Modulation of chromosome damage localization by DNA replication timing

Abstract

Purpose: Non-random occurrence of induced chromosome breakpoints (BP) has been repeatedly reported. DNA synthesis and chromatin remodeling may influence chromosome BP localization. The CHO9 X chromosome exhibits an early replicating short euchromatic arm (Xp_e) and a late replicating long heterochromatic arm (Xq_h). We investigated the role played by DNA replication and related chromatin remodeling processes on BP distribution in eu/heterochromatin using the CHO9 X chromosome as a model.

Materials and methods: BP induced by etoposide, a topoisomerase II inhibitor, as well as by the S-dependent clastogens ultraviolet-C light (UV-C) and methyl methanesulfonate (MMS) were mapped to CHO9 X chromosome arms. The base analogue 5-bromo-2′-deoxyuridine (BrdUrd) was pulse-added immediately after UV-C irradiation or during etoposide and MMS treatments (40 min) to identify cells in early S-phase (Xp_e labeled) or late S-phase (Xq_h labeled) after indirect BrdUrd immunodetection in metaphase spreads using primary anti-BrdUrd and secondary fluorochrome-tagged antibodies.

Results: During early S-phase, BP induced by etoposide and MMS mapped preferentially to Xp_e while BP produced by UV-C localized randomly. BP induced by all agents during late S-phase clustered in Xq_h.

Conclusions: Results obtained suggest that replication time of eu/heterochromatin as well as chromatin remodeling may determine BP localization on the CHO9 X chromosome.

Keywords:

• DNA replication time
• eu/heterochromatin
• topoisomerase II inhibitor
• S-dependent clastogens
• chromosome breakpoint distribution
• chromatin remodeling