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Stepwise loading of yeast clamp revealed by ensemble and single-molecule studies.

ABSTRACT: In ensemble and single-molecule experiments using the yeast proliferating cell nuclear antigen (PCNA, clamp) and replication factor C (RFC, clamp loader), we have examined the assembly of the RFC·PCNA·DNA complex and its progression to holoenzyme upon addition of polymerase ? (pol?). We obtained data that indicate (i) PCNA loading on DNA proceeds through multiple conformational intermediates and is successful after several failed attempts; (ii) RFC does not act catalytically on a primed 45-mer templated fork; (iii) the RFC·PCNA·DNA complex formed in the presence of ATP is derived from at least two kinetically distinguishable species; (iv) these species disassemble through either unloading of RFC·PCNA from DNA or dissociation of PCNA into its component subunits; and (v) in the presence of pol? only one species converts to the RFC·PCNA·DNA·pol? holoenzyme. These findings redefine and deepen our understanding of the clamp-loading process and reveal that it is surprisingly one of trial and error to arrive at a heuristic solution.

SUBMITTER: Kumar R

PROVIDER: S-EPMC2993353 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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Stepwise loading of yeast clamp revealed by ensemble and single-molecule studies.

Kumar Ravindra R Nashine Vishal C VC Mishra Padmaja P PP Benkovic Stephen J SJ Lee Tae-Hee TH

Proceedings of the National Academy of Sciences of the United States of America 20101101 46

In ensemble and single-molecule experiments using the yeast proliferating cell nuclear antigen (PCNA, clamp) and replication factor C (RFC, clamp loader), we have examined the assembly of the RFC·PCNA·DNA complex and its progression to holoenzyme upon addition of polymerase δ (polδ). We obtained data that indicate (i) PCNA loading on DNA proceeds through multiple conformational intermediates and is successful after several failed attempts; (ii) RFC does not act catalytically on a primed 45-mer t ...[more]

PMID: 21041673

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Project description:A major hallmark of Alzheimer's disease is the misfolding and aggregation of the amyloid- β peptide (Aβ). While early research pointed towards large fibrillar- and plaque-like aggregates as being the most toxic species, recent evidence now implicates small soluble Aβ oligomers as being orders of magnitude more harmful. Techniques capable of characterizing oligomer stoichiometry and assembly are thus critical for a deeper understanding of the earliest stages of neurodegeneration and for rationally testing next-generation oligomer inhibitors. While the fluorescence response of extrinsic fluorescent probes such as Thioflavin-T have become workhorse tools for characterizing large Aβ aggregates in solution, it is widely accepted that these methods suffer from many important drawbacks, including an insensitivity to oligomeric species. Here, we integrate several biophysics techniques to gain new insight into oligomer formation at the single-molecule level. We showcase single-molecule stepwise photobleaching of fluorescent dye molecules as a powerful method to bypass many of the traditional limitations, and provide a step-by-step guide to implementing the technique in vitro. By collecting fluorescence emission from single Aβ(1-42) peptides labelled at the N-terminal position with HiLyte Fluor 555 via wide-field total internal reflection fluorescence (TIRF) imaging, we demonstrate how to characterize the number of peptides per single immobile oligomer and reveal heterogeneity within sample populations. Importantly, fluorescence emerging from Aβ oligomers cannot be easily investigated using diffraction-limited optical microscopy tools. To assay oligomer activity, we also demonstrate the implementation of another biophysical method involving the ratiometric imaging of Fura-2-AM loaded cells which quantifies the rate of oligomer-induced dysregulation of intracellular Ca2+ homeostasis. We anticipate that the integrated single-molecule biophysics approaches highlighted here will develop further and in principle may be extended to the investigation of other protein aggregation systems under controlled experimental conditions.

Dataset Information

Stepwise loading of yeast clamp revealed by ensemble and single-molecule studies.

Publications

Stepwise loading of yeast clamp revealed by ensemble and single-molecule studies.

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