Ligand-linked Structural Changes in the Escherichia coli Biotin Repressor: The Significance of Surface Loops for Binding and Allostery

AbstractDeamidation is a major age-related modification in the human lens that is highly prevalent in crystallins isolated from cataractous lenses. However, the mechanism by which deamidation causes proteins to become insoluble is not known, because of only subtle structural changes observed in vitro. We have identified Asn14 and Asn76 of γS-crystallin as highly deamidated in insoluble proteins. These sites are on the surface of the N-terminal domain and were mimicked by replacing the Asn with Asp residues. We used heteronuclear NMR spectroscopy to measure their amide hydrogen exchange and 15N relaxation dynamics to identify regions with significantly increased dynamics compared to wildtype-γS. Changes in dynamics were localized to the C-terminal domain, particularly to helix and surface loops distant from the mutation sites. Thus, a potential mechanism for γS deamidation-induced insolubilization in cataractous lenses is altered dynamics due to local regions of unfolding and increased flexibility.

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A gatekeeping function of the replicative polymerase controls pathway choice in the resolution of lesion-stalled replisomes

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1914485116 ◽

2019 ◽

Vol 116 (51) ◽

pp. 25591-25601 ◽

Cited By ~ 5

Author(s):

Seungwoo Chang ◽

Karel Naiman ◽

Elizabeth S. Thrall ◽

James E. Kath ◽

Slobodan Jergic ◽

...

Keyword(s):

Escherichia Coli ◽

Dna Synthesis ◽

Genome Stability ◽

Structural Changes ◽

Translesion Synthesis ◽

Dynamic Interaction ◽

Dna Lesions ◽

Exonuclease Activity ◽

Replication Fidelity ◽

Proper Resolution

DNA lesions stall the replisome and proper resolution of these obstructions is critical for genome stability. Replisomes can directly replicate past a lesion by error-prone translesion synthesis. Alternatively, replisomes can reprime DNA synthesis downstream of the lesion, creating a single-stranded DNA gap that is repaired primarily in an error-free, homology-directed manner. Here we demonstrate how structural changes within theEscherichia colireplisome determine the resolution pathway of lesion-stalled replisomes. This pathway selection is controlled by a dynamic interaction between the proofreading subunit of the replicative polymerase and the processivity clamp, which sets a kinetic barrier to restrict access of translesion synthesis (TLS) polymerases to the primer/template junction. Failure of TLS polymerases to overcome this barrier leads to repriming, which competes kinetically with TLS. Our results demonstrate that independent of its exonuclease activity, the proofreading subunit of the replisome acts as a gatekeeper and influences replication fidelity during the resolution of lesion-stalled replisomes.

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Bacteriophage ФX 174

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100099908 ◽

1968 ◽

Vol 26 ◽

pp. 32-33

Author(s):

M. E. Bayer

Keyword(s):

Escherichia Coli ◽

Nutrient Medium ◽

Structural Changes ◽

Uranyl Acetate ◽

Dense Material ◽

Medium Ph ◽

Electron Dense Material ◽

Virus Particles ◽

Ultrathin Sections

Cultures of Escherichia coli CIA (Bertani) growing logarithmically in nutrient medium were infected with 5 to 200 bacteriophage §X 174 per cell. At various times after infection the cultures were fixed for 1 hour in nutrient medium containing 5% formaldehyde pH 7, then pelleted and resuspended in 1% OSO4 in L-medium pH 7 for 1 hour; the material was pelleted again and resuspended in a mixture of 1% OsO4 and 1% uranyl-acetate in water; in this mixture the cells were fixed for 10 hours at 20° C; after fixation they were dehydrated in acetone and embedded in Vestopal W. In ultrathin sections the first structural changes became visible 15 minutes after infection when some of the cells seemed to swell and round up. In the cells small aggregates of electron dense material were observed in the chromosomal areas, and sometimes virus particles were also seen within the area of the cells’ chromosome.

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