rate and direction-of specific telomeres/subtelomeres of individual human chromosomes in two embryonic stem (ES) cell lines and two primary somatic cell types. We observe that replication can initiate within human telomere repeats but was most frequently accomplished by replisomes originating in the subtelomere. No major delay or pausing in fork progression was detected that might lead to telomere/subtelomere fragility. In addition, telomeres from different chromosomes from the same cell type displayed chromosome-specific replication programs rather than a universal program. Importantly, although there was some variation in the replication program of the same telomere in different cell types, the basic features of the program of a specific chromosome end appear to be conserved.

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. Complete ablation of SA1 results in embryonic lethality, while heterozygous animals have shorter lifespan and earlier onset of tumourigenesis. SA1-null mouse embryonic fibroblasts show decreased proliferation and increased aneuploidy as a result of chromosome segregation defects. These defects are not caused by impaired centromeric cohesion, which depends on cohesin-SA2. Instead, they arise from defective telomere replication, which requires cohesion mediated specifically by cohesin-SA1. We propose a novel mechanism for aneuploidy generation that involves impaired telomere replication upon loss of cohesin-SA1, with clear implications in tumourigenesis.

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 extension rate increases were independent of template sequence composition and only moderately correlated to telomerase RNA (TR) binding. Importantly, analysis of substrate primer elongation showed that the extension rate increases primarily resulted from increases in the repeat (type II) translocation rate. Our findings indicate a participatory role for the T motif in repeat translocation, an obligatory event for repetitive telomeric DNA synthesis. Thus, the T motif serves as a restrictive determinant of repetitive reverse transcription.

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. In this study, we show that substitutions at Val867 lead to significant changes in overall enzyme activity and telomere repeat extension rate, but have little effect on polymerase processivity. All Val867 substitutions examined (Ala, Met, Thr) led to reduced repeat extension rates, ranging from approximately 20 to 50% of the wild-type rate. Reconstitution of V867M hTERT and telomerase RNAs (TRs) with mutated template sequences revealed the effect on extension rate was associated with a template copying defect specific to template A residues. Furthermore, the Val867 hTERT mutants also displayed increased nucleotide incorporation fidelity, implicating Val867 as a determinant of telomerase fidelity. These findings suggest that by evolving to have a valine at position 867, the wild-type hTERT protein may have partially compromised polymerase fidelity for optimal and rapid repeat synthesis.

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. Changes in extension rate could not be attributed solely to altered heteroduplex melting, strongly suggesting that specific interactions between telomerase template, protein, and products contribute significantly in determining repeat extension rate. Furthermore, some substitutions that had no effect on extension rate led to striking increases in repeat processivity, indicating that processivity and extension rates can be regulated independently of each other. Our results suggest that telomerase RNA template sequence is a key determinant of the contribution of telomerase to telomere length regulation.