Major Domain Swiveling Revealed by the Crystal Structures of Complexes of E. coli Rep Helicase Bound to Single-Stranded DNA and ADP

Abstract During DNA replication in E. coli, a switch between DnaG primase and DNA polymerase III holoenzyme (pol III) activities has to occur every time when the synthesis of a new Okazaki fragment starts. As both primase and the χ subunit of pol III interact with the highly conserved C-terminus of single-stranded DNA-binding protein (SSB), it had been proposed that the binding of both proteins to SSB is mutually exclusive. Using a replication system containing the origin of replication of the single-stranded DNA phage G4 (G4ori) saturated with SSB, we tested whether DnaG and pol III can bind concurrently to the primed template. We found that the addition of pol III does not lead to a displacement of primase, but to the formation of higher complexes. Even pol III-mediated primer elongation by one or several DNA nucleotides does not result in the dissociation of DnaG. About 10 nucleotides have to be added in order to displace one of the two primase molecules bound to SSB-saturated G4ori. The concurrent binding of primase and pol III is highly plausible, since even the SSB tetramer situated directly next to the 3′-terminus of the primer provides four C-termini for protein-protein interactions.

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E. coli Single-Stranded DNA Binding (SSB) Protein Undergoes Dynamic Liquid-Liquid Phase Separation Controlled via Protein-Protein and Protein-DNA Interactions

Biophysical Journal ◽

10.1016/j.bpj.2019.11.2680 ◽

2020 ◽

Vol 118 (3) ◽

pp. 484a

Author(s):

Gabor Harami ◽

Zoltan J. Kovacs ◽

János Pálinkás ◽

Rita Pancsa ◽

Veronika Baráth ◽

...

Keyword(s):

Phase Separation ◽

Liquid Phase ◽

Dna Binding ◽

Liquid Phase Separation ◽

Dna Interactions ◽

Single Stranded Dna ◽

E Coli ◽

Protein Dna Interactions ◽

Liquid Liquid Phase Separation

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The Mechanism of Replication of Single-Stranded DNA. VI. Requirements for Ribonucleoside Triphosphates and DNA Polymerase III

Canadian Journal of Biochemistry ◽

10.1139/o73-214 ◽

1973 ◽

Vol 51 (12) ◽

pp. 1588-1597 ◽

Cited By ~ 7

Author(s):

David T. Denhardt ◽

Makoto Iwaya ◽

Grant McFadden ◽

Gerald Schochetman

Keyword(s):

Dna Polymerase ◽

Dna Polymerases ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Dna Polymerase Iii ◽

Single Stranded Dna ◽

E Coli ◽

Polymerase Iii

Evidence is presented that in Escherichia coli made permeable to nucleotides by exposure to toluene, the synthesis of a DNA chain complementary to the infecting single-stranded DNA of bacteriophage [Formula: see text] requires ATP as well as the four deoxyribonucleoside triphosphates. This synthesis results in the formation of the parental double-stranded replicative-form (RF) molecule. The ATP is not required simply to prevent degradation of the ribonucleoside or deoxyribonucleoside triphosphates; it can be partially substituted for by other ribonucleoside triphosphates.No single one of the known E. coli DNA polymerases appears to be uniquely responsible in vivo for the formation of the parental RF. Since [Formula: see text] replicates well in strains lacking all, or almost all, of the in-vitro activities of DNA polymerases I and II, neither of these two enzymes would seem essential; and in a temperature-sensitive E. coli mutant (dnaEts) deficient in DNA polmerase-I activity and possessing a temperature-sensitive DNA polymerase III, the viral single-stranded DNA is efficiently incorporated into an RF molecule at the restrictive temperature. In contrast, both RF replication and progeny single-stranded DNA synthesis are dependent upon DNA polymerase III activity.

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High-resolution crystal structures of Escherichia coli FtsZ bound to GDP and GTP

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x20001132 ◽

2020 ◽

Vol 76 (2) ◽

pp. 94-102 ◽

Cited By ~ 2

Author(s):

Maria A. Schumacher ◽

Tomoo Ohashi ◽

Lauren Corbin ◽

Harold P. Erickson

Keyword(s):

Escherichia Coli ◽

Staphylococcus Aureus ◽

Cell Division ◽

High Resolution ◽

Crystal Structures ◽

Bacterial Cell ◽

Model System ◽

E Coli ◽

Z Ring ◽

Main Model

Bacterial cytokinesis is mediated by the Z-ring, which is formed by the prokaryotic tubulin homolog FtsZ. Recent data indicate that the Z-ring is composed of small patches of FtsZ protofilaments that travel around the bacterial cell by treadmilling. Treadmilling involves a switch from a relaxed (R) state, favored for monomers, to a tense (T) conformation, which is favored upon association into filaments. The R conformation has been observed in numerous monomeric FtsZ crystal structures and the T conformation in Staphylococcus aureus FtsZ crystallized as assembled filaments. However, while Escherichia coli has served as a main model system for the study of the Z-ring and the associated divisome, a structure has not yet been reported for E. coli FtsZ. To address this gap, structures were determined of the E. coli FtsZ mutant FtsZ(L178E) with GDP and GTP bound to 1.35 and 1.40 Å resolution, respectively. The E. coli FtsZ(L178E) structures both crystallized as straight filaments with subunits in the R conformation. These high-resolution structures can be employed to facilitate experimental cell-division studies and their interpretation in E. coli.

