Asynchronously Replicating Eu/Heterochromatic Regions Shape Chromosome Damage

Abstract

In order to shed more light on the influence of DNA replication on the formation and distribution of chromosome aberrations, breakpoints (BP) produced by UV-C and AluI were assigned either to the early replicating short euchromatic arm (Xp_e) or to the late replicating long heterochromatic arm (Xq_h) of the Chinese hamster (CHO9) X chromosome. Early (ES) or late (LS) S-phase cells were assessed by pulse incorporating the base analogue 5-bromo-2′-deoxyuridine (BrdU) immediately after UV-C irradiation (30 J/m²) or AluI (20 U) poration followed by BrdU immunodetection with FITC-tagged antibodies in metaphase spreads. Short (30 s) UV-C exposures (1 J/m²/s) induced BP preferentially in Xq_h in LS cells and a random distribution of BP along Xp_e and Xq_h in ES cells. However, BP induced by long (5 min) UV-C exposures (0.1 J/m²/s) clustered according to arm replication time (Xp_e during ES and Xq_h along LS). Giemsa-stained metaphases showed elevated frequencies of UV-C induced chromatid-type aberrations and gaps, especially in cells exposed to longer UV-C irradiation. BP induced by AluI clustered in Xp_e in ES but distributed randomly during LS. In contrast to UV-C, AluI did not produce an increase in the yield of gaps, neither in ES nor in LS cells. Putative mechanisms underlying the observed distributions of chromosome damage in replicating CHO9 cells exposed to UV-C and AluI are discussed.

Spontaneous and Bleomycin-Induced <i>γ</i>H2AX Signals in CHO9 Metaphase Chromosomes

“…Chromosome segments close to centromeres showed low γH2AX signaling (sign-test, p < 0.0001) with a tendency to increase towards distal regions (Figure 3). This uneven γH2AX signal distribution between chromosomes and chromosome regions is in agreement with the inter-and intra-chromosomal variability of the induced-damage reported by researchers of our laboratory in CHO cells [46]- [52] and others [53]- [61] both in human and in other mammalian cells.…”

Chromatin Damage Patterns Shift According to Eu/ Heterochromatin Replication

“…Xp/Xq BP localization showed that when Xp replicates, BP produced by either MMS, UV-C or etoposide clustered in Xp. On the other hand, during Xq replication, BP induced by the clastogens concentrated in Xq [91,92] (Figure 6). Since UV-C and MMS are S-phase dependent clastogens, the observed predominance of BP produced in Xp or Xq according to replication timing could be explained based on their requirement of DNA synthesis to produce CA.…”

“…Besides, Xq replicates in late S-phase whereas the euchromatin of the short arm (Xp) and the Xq secondary constriction duplicates during early S-phase ( Figure 5) [89,90]. Differential replication timing of Xp and Xq of CHO cells provided a valuable experimental model to analyze the relationship between eu/heterochromatin DNA replication and CA induced by different types of clastogens: UV-C light, the methylating agent methylmethane sulphonate (MMS) and the anti-topoisomerase II inhibitor etoposide (a cleavable complex trapper) in BrdU pulse-labeled CHO9 chromosomes [91,92].…”

“…Incorporation of BrdU in Xp or Xq was disclosed by immunolabeling either treated or control CHO9 metaphases with anti-BrdU antibodies coupled to FITC. The relationship between replication timing, chromatin conformation and genetic damage was investigated by mapping induced BP in Xp and Xq in cells treated both in early and late S-phase [91,92].…”

“…Furthermore, two nearby SSB in each DNA strand may behave as a DSB [98]. The DSB generated could be ultimately processed and transformed in CA [91,92].…”