Nicola J. Royle

Clustering of hypervariable minisatellites in the proterminal regions of human autosomes

Six of the human minisatellites detected by DNA fingerprint probes have been localized by in situ hybridization to human metaphase chromosomes. These hypervariable loci are not dispersed at random in the human genome, but show preferential, though not exclusive, localization to terminal G-bands of human autosomes. Two of the proterminal minisatellites are very closely linked to other variable loci. Sequence analysis of one of these additional minisatellites suggests that the two linked minisatellites arose by independent amplification of different repeat units. The proterminal regions of human autosomes may therefore be rich in minisatellites, analogous to the pseudoautosomal terminal pairing region of human sex chromosomes that is similarly abundant in hypervariable minisatellites.

Molecular characterization of inter-telomere and intra-telomere mutations in human ALT cells

Telomeres in most immortal cells are maintained by the enzyme telomerase, allowing cells to divide indefinitely. Some telomerase-negative tumors and immortal cell lines maintain long heterogeneous telomeres by the ALT (alternative lengthening of telomeres) mechanism; such tumors are expected to be resistant to anti-telomerase drug therapies. Occasionally telomerase-negative Saccharomyces cerevisiae mutants survive, and 10% of them (type II survivors) have unstable telomeres. As in human ALT+ cells, short telomeres in yeast type II survivors lengthen abruptly; in yeast, this is dependent on the recombination proteins Rad52p and Rad50p. In human cells, ALT involves copying of sequence from a donor to a recipient telomere. We have characterized for the first time a class of complex telomere mutations seen only in ALT+ cells. The mutant telomeres are defined by the replacement of the progenitor telomere at a discrete point (fusion point) with a different telomere repeat array. Among 19 characterized fusion points, one occurred within the first six repeats of the telomere, indicating that these recombination-like events can occur anywhere within the telomere. One mutant telomere may have been involved in a secondary recombination-like mutation event, suggesting that these mutations are sporadic but ongoing in ALT+ cells. We also identified simple intra-allelic mutations at high frequency, which evidently contribute to telomere instability in ALT+ cells.

Mechanisms underlying telomere repeat turnover, revealed by hypervariable variant repeat distribution patterns in the human Xp/Yp telomere.

Sequences immediately adjacent to the human Xp/Yp telomere exhibit a high frequency of base substitutional polymorphisms, together with almost complete linkage disequilibrium, to create only a few diverged haplotypes. This sequence divergence has been used to develop a PCR‐based system for mapping the distribution of the telomere (TTAGGG) and variant repeats (TGAGGG and TCAGGG) at the proximal end of the telomere repeat array. The distribution of these repeats is extremely variable. Almost all Xp/Yp telomeres are different, indicating a high mutation rate. Some telomere maps associated with the same flanking haplotype show similarities, identifying subsets of telomeres that share a recent common ancestry. Mechanisms underlying the rapid turnover of repeats at the proximal end of the Xp/Yp telomere include intra‐allelic processes, such as slippage during replication. Inter‐allelic exchanges may occur occasionally, but telomerase activity probably plays only a minor role in the germline turnover of proximally located telomere and variant repeats.

Sequence variant (CTAGGG)n in the human telomere favors a G-quadruplex structure containing a G·C·G·C tetrad

Short contiguous arrays of variant CTAGGG repeats in the human telomere are unstable in the male germline and somatic cells, suggesting formation of unusual structures by this repeat type. Here, we report on the structure of an intramolecular G-quadruplex formed by DNA sequences containing four human telomeric variant CTAGGG repeats in potassium solution. Our results reveal a new robust antiparallel G-quadruplex fold involving two G-tetrads sandwiched between a G·C base pair and a G·C·G·C tetrad, which could represent a new platform for drug design targeted to human telomeric DNA.

High levels of sequence polymorphism and linkage disequilibrium at the telomere of 12q: implications for telomere biology and human evolution.

The human Xp/Yp telomere-junction region exhibits high levels of sequence polymorphism and linkage disequilibrium. To determine whether this is a general feature of human telomeres, we have undertaken sequence analysis at the 12q telomere and have extended the analysis at Xp/Yp. A total of 22 single-nucleotide polymorphisms (SNPs) and one 30-bp duplication were detected in the 1,870 bp adjacent to the 12q telomere. Twenty polymorphic positions were in almost complete linkage disequilibrium, creating three common diverged haplotypes accounting for 80% of 12q telomeres in the white population. A further 6% of 12q telomeres contained a 1,439-bp deletion in the DNA flanking the telomere. The remaining 13% of 12q telomeres did not amplify with the primers used (nulls). The distribution of telomere (TTAGGG) and variant repeats within 12q telomeres was hypervariable, but alleles with similar distribution patterns were associated with the same haplotype in the telomere-adjacent DNA. These data suggest that 12q telomeres, like Xp/Yp telomeres, exhibit low levels of homologous recombination and evolve along haploid lineages. In contrast, high levels of homologous recombination occur in the adjacent proterminal regions of human chromosomes. This suggests that there is a localized telomere-mediated suppression of recombination. In addition, the genetic characteristics of these regions may provide a source of deep lineages for the study of early human evolution, unaffected by both natural selection and recombination. To explain the presence of a few diverged haplotypes adjacent to the Xp/Yp and 12q telomeres, we propose a model that involves the hybridization of two archaic hominoid lineages ultimately giving rise to modern Homo sapiens.

