Pot1b -/- tumors activate G-quadruplex-induced DNA damage to promote telomere hyper-elongation.

Malignant cancers must activate telomere maintenance mechanisms to achieve replicative immortality. Mutations in the human Protection of Telomeres 1 (POT1) gene are frequently detected in cancers with abnormally long telomeres, suggesting that the loss of POT1 function disrupts the regulation of telomere length homeostasis to promote telomere elongation. However, our understanding of the mechanisms leading to elongated telomeres remains incomplete. The mouse genome encodes two POT1 proteins, POT1a and POT1b possessing separation of hPOT1 functions. We performed serial transplantation of Pot1b-/- sarcomas to better understand the role of POT1b in regulating telomere length maintenance. While early-generation Pot1b-/- sarcomas initially possessed shortened telomeres, late-generation Pot1b-/- cells display markedly hyper-elongated telomeres that were recognized as damaged DNA by the Replication Protein A (RPA) complex. The RPA-ATR-dependent DNA damage response at telomeres promotes telomerase recruitment to facilitate telomere hyper-elongation. POT1b, but not POT1a, was able to unfold G-quadruplex present in hyper-elongated telomeres to repress the DNA damage response. Our findings demonstrate that the repression of the RPA-ATR DDR is conserved between POT1b and human POT1, suggesting that similar mechanisms may underly the phenotypes observed in human cancers harboring human POT1 mutations.

Distinct functions of POT1 proteins contribute to the regulation of telomerase recruitment to telomeres.

Human shelterin components POT1 and TPP1 form a stable heterodimer that protects telomere ends from ATR-dependent DNA damage responses and regulates telomerase-dependent telomere extension. Mice possess two functionally distinct POT1 proteins. POT1a represses ATR/CHK1 DNA damage responses and the alternative non-homologous end-joining DNA repair pathway while POT1b regulates C-strand resection and recruits the CTC1-STN1-TEN1 (CST) complex to telomeres to mediate C-strand fill-in synthesis. Whether POT1a and POT1b are involved in regulating the length of the telomeric G-strand is unclear. Here we demonstrate that POT1b, independent of its CST function, enhances recruitment of telomerase to telomeres through three amino acids in its TPP1 interacting C-terminus. POT1b thus coordinates the synthesis of both telomeric G- and C-strands. In contrast, POT1a negatively regulates telomere length by inhibiting telomerase recruitment to telomeres. The identification of unique amino acids between POT1a and POT1b helps us understand mechanistically how human POT1 switches between end protective functions and promoting telomerase recruitment.

Absence of XRCC4 and its paralogs in human cells reveal differences in outcomes for DNA repair and V(D)J recombination.

The repair of DNA double-stranded breaks (DSBs) is an essential function performed by the Classical Non-Homologous End-Joining (C-NHEJ) pathway in higher eukaryotes. C-NHEJ, in fact, does double duty as it is also required for the repair of the intermediates formed during lymphoid B- and T-cell recombination. Consequently, the failure to properly repair DSBs leads to both genomic instability and immunodeficiency. A critical DSB protein required for C-NHEJ is the DNA Ligase IV (LIGIV) accessory factor, X-Ray Cross Complementing 4 (XRCC4). XRCC4 is believed to stabilize LIGIV, participate in LIGIV activation, and to help tether the broken DSB ends together. XRCC4's role in these processes has been muddied by the identification of two additional XRCC4 paralogs, XRCC4-Like Factor (XLF), and Paralog of XRCC4 and XLF (PAXX). The roles that these paralogs play in C-NHEJ is partially understood, but, in turn, has itself been obscured by species-specific differences observed in the absence of one or the other paralogs. In order to investigate the role(s) that XRCC4 may play, with or without XLF and/or PAXX, in lymphoid variable(diversity)joining [V(D)J] recombination as well as in DNA DSB repair in human somatic cells, we utilized gene targeting to inactivate the XRCC4 gene in both parental and XLF- HCT116 cells and then inactivated PAXX in those same cell lines. The loss of XRCC4 expression by itself led, as anticipated, to increased sensitivity to DNA damaging agents as well as an increased dependence on microhomology-mediated DNA repair whether in the context of DSB repair or during V(D)J recombination. The additional loss of XLF in these cell lines sensitized the cells even more whereas the presence or absence of PAXX was scarcely negligible. These studies demonstrate that, of the three LIG4 accessory factor paralogs, the absence of XRCC4 influences DNA repair and recombination the most in human cells.

