Long inverted repeat transiently stalls DNA replication by forming hairpin structures on both leading and lagging strands

Abstract

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.

Unprecedented large inverted repeats at the replication terminus of circular bacterial chromosomes suggest a novel mode of chromosome rescue

“…Not only is the presence of such a naturally occurring very large IR per se unprecedented in circular bacterial genomes, where even small IRs are generally unstable67, but the repeated sequence also includes the putative dif site, the recombination site involved in the resolution of chromosome dimers8. Consequently, the chromosome contains two putative dif sites in opposite orientation instead of the unique site found in other circular bacterial chromosomes, a configuration expected to complicate the resolution of dimers that regularly form during chromosomal replication9 as recombination between two dif sites of opposite orientation (among the four dif sites in an L. bulgaricus chromosome dimer) would result in the inversion of a part of the dimer rather than its resolution510.…”

Acquired Chromosomal Abnormalities and Their Potential Formation Mechanisms in Solid Tumours

“…Of 5′ boundaries in 56 amplicon units, 49 (87.5%) have at least one LIR with stem length ≥20 bp, stem identity ≥70%, and loop length of 0-10 kb, while 47 (83.93%) of 3′ boundaries include at least one LIR with same features ( Chromosomal Abnormalities -A Hallmark Manifestation of Genomic Instability unit, respectively, suggesting that LIRs inside the amplicon unit could potentially generate a hairpin at single-stranded DNA and break the DNA strand during replication. Hairpin structure was shown to form at an interrupted LIR with 111-bp stem and 24-bp internal spacer on both leading and lagging strand templates during replication [110]. In addition, a singlestrand DNA break may cause replication fork stalling or collapse [48].…”

“…In addition, a singlestrand DNA break may cause replication fork stalling or collapse [48]. However, LIRs outside a replicon, near the rearrangement boundaries, may also cause a replication fork stalling through formation of cruciform extrusion [110].…”

“…Hairpin structures were shown to form at an interrupted LIR with 111-bp stem and 24-bp spacer at the frequencies of 32-37% on both leading and lagging strand templates, respectively, suggesting that hairpins were extruded simultaneously by palindrome on both leading and lagging strand templates during replication [110]. However, another study showed that an interrupted palindrome with 230-bp stem and 20-bp spacer formed a hairpin only on the lagging-strand template in Escherichia coli, whereas perfect palindrome generated hairpin on both leading and lagging strand templates during replication [111].…”

Collision with duplex DNA renders Escherichia coli DNA polymerase III holoenzyme susceptible to DNA polymerase IV-mediated polymerase switching on the sliding clamp

“…SSB, wild-type or dnaE173 Pol III* was purified with CBP-tag on ψ subunit as previously described 48 . The γ complex was a generous gift from Dr T. Katayama (Kyushu University).…”