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. 2018 Jun 13;46(13):6670–6682. doi: 10.1093/nar/gky463

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© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 3. — RecD is not required for recombination intermediate formation. (A, B and D) Analysis of the state of a 174 Kb DNA fragment containing the DSB locus (intact, broken or branched) by pulsed-field gel electrophoresis and Southern blot, probed for both sides of the DSB locus (proximal and distal to the origin of replication oriC) following induction of SbcCD expression for 60 min. DNA from a strain harbouring an I-SceI cleavage site in the locus in which the palindrome is integrated was digested with I-SceI in vitro to act as marker for broken DNA (‘in vitro digest’). In vitro digestion with I-SceI results in low frequency cleavage at an endogenous chromosomal site (I-SceI_cs*). (C) Normalized enrichment of DNA in an 80 kb region containing the DSB locus following RecA chromatin immunoprecipitation (ChIP) in RecBCD+ and recD mutants with and without a palindrome at lacZ, following induction of SbcCD expression for 60 min. To account for variability in sequencing depth, data were normalized using a median of ratios scaling factor. The data was smoothed using a loess filter with a 2 kb window. All panels show results from single, representative experiments in which DNA replication proceeds from left to right. See also Supplementary Figure S3.