CTC1-STN1 coordinates G- and C-strand synthesis to regulate telomere length.

Coats plus (CP) is a rare autosomal recessive disorder caused by mutations in CTC1, a component of the CST (CTC1, STN1, and TEN1) complex important for telomere length maintenance. The molecular basis of how CP mutations impact upon telomere length remains unclear. The CP CTC1L1142H mutation has been previously shown to disrupt telomere maintenance. In this study, we used CRISPR/Cas9 to engineer this mutation into both alleles of HCT116 and RPE cells to demonstrate that CTC1:STN1 interaction is required to repress telomerase activity. CTC1L1142H interacts poorly with STN1, leading to telomerase-mediated telomere elongation. Impaired interaction between CTC1L1142H :STN1 and DNA Pol-α results in increased telomerase recruitment to telomeres and further telomere elongation, revealing that C:S binding to DNA Pol-α is required to fully repress telomerase activity. CP CTC1 mutants that fail to interact with DNA Pol-α resulted in loss of C-strand maintenance and catastrophic telomere shortening. Our findings place the CST complex as an important regulator of both G-strand extensions by telomerase and C-strand synthesis by DNA Pol-α.

Mechanisms of precise genome editing using oligonucleotide donors.

The use of programmable meganucleases is transforming genome editing and functional genomics. CRISPR/Cas9 was developed such that targeted genomic lesions could be introduced in vivo with unprecedented ease. In the presence of homology donors, these lesions facilitate high-efficiency precise genome editing (PGE) via homology-directed repair (HDR) pathways. However, the identity and hierarchy of the HDR (sub)pathways leading to the formation of PGE products remain elusive. Here, we established a green to blue fluorescent protein conversion system to systematically characterize oligodeoxynucleotide (ODN)-mediated PGE using Cas9 and its nickase variants in human cells. We demonstrate that, unlike double-stranded DNA (dsDNA) donors with central heterologies, ODNs generated short conversion tracts with Gaussian-like distributions. Interestingly, single-nick-induced PGE using ODN donors produced conversion tracts biased either mostly uni- or bidirectional depending on the relative strandedness of the ODNs and the nick. Moreover, the ODNs were physically incorporated into the genome only in the bidirectional, but not in the unidirectional, conversion pathway. In the presence of double-stranded genomic lesions, the unidirectional conversion pathway was preferentially utilized even though the knock-in mutation could theoretically have been converted by both pathways. Collectively, our results suggest that ODN-mediated PGE utilizes synthesis-dependent strand annealing and single-stranded DNA incorporation pathways. Both of these pathways generate short conversion tracts with Gaussian-like distributions. Although synthesis-dependent strand annealing is preferentially utilized, our work unequivocally establishes the existence of a single-stranded DNA incorporation pathway in human cells. This work extends the paradigms of HDR-mediated gene conversion and establishes guidelines for PGE in human cells.

Sister chromatid telomere fusions, but not NHEJ-mediated inter-chromosomal telomere fusions, occur independently of DNA ligases 3 and 4.

Telomeres shorten with each cell division and can ultimately become substrates for nonhomologous end-joining repair, leading to large-scale genomic rearrangements of the kind frequently observed in human cancers. We have characterized more than 1400 telomere fusion events at the single-molecule level, using a combination of high-throughput sequence analysis together with experimentally induced telomeric double-stranded DNA breaks. We show that a single chromosomal dysfunctional telomere can fuse with diverse nontelomeric genomic loci, even in the presence of an otherwise stable genome, and that fusion predominates in coding regions. Fusion frequency was markedly increased in the absence of TP53 checkpoint control and significantly modulated by the cellular capacity for classical, versus alternative, nonhomologous end joining (NHEJ). We observed a striking reduction in inter-chromosomal fusion events in cells lacking DNA ligase 4, in contrast to a remarkably consistent profile of intra-chromosomal fusion in the context of multiple genetic knockouts, including DNA ligase 3 and 4 double-knockouts. We reveal distinct mutational signatures associated with classical NHEJ-mediated inter-chromosomal, as opposed to alternative NHEJ-mediated intra-chromosomal, telomere fusions and evidence for an unanticipated sufficiency of DNA ligase 1 for these intra-chromosomal events. Our findings have implications for mechanisms driving cancer genome evolution.