Douglas Miller

Telomerase-dependent and -independent chromosome healing in mouse embryonic stem cells

Telomeres play an important role in protecting the ends of chromosomes and preventing chromosome fusion. We have previously demonstrated that double-strand breaks near telomeres in mammalian cells result in either the addition of a new telomere at the site of the break, termed chromosome healing, or sister chromatid fusion that initiates chromosome instability. In the present study, we have investigated the role of telomerase in chromosome healing and the importance of chromosome healing in preventing chromosome instability. In embryonic stem cell lines that are wild type for the catalytic subunit of telomerase (TERT), chromosome healing at I-SceI-induced double-strand breaks near telomeres accounted for 22 of 35 rearrangements, with the new telomeres added directly at the site of the break in all but one instance. In contrast, in two TERT-knockout embryonic stem cell lines, chromosome healing accounted for only 1 of 62 rearrangements, with a 23 bp insertion at the site of the sole chromosome-healing event. However, in a third TERT-knockout embryonic stem cell line, 10PTKO-A, chromosome healing was a common event that accounted for 20 of 34 rearrangements. Although this chromosome healing also occurred at the I-SceI site, differences in the microhomology at the site of telomere addition demonstrated that the mechanism was distinct from that in wild-type embryonic stem cell lines. In addition, the newly added telomeres in 10PTKO-A shortened with time in culture, eventually resulting in either telomere elongation through a telomerase-independent mechanism or loss of the subtelomeric plasmid sequences entirely. The combined results demonstrate that chromosome healing can occur through both telomerase-dependent and -independent mechanisms, and that although both mechanisms can prevent degradation and sister chromatid fusion, neither mechanism is efficient enough to prevent sister chromatid fusion from occurring in many cells experiencing double-strand breaks near telomeres.

Effect of Telomere Proximity on Telomere Position Effect, Chromosome Healing, and Sensitivity to DNA Double-Strand Breaks in a Human Tumor Cell Line

ABSTRACT The ends of chromosomes, called telomeres, are composed of a DNA repeat sequence and associated proteins, which prevent DNA degradation and chromosome fusion. We have previously used plasmid sequences integrated adjacent to a telomere to demonstrate that mammalian telomeres suppress gene expression, called telomere position effect (TPE). We have also shown that subtelomeric regions are highly sensitive to double-strand breaks, leading to chromosome instability, and that this instability can be prevented by the addition of a new telomere to the break, a process called chromosome healing. We have now targeted the same plasmid sequences to a site 100 kb from a telomere in a human carcinoma cell line to address the effect of telomere proximity on telomere position effect, chromosome healing, and sensitivity to double-strand breaks. The results demonstrate a substantial decrease in TPE 100 kb from the telomere, demonstrating that TPE is very limited in range. Chromosome healing was also diminished 100 kb from the telomere, consistent with our model that chromosome healing serves as a repair process for restoring lost telomeres. Conversely, the region 100 kb from the telomere was highly sensitive to double-strand breaks, demonstrating that the sensitive region is a relatively large target for ionizing radiation-induced chromosome instability.

Increased sensitivity of subtelomeric regions to DNA double-strand breaks in a human cancer cell line.

We previously reported that a single DNA double-strand break (DSB) near a telomere in mouse embryonic stem cells can result in chromosome instability. We have observed this same type of instability as a result of spontaneous telomere loss in human tumor cell lines, suggesting that a deficiency in the repair of DSBs near telomeres has a role in chromosome instability in human cancer. We have now investigated the frequency of the chromosome instability resulting from DSBs near telomeres in the EJ-30 human bladder carcinoma cell line to determine whether subtelomeric regions are sensitive to DSBs, as previously reported in yeast. These studies involved determining the frequency of large deletions, chromosome rearrangements, and chromosome instability resulting from I-SceI endonuclease-induced DSBs at interstitial and telomeric sites. As an internal control, we also analyzed the frequency of small deletions, which have been shown to be the most common type of mutation resulting from I-SceI-induced DSBs at interstitial sites. The results demonstrate that although the frequency of small deletions is similar at interstitial and telomeric DSBs, the frequency of large deletions and chromosome rearrangements is much greater at telomeric DSBs. DSB-induced chromosome rearrangements at telomeric sites also resulted in prolonged periods of chromosome instability. Telomeric regions in mammalian cells are therefore highly sensitive to DSBs, suggesting that spontaneous or ionizing radiation-induced DSBs at these locations may be responsible for many of the chromosome rearrangements that are associated with human cancer.

