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Safeguarding mitochondrial genomes in higher eukaryotes
Fu, Yi; Tigano, Marco; Sfeir, Agnel
Mitochondria respond to DNA damage and preserve their own genetic material in a manner distinct from that of the nucleus but that requires organized mito-nuclear communication. Failure to resolve mtDNA breaks leads to mitochondrial dysfunction and affects host cells and tissues. Here, we review the pathways that safeguard mitochondrial genomes and examine the insights gained from studies of cellular and tissue-wide responses to mtDNA damage and mito-nuclear genome incompatibility.
PMID: 32764737
ISSN: 1545-9985
CID: 4560672
Single-Molecule Imaging of Telomerase RNA Reveals a Recruitment-Retention Model for Telomere Elongation
Laprade, Hadrien; Querido, Emmanuelle; Smith, Michael Joseph; Guérit, David; Crimmins, Hannah; Conomos, Dimitri; Pourret, Emilie; Chartrand, Pascal; Sfeir, Agnel
Extension of telomeres is a critical step in the immortalization of cancer cells. This complex reaction requires proper spatiotemporal coordination of telomerase and telomeres and remains poorly understood at the cellular level. To understand how cancer cells execute this process, we combine CRISPR genome editing and MS2 RNA tagging to image single molecules of telomerase RNA (hTR). Real-time dynamics and photoactivation experiments of hTR in Cajal bodies (CBs) reveal that hTERT controls the exit of hTR from CBs. Single-molecule tracking of hTR at telomeres shows that TPP1-mediated recruitment results in short telomere-telomerase scanning interactions, and then base pairing between hTR and telomere ssDNA promotes long interactions required for stable telomerase retention. Interestingly, POT1 OB-fold mutations that result in abnormally long telomeres in cancers act by enhancing this retention step. In summary, single-molecule imaging unveils the life cycle of telomerase RNA and provides a framework to reveal how cancer-associated mutations mechanistically drive defects in telomere homeostasis.
PMID: 32497497
ISSN: 1097-4164
CID: 4476762
DNA polymerase theta (Polθ) - an error-prone polymerase necessary for genome stability
Brambati, Alessandra; Barry, Raymond Mario; Sfeir, Agnel
Mammalian cells have evolved multiple pathways to repair DNA double strand breaks (DSBs) and ensure genome stability. In addition to non-homologous end-joining (NHEJ) and homologous recombination (HR), cells evolved an error-prone repair pathway termed microhomology-mediated end joining (MMEJ). The mutagenic outcome of MMEJ derives from the activity of DNA polymerase theta (Polθ) - a multidomain enzyme that is minimally expressed in normal tissue but overexpressed in tumors. Polθ expression is particularly crucial for the proliferation of HR deficient cancer cells. As a result, this mutagenic repair emerged as an attractive target for cancer therapy, and inhibitors are currently in pre-clinical development. Here, we review the multifunctionality of this enigmatic polymerase, focusing on its role during DSB repair in mammalian cells and its impact on cancer genomes.
PMID: 32302896
ISSN: 1879-0380
CID: 4386682
A single-molecule view of telomerase regulation at telomeres
Chartrand, Pascal; Sfeir, Agnel
Telomerase plays a key role in the immortalization of cancer cells by maintaining telomeres length. Using single-molecule imaging of telomerase RNA molecules in cancer cells, we recently reported novel insights into the role of Cajal bodies in telomerase biogenesis and the regulation of telomerase recruitment to telomeres.
PMCID:7671040
PMID: 33241110
ISSN: 2372-3556
CID: 4681472
Single-molecule analysis of mtDNA replication with high resolution
Tigano, Marco; Phillips, Aaron Fraser; Sfeir, Agnel
DNA combing technology is a powerful methodology for the study of DNA replication in vivo. This tool can be used to identify origins of replication, assess of directionality of forks, and measure fork speed. Over the years, the method has been used extensively to study nuclear DNA replication. The first step involves the incorporation of thymidine analogs (CldU and IdU) into nascent DNA chains and followed by their visualization with immunofluorescence using antibodies that can distinguish the two analogs. Recently, we adapted and fine-tuned DNA combing technology to the specifics of mitochondrial DNA (Phillips et al., 2017, p. 155). The protocol, which we termed mito-SMARD (mitochondrial single molecule analysis of replication DNA), provides in vivo insight into mitochondrial DNA (mtDNA) replication with high resolution.
