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DNA Replication Initiation Is Blocked by a Distant Chromosome-Membrane Attachment.

ABSTRACT: Although it has been recognized for several decades that chromosome structure regulates the capacity of replication origins to initiate, very little is known about how or if cells actively regulate structure to direct initiation. We report that a localized inducible protein tether between the chromosome and cell membrane in E. coli cells imparts a rapid and complete block to replication initiation. Tethers, composed of a trans-membrane and transcription repressor fusion protein bound to an array of operator sequences, can be placed up to 1 Mb from the origin with no loss of penetrance. Tether-induced initiation blocking has no effect on elongation at pre-existing replication forks and does not cause cell or DNA damage. Whole-genome and site-specific fluorescent DNA labeling in tethered cells indicates that global nucleoid structure and chromosome organization are disrupted. Gene expression patterns, assayed by RNA sequencing, show that tethering induces global supercoiling changes, which are likely incompatible with replication initiation. Parallels between tether-induced initiation blocking and rifampicin treatment and the role of programmed changes in chromosome structure in replication control are discussed.

SUBMITTER: Magnan D

PROVIDER: S-EPMC4546506 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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DNA Replication Initiation Is Blocked by a Distant Chromosome-Membrane Attachment.

Magnan David D Joshi Mohan C MC Barker Anna K AK Visser Bryan J BJ Bates David D

Current biology : CB 20150806 16

Although it has been recognized for several decades that chromosome structure regulates the capacity of replication origins to initiate, very little is known about how or if cells actively regulate structure to direct initiation. We report that a localized inducible protein tether between the chromosome and cell membrane in E. coli cells imparts a rapid and complete block to replication initiation. Tethers, composed of a trans-membrane and transcription repressor fusion protein bound to an array ...[more]

PMID: 26255849

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Project description:Eukaryotic DNA replication initiates from multiple sites on each chromosome called replication origins. In the budding yeast Saccharomyces cerevisiae, origins are defined at discrete sites. Regular spacing and diverse firing characteristics of origins are thought to be required for efficient completion of replication, especially in the presence of replication stress. However, a S. cerevisiae chromosome III harboring multiple origin deletions has been reported to replicate relatively normally, and yet how an origin-deficient chromosome could accomplish successful replication remains unkown. To address this issue, we deleted seven well-characterized origins from chromosome VI, and found that thsese deletions do not cause gross growth defects even in the presence of replication inhibitors. We demonstrated that the origin deletions do cause a strong decrease in the binding of the origin recognition complex. Unexpectedly, replication profiling of this chromosome showed that DNA replication initiates from non-canonical loci around deleted origins in yeast. These results suggest that replication initiation can be unexpectedly flexible in this organism. In this study, we aimed to establish an independent system to investigate how an origin-deficient chromosome is replicated. To this end, we systematically deleted seven well-characterized origins on the left arm of S. cerevisiae chromosome VI and analyzed (1) Orc2 localization during G2/M arrest and (2) BrdU incorporation during synchronous release from G1 arrest into S-phase, and compared the results to wild-type cell signals. For Orc2 ChIP-Chip experiments, Orc-bound DNA was isolated from Orc2-2Xlinker-3XFlag epitope-tagged cells arrested in G2/M using antibodies against Flag. For BrdU ChIP-Chip experiments, actively replicating DNA was isolated from cells harboring a single integrated BrdU incorporation vector released synchronously into 200mM HU using antibodies against BrdU. Immunoprecipitated and input (Orc2) or G1 (BrdU) DNA was then amplified and competitively hybridized to high-resolution strand-specific microarrays covering chromosomes III, VI, and XII.

Dataset Information

DNA Replication Initiation Is Blocked by a Distant Chromosome-Membrane Attachment.

Publications

DNA Replication Initiation Is Blocked by a Distant Chromosome-Membrane Attachment.

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