Structure of the SSB-DNA polymerase III interface and its role in DNA replication

Marceau, Aimee H.; Bahng, Soon; Massoni, Shawn C.; George, Nicholas P.; Sandler, Steven J.; Marians, Kenneth J.; Keck, James L.

doi:10.1038/emboj.2011.305

Cited by 138 publications

(144 citation statements)

References 54 publications

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“…3A). In other proteins, sequence changes at the α-carboxyl interaction position dramatically destabilize their interactions with SSB (38)(39)(40)(41).…”

Section: Resultsmentioning

confidence: 99%

Structural mechanisms of PriA-mediated DNA replication restart

Bhattacharyya

George

Thurmes

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

205

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Collisions between cellular DNA replication machinery (replisomes) and damaged DNA or immovable protein complexes can dissociate replisomes before the completion of replication. This potentially lethal problem is resolved by cellular "replication restart" reactions that recognize the structures of prematurely abandoned replication forks and mediate replisomal reloading. In bacteria, this essential activity is orchestrated by the PriA DNA helicase, which identifies replication forks via structure-specific DNA binding and interactions with fork-associated ssDNA-binding proteins (SSBs). However, the mechanisms by which PriA binds replication fork DNA and coordinates subsequent replication restart reactions have remained unclear due to the dearth of high-resolution structural information available for the protein. Here, we describe the crystal structures of full-length PriA and PriA bound to SSB. The structures reveal a modular arrangement for PriA in which several DNA-binding domains surround its helicase core in a manner that appears to be poised for binding to branched replication fork DNA structures while simultaneously allowing complex formation with SSB. PriA interaction with SSB is shown to modulate SSB/DNA complexes in a manner that exposes a potential replication initiation site. From these observations, a model emerges to explain how PriA links recognition of diverse replication forks to replication restart.X-ray crystal structure | single-molecule FRET

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“…3A). In other proteins, sequence changes at the α-carboxyl interaction position dramatically destabilize their interactions with SSB (38)(39)(40)(41).…”

Section: Resultsmentioning

confidence: 99%

Structural mechanisms of PriA-mediated DNA replication restart

Bhattacharyya

George

Thurmes

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

205

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“…For nucleoid imaging, mCherry fused to the ␣-subunit of the HU protein and expressed from the endogenous chromosomal promoter was used (hupA100::mCherry) (kindly provided by S. Sandler) (67). hupA100::mCherry was transferred into AB1157 by P1 transduction (62) to obtain EH20 (see Table S1 in the supplemental material).…”

Section: Methodsmentioning

confidence: 99%

Lack of the H-NS Protein Results in Extended and Aberrantly Positioned DNA during Chromosome Replication and Segregation in Escherichia coli

2016

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The architectural protein H-NS binds nonspecifically to hundreds of sites throughout the chromosome and can multimerize to stiffen segments of DNA as well as to form DNA-protein-DNA bridges. H-NS has been suggested to contribute to the orderly folding of the Escherichia coli chromosome in the highly compacted nucleoid. In this study, we investigated the positioning and dynamics of the origins, the replisomes, and the SeqA structures trailing the replication forks in cells lacking the H-NS protein.In H-NS mutant cells, foci of SeqA, replisomes, and origins were irregularly positioned in the cell. Further analysis showed that the average distance between the SeqA structures and the replisome was increased by ϳ100 nm compared to that in wild-type cells, whereas the colocalization of SeqA-bound sister DNA behind replication forks was not affected. This result may suggest that H-NS contributes to the folding of DNA along adjacent segments. H-NS mutant cells were found to be incapable of adopting the distinct and condensed nucleoid structures characteristic of E. coli cells growing rapidly in rich medium. It appears as if H-NS mutant cells adopt a "slow-growth" type of chromosome organization under nutrient-rich conditions, which leads to a decreased cellular DNA content. IMPORTANCE It is not fully understood how and to what extent nucleoid-associated proteins contribute to chromosome folding and organization during replication and segregation in Escherichia coli. In this work, we find in vivo indications that cells lacking the nucleoid-associated protein H-NS have a lower degree of DNA condensation than wild-type cells. Our work suggests that H-NS is in-volved in condensing the DNA along adjacent segments on the chromosome and is not likely to tether newly replicated strands of sister DNA. We also find indications that H-NS is required for rapid growth with high DNA content and for the formation of a highly condensed nucleoid structure under such conditions. A cross all domains of life, it is crucial that genomes are structurally organized in a way that compacts DNA to fit inside the confined space of a cell and at the same time allows for interaction with key proteins performing replication, transcription, recombination, and repair (1-7). Unlike eukaryotic cells, bacterial cells do not possess an envelope-enclosed organelle for storage and handling of genomic DNA. The DNA is instead organized into compact bodies called nucleoids (3)(4)(5)8). These nucleoids are highly complex, and the underlying organizational mechanisms appear to be remarkably similar to that of eukaryotic cells (3, 9). The nucleoid occupies the central part of the bacterial cell (8), and its shape is dependent on a variety of factors, such as environmental conditions or genetic mutations (7, 10-13). For example, significant nucleoid compaction occurs after exposure of Escherichia coli to UV light, due to a global reorganization in response to DNA damage and the activation of the SOS response (12, 13).Certain types of proteins, called nucle...