Subtelomeric regions in mammalian cells are deficient in DNA double-strand break repair

We have previously demonstrated that double-strand breaks (DSBs) in regions near telomeres are much more likely to result in large deletions, gross chromosome rearrangements, and chromosome instability than DSBs at interstitial sites within chromosomes. In the present study, we investigated whether this response of subtelomeric regions to DSBs is a result of a deficiency in DSB repair by comparing the frequency of homologous recombination repair (HRR) and nonhomologous end joining (NHEJ) at interstitial and telomeric sites following the introduction of DSBs by I-SceI endonuclease. We also monitored the frequency of small deletions, which have been shown to be the most common mutation at I-SceI-induced DSBs at interstitial sites. We observed no difference in the frequency of small deletions or HRR at interstitial and subtelomeric DSBs. However, the frequency of NHEJ was significantly lower at DSBs near telomeres compared to interstitial sites. The frequency of NHEJ was also lower at DSBs occurring at interstitial sites containing telomeric repeat sequences. We propose that regions near telomeres are deficient in classical NHEJ as a result of the presence of cis-acting telomere-binding proteins that cause DSBs to be processed as though they were telomeres, resulting in excessive resection, telomere loss, and eventual chromosome rearrangements by alternative NHEJ.

Formation of extrachromosomal circular DNA in HeLa cells by nonhomologous recombination

Extrachromosomal circular DNA (eccDNA) generated from chromosomal DNA is found in all mammalian cells and increases with cell stress or aging. Studies of eccDNA structure and mode of formation provide insight into mechanisms of instability of the mammalian genome. Previous studies have suggested that eccDNA is generated through a process involving recombination between repetitive sequences. However, we observed that approximately one half of the small eccDNA fragments cloned from HeLa S3 cells were composed entirely of nonrepetitive or low-copy DNA sequences. We analyzed four of these fragments by polymerase chain reaction and nucleotide sequencing and found that they were complete eccDNAs. We then screened a human genomic library with the eccDNAs to isolate the complementary chromosomal sequences. Comparing the recombination junctions within the eccDNAs with the chromosomal sequences from which they were derived revealed that nonhomologous recombination was involved in their formation. One of the eccDNAs was composed of two separate sequences from different parts of the genome. These results suggest that rejoining of ends of fragmented DNA is responsible for the generation of a substantial portion of the eccDNAs found in HeLa S3 cells.

The Role of ATM in the Deficiency in Nonhomologous End-Joining near Telomeres in a Human Cancer Cell Line

