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. Author manuscript; available in PMC: 2018 Jun 22.
Published in final edited form as: Cell Rep. 2015 Mar 12;10(10):1674–1680. doi: 10.1016/j.celrep.2015.02.039

Figure 2. Replication-related and -independent mechanisms of IR-induced DSBs.

Figure 2

(A) Mapping of IR-induced DSBs in replication competent COS-7 cells. LM-PCR amplified regions between the upstream specific primer and the linker IR-induced breakpoints were separated on 1.8% agarose gels. Breakpoint hotspots 1 and 2 (BH 1&2) are indicated. (B) DSBs identified by LM-PCR on plasmids recovered from replication-proficient (SV40+) or -deficient (SV40−) extracts. (C) Mapping of breakpoints by sequencing of cloned PCR products. Gray lines represent the incidence of breaks at this position. (D) IRs stall replication forks in COS-7 cells. Upper panel: schematic of a smooth Y-arc from 2-D gel electrophoresis for the control plasmid (left), and the bulge on the Y-arc, as well as the double-Y shaped replication intermediates caused by a replication barrier from the IR-containing plasmid. Lower panel: short IRs stall replication. The arrows designate the bulge on the Y-shaped arc, indicative of stalled replication intermediates, and the double Y-shaped replication intermediates containing IR-stalled replication forks colliding with forks progressing from the opposite direction. A representative image of 4 independent analyses is shown.