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“…Characterization of many of the SSBprotein interactions has shown in each case that the C-terminus is the site of interaction with its protein binding partners, which include the χ subunit of the replicative clamp loader complex, the replication restart helicase PriA, and exonuclease I [8]. Structural information about several of these interactions suggests that SSB-protein interactions share a conserved binding mechanism that relies on a mixture of hydrophobic and electrostatic contacts [18,20,21].…”

Section: Introductionmentioning

confidence: 99%

Escherichia coli Single-Stranded DNA-Binding Protein: NanoESI-MS Studies of Salt-Modulated Subunit Exchange and DNA Binding Transactions

Mason

Jergic

et al. 2013

J. Am. Soc. Mass Spectrom.

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Escherichia coli single-stranded DNA-binding protein: nanoESI-MS studies of salt-modulated subunit exchange and DNA binding transactions AbstractSingle-stranded DNA-binding proteins (SSBs) are ubiquitous oligomeric proteins that bind with very high affinity to single-stranded DNA and have a variety of essential roles in DNA metabolism. Nanoelectrospray ionization mass spectrometry (nanoESI-MS) was used to monitor subunit exchange in full-length and truncated forms of the homotetrameric SSB from Escherichia coli. Subunit exchange in the native protein was found to occur slowly over a period of hours, but was significantly more rapid in a truncated variant of SSB from which the eight C-terminal residues were deleted. This effect is proposed to result from C-terminus mediated stabilization of the SSB tetramer, in which the C-termini interact with the DNA-binding cores of adjacent subunits. NanoESI-MS was also used to examine DNA binding to the SSB tetramer. Binding of single-stranded oligonucleotides [one molecule of (dT)70, one molecule of (dT)35, or two molecules of (dT)35] was found to prevent SSB subunit exchange. Transfer of SSB tetramers between discrete oligonucleotides was also observed and is consistent with predictions from solution-phase studies, suggesting that SSB-DNA complexes can be reliably analyzed by ESI mass spectrometry. Escherichia coli. Subunit exchange in the native protein was found to occur slowly over a period of hours, but was significantly more rapid in a truncated variant of SSB from which the eight C-terminal residues were deleted. This effect is proposed to result from C-terminus mediated stabilization of the SSB tetramer, in which the C-termini interact with the DNAbinding cores of adjacent subunits. NanoESI-MS was also used to examine DNA binding to the SSB tetramer. Binding of single-stranded oligonucleotides [one molecule of (dT) 70 , one molecule of (dT) 35 or two molecules of (dT) 35 ] was found to prevent SSB subunit exchange.Transfer of SSB tetramers between discrete oligonucleotides was also observed and is consistent with predictions from solution-phase studies, suggesting that SSB-DNA complexes can be reliably analyzed by ESI mass spectrometry.

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Structure of the SSB-DNA polymerase III interface and its role in DNA replication

Cited by 138 publications

References 54 publications

Structural mechanisms of PriA-mediated DNA replication restart

Structural mechanisms of PriA-mediated DNA replication restart

Lack of the H-NS Protein Results in Extended and Aberrantly Positioned DNA during Chromosome Replication and Segregation in Escherichia coli

Escherichia coli Single-Stranded DNA-Binding Protein: NanoESI-MS Studies of Salt-Modulated Subunit Exchange and DNA Binding Transactions

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