Telomeres distinguish chromosome ends from double-strand breaks (DSBs) and prevent chromosome fusion. However, telomeres can also interfere with DNA repair, as shown by a deficiency in nonhomologous end joining (NHEJ) and an increase in large deletions at telomeric DSBs. The sensitivity of telomeric regions to DSBs is important in the cellular response to ionizing radiation and oncogene-induced replication stress, either by preventing cell division in normal cells, or by promoting chromosome instability in cancer cells. We have previously proposed that the telomeric protein TRF2 causes the sensitivity of telomeric regions to DSBs, either through its inhibition of ATM, or by promoting the processing of DSBs as though they are telomeres, which is independent of ATM. Our current study addresses the mechanism responsible for the deficiency in repair of DSBs near telomeres by combining assays for large deletions, NHEJ, small deletions, and gross chromosome rearrangements (GCRs) to compare the types of events resulting from DSBs at interstitial and telomeric DSBs. Our results confirm the sensitivity of telomeric regions to DSBs by demonstrating that the frequency of GCRs is greatly increased at DSBs near telomeres and that the role of ATM in DSB repair is very different at interstitial and telomeric DSBs. Unlike at interstitial DSBs, a deficiency in ATM decreases NHEJ and small deletions at telomeric DSBs, while it increases large deletions. These results strongly suggest that ATM is functional near telomeres and is involved in end protection at telomeric DSBs, but is not required for the extensive resection at telomeric DSBs. The results support our model in which the deficiency in DSB repair near telomeres is a result of ATM-independent processing of DSBs as though they are telomeres, leading to extensive resection, telomere loss, and GCRs involving alternative NHEJ.

Processing by MRE11 is involved in the sensitivity of subtelomeric regions to DNA double-strand breaks

The caps on the ends of chromosomes, called telomeres, keep the ends of chromosomes from appearing as DNA double-strand breaks (DSBs) and prevent chromosome fusion. However, subtelomeric regions are sensitive to DSBs, which in normal cells is responsible for ionizing radiation-induced cell senescence and protection against oncogene-induced replication stress, but promotes chromosome instability in cancer cells that lack cell cycle checkpoints. We have previously reported that I-SceI endonuclease-induced DSBs near telomeres in a human cancer cell line are much more likely to generate large deletions and gross chromosome rearrangements (GCRs) than interstitial DSBs, but found no difference in the frequency of I-SceI-induced small deletions at interstitial and subtelomeric DSBs. We now show that inhibition of MRE11 3′–5′ exonuclease activity with Mirin reduces the frequency of large deletions and GCRs at both interstitial and subtelomeric DSBs, but has little effect on the frequency of small deletions. We conclude that large deletions and GCRs are due to excessive processing of DSBs, while most small deletions occur during classical nonhomologous end joining (C-NHEJ). The sensitivity of subtelomeric regions to DSBs is therefore because they are prone to undergo excessive processing, and not because of a deficiency in C-NHEJ in subtelomeric regions.

PIF1 disruption or NBS1 hypomorphism does not affect chromosome healing or fusion resulting from double-strand breaks near telomeres in murine embryonic stem cells.

Telomerase serves to maintain telomeric repeat sequences at the ends of chromosomes. However, telomerase can also add telomeric repeat sequences at DNA double-strand breaks (DSBs), a process called chromosome healing. Here, we employed a method of inducing DSBs near telomeres to query the role of two proteins, PIF1 and NBS1, in chromosome healing in mammalian cells. PIF1 was investigated because the PIF1 homolog in Saccharomyces cerevisiae inhibits chromosome healing, as shown by a 1000-fold increase in chromosome in PIF1-deficient cells. NBS1 was investigated because the functional homolog of NBS1 in S. cerevisiae, Xrs2, is part of the Mre11/Rad50/Xrs2 complex that is required for chromosome healing due to its role in the processing of DSBs and recruitment of telomerase. We found that disruption of mPif1 had no detectable effect on the frequency of chromosome healing at DSBs near telomeres in murine embryonic stem cells. Moreover, the Nbs1(ΔB) hypomorph, which is defective in the processing of DSBs, also had no detectable effect on the frequency of chromosome healing, DNA degradation, or gross chromosome rearrangements (GCRs) that result from telomeric DSBs. Although we cannot rule out small changes in chromosome healing using this system, it is clear from our results that knockout of PIF1 or the Nbs1(ΔB) hypomorph does not result in large differences in chromosome healing in murine cells. These results represent the first genetic assessment of the role of these proteins in chromosome healing in mammals, and suggest that murine cells have evolved mechanisms to ensure the functional redundancy of Pif1 or Nbs1 in the regulation of chromosome healing.