When the Ends Justify the Means: Regulation of Telomere Addition at Double-Strand Breaks in Yeast

Telomeres, repetitive sequences located at the ends of most eukaryotic chromosomes, provide a mechanism to replenish terminal sequences lost during DNA replication, limit nucleolytic resection, and protect chromosome ends from engaging in double-strand break (DSB) repair. The ribonucleoprotein telomerase contains an RNA subunit that serves as the template for the synthesis of telomeric DNA. While telomere elongation is typically primed by a 3′ overhang at existing chromosome ends, telomerase can act upon internal non-telomeric sequences. Such de novo telomere addition can be programmed (for example, during chromosome fragmentation in ciliated protozoa) or can occur spontaneously in response to a chromosome break. Telomerase action at a DSB can interfere with conservative mechanisms of DNA repair and results in loss of distal sequences but may prevent additional nucleolytic resection and/or chromosome rearrangement through formation of a functional telomere (termed “chromosome healing”). Here, we review studies of spontaneous and induced DSBs in the yeast Saccharomyces cerevisiae that shed light on mechanisms that negatively regulate de novo telomere addition, in particular how the cell prevents telomerase action at DSBs while facilitating elongation of critically short telomeres. Much of our understanding comes from the use of perfect artificial telomeric tracts to “seed” de novo telomere addition. However, endogenous sequences that are enriched in thymine and guanine nucleotides on one strand (TG-rich) but do not perfectly match the telomere consensus sequence can also stimulate unusually high frequencies of telomere formation following a DSB. These observations suggest that some internal sites may fully or partially escape mechanisms that normally negatively regulate de novo telomere addition.

Maize Chromosome Abnormalities and Breakage-Fusion-Bridge Cycles in Callus Cultures

The maize karyotype was first characterized by the observation of pachytene chromosomes. The somatic chromosomes were identified by C-banding and FISH with repetitive DNA sequences. C-banding was useful for the identification of chromosome abnormalities in callus cultures. In the present review, we focus on the involvement of heterochromatic knobs on the occurrence of chromosome abnormalities in callus cultures. In a previous work we detected anaphase bridges resulting from delayed chromatid separation at knob regions and typical bridges derived from dicentric chromatids in cultures. The analysis of altered chromosomes showed they were derived from a chromatid-type breakage-fusion-bridge (BFB) cycle. Fluorescent in situ hybridization (FISH) showed signals of telomere sequences in the broken chromosome arm, thus giving evidence of de novo telomere formation on the broken chromosome end. Further observations of long- and short-term cultures have shown the presence of chromosome alterations derived from BFB cycles followed by chromosome healing. Additionally, the occurrence of unequal crossing over in a knob region was observed in callus culture. These results are of interest for studies on the mechanisms of chromosome alterations during evolution.

Homolog-Dependent Repair Following Dicentric Chromosome Breakage in Drosophila melanogaster

Double-strand DNA breaks are repaired by one of several mechanisms that rejoin two broken ends. However, cells are challenged when asked to repair a single broken end and respond by: (1) inducing programmed cell death; (2) healing the broken end by constructing a new telomere; (3) adapting to the broken end and resuming the mitotic cycle without repair; and (4) using information from the sister chromatid or homologous chromosome to restore a normal chromosome terminus. During one form of homolog-dependent repair in yeast, termed break-induced replication (BIR), a template chromosome can be copied for hundreds of kilobases. BIR efficiency depends on Pif1 helicase and Pol32, a nonessential subunit of DNA polymerase δ. To date, there is little evidence that BIR can be used for extensive chromosome repair in higher eukaryotes. We report that a dicentric chromosome broken in mitosis in the male germline of Drosophila melanogaster is usually repaired by healing, but can also be repaired in a homolog-dependent fashion, restoring at least 1.3 Mb of terminal sequence information. This mode of repair is significantly reduced in pif1 and pol32 mutants. Formally, the repaired chromosomes are recombinants. However, the absence of reciprocal recombinants and the dependence on Pif1 and Pol32 strongly support the hypothesis that BIR is the mechanism for restoration of the chromosome terminus. In contrast to yeast, pif1 mutants in Drosophila exhibit a reduced rate of chromosome healing, likely owing to fundamental differences in telomeres between these organisms.

Breakage–fusion–bridge cycles and de novo telomere formation on broken chromosomes in maize callus cultures

Breakpoints involved in chromosome alterations associated with heterochromatin have been detected in maize plants regenerated from callus culture. A cytogenetic analysis of plants regenerated from a maize callus was performed aiming to analyze the stability of a chromosome 7 bearing a deficiency-duplication (Df-Dp), which was interpreted as derived from a chromatid type breakage–fusion–bridge (BFB) cycle. The Df-Dp chromosome 7 was stable in mitotic and meiotic cells of the regenerated plants. Fluorescence in situ hybridization showed signals of telomeric sequences on the broken chromosome arm and provided evidence of de novo telomere formation. The stability of two types of altered chromosome 7 was investigated in C-banded metaphases from samples of the original callus that were collected during a period of 30–42 months after culture initiation. New alterations involving heterochromatic knobs of chromosomes 7 and 9 were observed. The aberrant chromosomes were stable in the subcultures, thus providing evidence of broken chromosome healing. The examination of anaphases showed the presence of bridges, which was consistent with the occurrence of BFB cycles. De novo telomere formation occurred in euchromatic and heterochromatic chromosome termini. The results point to events of chromosomal evolution that might occur in plants.

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.