PMID: 32183970
ISSN: 0091-679x
CID: 4353532
The helicase domain of Poltheta counteracts RPA to promote alt-NHEJ
Mateos-Gomez, Pedro A; Kent, Tatiana; Deng, Sarah K; McDevitt, Shane; Kashkina, Ekaterina; Hoang, Trung M; Pomerantz, Richard T; Sfeir, Agnel
Mammalian polymerase theta (Poltheta) is a multifunctional enzyme that promotes error-prone DNA repair by alternative nonhomologous end joining (alt-NHEJ). Here we present structure-function analyses that reveal that, in addition to the polymerase domain, Poltheta-helicase activity plays a central role during double-strand break (DSB) repair. Our results show that the helicase domain promotes chromosomal translocations by alt-NHEJ in mouse embryonic stem cells and also suppresses CRISPR-Cas9- mediated gene targeting by homologous recombination (HR). In vitro assays demonstrate that Poltheta-helicase activity facilitates the removal of RPA from resected DSBs to allow their annealing and subsequent joining by alt-NHEJ. Consistent with an antagonistic role for RPA during alt-NHEJ, inhibition of RPA1 enhances end joining and suppresses recombination. Taken together, our results reveal that the balance between HR and alt-NHEJ is controlled by opposing activities of Poltheta and RPA, providing further insight into the regulation of repair-pathway choice in mammalian cells.
PMCID:6047744
PMID: 29058711
ISSN: 1545-9985
CID: 2757502
Single-Molecule Analysis of mtDNA Replication Uncovers the Basis of the Common Deletion
Phillips, Aaron F; Millet, Armel R; Tigano, Marco; Dubois, Sonia M; Crimmins, Hannah; Babin, Loelia; Charpentier, Marine; Piganeau, Marion; Brunet, Erika; Sfeir, Agnel
Mutations in mtDNA lead to muscular and neurological diseases and are linked to aging. The most frequent aberrancy is the "common deletion" that involves a 4,977-bp region flanked by 13-bp repeats. To investigate the basis of this deletion, we developed a single-molecule mtDNA combing method. The analysis of replicating mtDNA molecules provided in vivo evidence in support of the asymmetric mode of replication. Furthermore, we observed frequent fork stalling at the junction of the common deletion, suggesting that impaired replication triggers the formation of this toxic lesion. In parallel experiments, we employed mito-TALENs to induce breaks in distinct loci of the mitochondrial genome and found that breaks adjacent to the 5' repeat trigger the common deletion. Interestingly, this process was mediated by the mitochondrial replisome independent of canonical DSB repair. Altogether, our data underscore a unique replication-dependent repair pathway that leads to the mitochondrial common deletion.
PMID: 28111015
ISSN: 1097-4164
CID: 2418222
Stressed telomeres without POT1 enhance tumorigenesis [Editorial]
Sfeir, Agnel; Denchi, Eros Lazzerini
PMCID:5216905
PMID: 27419638
ISSN: 1949-2553
CID: 2719212
Polymerase theta is a robust terminal transferase that oscillates between three different mechanisms during end-joining
Kent, Tatiana; Mateos-Gomez, Pedro A; Sfeir, Agnel; Pomerantz, Richard T
DNA polymerase theta (Poltheta) promotes insertion mutations during alternative end-joining (alt-EJ) by an unknown mechanism. Here, we discover that mammalian Poltheta transfers nucleotides to the 3' terminus of DNA during alt-EJ in vitro and in vivo by oscillating between three different modes of terminal transferase activity: non-templated extension, templated extension in cis, and templated extension in trans. This switching mechanism requires manganese as a co-factor for Poltheta template-independent activity and allows for random combinations of templated and non-templated nucleotide insertions. We further find that Poltheta terminal transferase activity is most efficient on DNA containing 3' overhangs, is facilitated by an insertion loop and conserved residues that hold the 3' primer terminus, and is surprisingly more proficient than terminal deoxynucleotidyl transferase. In summary, this report identifies an unprecedented switching mechanism used by Poltheta to generate genetic diversity during alt-EJ and characterizes Poltheta as among the most proficient terminal transferases known.
PMCID:4912351
PMID: 27311885
ISSN: 2050-084x
CID: 2145252
Telomere Replication Stress Induced by POT1 Inactivation Accelerates Tumorigenesis
Pinzaru, Alexandra M; Hom, Robert A; Beal, Angela; Phillips, Aaron F; Ni, Eric; Cardozo, Timothy; Nair, Nidhi; Choi, Jaehyuk; Wuttke, Deborah S; Sfeir, Agnel; Denchi, Eros Lazzerini
Genome sequencing studies have revealed a number of cancer-associated mutations in the telomere-binding factor POT1. Here, we show that when combined with p53 deficiency, depletion of murine POT1a in common lymphoid progenitor cells fosters genetic instability, accelerates the onset, and increases the severity of T cell lymphomas. In parallel, we examined human and mouse cells carrying POT1 mutations found in cutaneous T cell lymphoma (CTCL) patients. Inhibition of POT1 activates ATR-dependent DNA damage signaling and induces telomere fragility, replication fork stalling, and telomere elongation. Our data suggest that these phenotypes are linked to impaired CST (CTC1-STN1-TEN1) function at telomeres. Lastly, we show that proliferation of cancer cells lacking POT1 is enabled by the attenuation of the ATR kinase pathway. These results uncover a role for defective telomere replication during tumorigenesis.
PMCID:6145145
PMID: 27239034
ISSN: 2211-1247
CID: 2125042