Characterization of terminal deletions at 7q32 and 22q13.3 healed by De novo telomere addition.

We have developed a strategy for the isolation of terminal deletion breakpoints from any chromosome that has been healed by de novo addition of a telomere repeat array. Breakpoints at 7q32 and 22q13.3 have been isolated and characterized in two patients (patients FB336R and AJ). Both truncated chromosomes have been healed by the addition of a novel telomere, with such an addition possibly mediated by the enzyme telomerase. The breakpoint at 7q32 in patient FB336R shows a structure similar to that of breakpoints on other chromosomes that have been healed in this way. However, the breakpoint at 22q13.3 in patient AJ has 10 nucleotides of unknown origin inserted between the sequence unique to chromosome 22q and the start of the telomere repeat array. This unusual structure is suggestive of a multistep healing event resulting in de novo telomere addition at this breakpoint, and possible mechanisms are discussed.

Isolation of telomere junction fragments by anchored polymerase chain reaction

We describe a simple polymerase chain reaction (pcr )-based method for isolating short stretches of nontelomeric DNA adjacent to arrays of telomere repeat units, in principle applicable to any species for which the telomere repeat sequence is known. Application of this approach to hum an DNA resulted in the isolation of many candidate telomere junction clones, at least some of which were shown to be derived from telomere-adjacent regions. Most of the isolated clones detect multiple sequences in the human genome which represent one or a few sequence families present at the ends of most or all autosomes and variably truncated before the start of the telomere repeat array. Substantial sequence divergence between different members of these sequence families suggests a low rate of sequence homogenization by telomere exchange processes. The pseudoautosomal telomere junction has also been isolated and contains a shortened version of a recently described family of short interspersed repetitive elements (SINEs), only 14 base pairs (b.p.) from the start of the telomere.

Lack of TRF2 in ALT cells causes PML-dependent p53 activation and loss of telomeric DNA

Alternative lengthening of telomere (ALT) tumors maintain telomeres by a telomerase-independent mechanism and are characterized by a nuclear structure called the ALT-associated PML body (APB). TRF2 is a component of a telomeric DNA/protein complex called shelterin. However, TRF2 function in ALT cells remains elusive. In telomerase-positive tumor cells, TRF2 inactivation results in telomere de-protection, activation of ATM, and consequent induction of p53-dependent apoptosis. We show that in ALT cells this sequence of events is different. First, TRF2 inactivation/silencing does not induce cell death in p53-proficient ALT cells, but rather triggers cellular senescence. Second, ATM is constitutively activated in ALT cells and colocalizes with TRF2 into APBs. However, it is only following TRF2 silencing that the ATM target p53 is activated. In this context, PML is indispensable for p53-dependent p21 induction. Finally, we find a substantial loss of telomeric DNA upon stable TRF2 knockdown in ALT cells. Overall, we provide insight into the functional consequences of shelterin alterations in ALT cells.

Human telomeres that carry an integrated copy of human herpesvirus 6 are often short and unstable, facilitating release of the viral genome from the chromosome

Linear chromosomes are stabilized by telomeres, but the presence of short dysfunctional telomeres triggers cellular senescence in human somatic tissues, thus contributing to ageing. Approximately 1% of the population inherits a chromosomally integrated copy of human herpesvirus 6 (CI-HHV-6), but the consequences of integration for the virus and for the telomere with the insertion are unknown. Here we show that the telomere on the distal end of the integrated virus is frequently the shortest measured in somatic cells but not the germline. The telomere carrying the CI-HHV-6 is also prone to truncations that result in the formation of a short telomere at a novel location within the viral genome. We detected extra-chromosomal circular HHV-6 molecules, some surprisingly comprising the entire viral genome with a single fully reconstituted direct repeat region (DR) with both terminal cleavage and packaging elements (PAC1 and PAC2). Truncated CI-HHV-6 and extra-chromosomal circular molecules are likely reciprocal products that arise through excision of a telomere-loop (t-loop) formed within the CI-HHV-6 genome. In summary, we show that the CI-HHV-6 genome disrupts stability of the associated telomere and this facilitates the release of viral sequences as circular molecules, some of which have the potential to become fully functioning viruses.

A novel human DNA polymorphism resulting from transfer of DNA from chromosome 6 to chromosome 16

A cloned minisatellite, termed lambda MS29, that is unusual because it detects two variable loci in human DNA has been isolated. One locus, DNF21S1, located in the terminal region of the short arm of human chromosome 6, is also present in great apes. The second minisatellite locus, DNF21S2, is located interstitially on chromosome 16p11 and is absent both from non-human primates and from some humans. Physical mapping and sequencing show that the second locus has arisen recently in evolution by duplication of a large (greater than 15 kb) segment of chromosome 6 DNA containing a minisatellite and transposition onto chromosome 16 into a member of a novel low-copy-number repetitive DNA family. This unusual duplication/transposition event appears to represent the first example of a human DNA polymorphism arising through DNA-mediated, rather than RNA-mediated, transfer between autosomes.