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Probing E. coli SSB Protein-DNA topology by reversing DNA backbone polarity

10.1101/2020.12.05.412478 ◽

2020 ◽

Author(s):

Alexander G. Kozlov ◽

Timothy M. Lohman

Keyword(s):

Dna Binding ◽

Crystal Structures ◽

Nearest Neighbor ◽

Genome Maintenance ◽

Binding Properties ◽

Ssdna Binding ◽

E Coli ◽

Phosphodiester Linkage ◽

The Individual ◽

Dna Binding Properties

AbstractE. coli single strand (ss) DNA binding protein (SSB) is an essential protein that binds ssDNA intermediates formed during genome maintenance. SSB homo-tetramers bind ssDNA in two major modes differing in occluded site size and cooperativity. The (SSB)35 mode in which ssDNA wraps on average around two subunits is favored at low [NaCl] and high SSB to DNA ratios and displays high “unlimited”, nearest-neighbor cooperativity forming long protein clusters. The (SSB)65 mode, in which ssDNA wraps completely around four subunits of the tetramer, is favored at higher [NaCl] (> 200 mM) and displays “limited” low cooperativity. Crystal structures of E. coli SSB and P. falciparum SSB show ssDNA bound to the SSB subunits (OB-folds) with opposite polarities of the sugar phosphate backbones. To investigate whether SSB subunits show a polarity preference for binding ssDNA, we examined EcSSB and PfSSB binding to a series of (dT)70 constructs in which the backbone polarity was switched in the middle of the DNA by incorporating a reverse polarity (RP) phosphodiester linkage, either 3’-3’ or 5’-5’. We find only minor effects on the DNA binding properties for these RP constructs, although (dT)70 with a 3’-3’ polarity switch shows decreased affinity for EcSSB in the (SSB)65 mode and lower cooperativity in the (SSB)35 mode. However, (dT)70 in which every phosphodiester linkage is reversed, does not form a completely wrapped (SSB)65 mode, but rather binds EcSSB in the (SSB)35 mode, with little cooperativity. In contrast, PfSSB, which binds ssDNA only in an (SSB)65 mode and with opposite backbone polarity and different topology, shows little effect of backbone polarity on its DNA binding properties. We present structural models suggesting that strict backbone polarity can be maintained for ssDNA binding to the individual OB-folds if there is a change in ssDNA wrapping topology of the RP ssDNA.Statement of SignificanceSingle stranded (ss) DNA binding (SSB) proteins are essential for genome maintenance. Usually homo-tetrameric, bacterial SSBs bind ssDNA in multiple modes, one of which involves wrapping 65 nucleotides of ssDNA around all four subunits. Crystal structures of E. coli and P. falciparum SSB-ssDNA complexes show ssDNA bound with different backbone polarity orientations raising the question of whether these SSBs maintain strict backbone polarity in binding ssDNA. We show that both E. coli and P. falciparum SSBs can still form high affinity fully wrapped complexes with non-natural DNA containing internal reversals of the backbone polarity. These results suggest that both proteins maintain a strict backbone polarity preference, but adopt an alternate ssDNA wrapping topology.

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Analysis of crystalline and solution states of ligand-free spermidine N-acetyltransferase (SpeG) from Escherichia coli

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798319006545 ◽

2019 ◽

Vol 75 (6) ◽

pp. 545-553 ◽

Cited By ~ 1

Author(s):

Ekaterina V. Filippova ◽

Steven Weigand ◽

Olga Kiryukhina ◽

Alan J. Wolfe ◽

Wayne F. Anderson

Keyword(s):

Escherichia Coli ◽

Vibrio Cholerae ◽

Crystal Structures ◽

Coenzyme A ◽

Amino Group ◽

Acetyl Coenzyme A ◽

E Coli ◽

Ligand Free ◽

Acetyl Group ◽

Terminal Amino

Spermidine N-acetyltransferase (SpeG) transfers an acetyl group from acetyl-coenzyme A to an N-terminal amino group of intracellular spermidine. This acetylation inactivates spermidine, reducing the polyamine toxicity that tends to occur under certain chemical and physical stresses. The structure of the SpeG protein from Vibrio cholerae has been characterized: while the monomer possesses a structural fold similar to those of other Gcn5-related N-acetyltransferase superfamily members, its dodecameric structure remains exceptional. In this paper, structural analyses of SpeG isolated from Escherichia coli are described. Like V. cholerae SpeG, E. coli SpeG forms dodecamers, as revealed by two crystal structures of the ligand-free E. coli SpeG dodecamer determined at 1.75 and 2.9 Å resolution. Although both V. cholerae SpeG and E. coli SpeG can adopt an asymmetric open dodecameric state, solution analysis showed that the oligomeric composition of ligand-free E. coli SpeG differs from that of ligand-free V. cholerae SpeG. Based on these data, it is proposed that the equilibrium balance of SpeG oligomers in the absence of ligands differs from one species to another and thus might be important for SpeG function.

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