Long inverted repeats (LIRs), often found in eukaryotic genomes, are unstable in Escherichia coli where they are recognised by the SbcCD (the bacterial Mre11/Rad50 homolog), an endonuclease/exonuclease capable of cleaving hairpin DNA. It has long been postulated that LIRs form hairpin structures exclusively on the lagging-strand template during DNA replication, and SbcCD cleaves these hairpin-containing lagging-strands to generate DNA double-strand breaks. Using a reconstituted oriC plasmid DNA replication system, we have examined how a replication fork behaves when it meets a LIR on DNA. We have shown that leading-strand synthesis stalls transiently within the upstream half of the LIR. Pausing of lagging-strand synthesis at the LIR was not clearly observed, but the pattern of priming sites for Okazaki fragment synthesis was altered within the downstream half of the LIR. We have found that the LIR on a replicating plasmid was cleaved by SbcCD with almost equal frequency on both the leading-and lagging-strand templates. These data strongly suggest that the LIR is readily converted to a cruciform DNA, before the arrival of the fork, creating SbcCDsensitive hairpin structures on both leading-and lagging-strands. We propose a model for the Author ManuscriptThis article is protected by copyright. All rights reserved replication-dependent extrusion of LIRs to form cruciform structures that transiently impede replication fork movement.
The Mre11/Rad50 complex is a central player in various genome maintenance pathways. Here, we report a novel mode of nuclease action found for the Escherichia coli Mre11/Rad50 complex, SbcC2/D2 complex (SbcCD). SbcCD cuts off the top of a cruciform DNA by making incisions on both strands and continues cleaving the dsDNA stem at ∼10-bp intervals. Using linear-shaped DNA substrates, we observed that SbcCD cleaved dsDNA using this activity when the substrate was 110 bp long, but that on shorter substrates the cutting pattern was changed to that predicted for the activity of a 3′-5′ exonuclease. Our results suggest that SbcCD processes hairpin and linear dsDNA ends with this novel DNA end-dependent binary endonuclease activity in response to substrate length rather than using previously reported activities. We propose a model for this mode of nuclease action, which provides new insight into SbcCD activity at a dsDNA end.
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