Long inverted repeats (LIRs), often found in eukaryotic genomes, are unstable in Escherichia coli where they are recognized by the SbcCD (the bacterial Mre11/Rad50 homologue), 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 SbcCD-sensitive hairpin structures on both leading and lagging strands. We propose a model for the replication-dependent extrusion of LIRs to form cruciform structures that transiently impede replication fork movement. Using a reconstituted oriC plasmid DNA replication system, we have examined how a replication fork behaves when it meets a long inverted repeats. Our data show that the leading-strand synthesis stalls transiently at the inverted repeats. We have found that the repeat was cleaved by SbcCD, the bacterial Mre11/Rad50 homolog, on both the leading and lagging strands. These data suggest that a cruciform DNA is formed at the repeat when the fork approaches, creating SbcCD-sensitive structures on template DNA.
All Science Journal Classification (ASJC) codes
- Cell Biology