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1.
Methods Mol Biol ; 1665: 217-239, 2018.
Article in English | MEDLINE | ID: mdl-28940072

ABSTRACT

The complex binding dynamics between DNA and proteins are often obscured by ensemble averaging effects in conventional biochemical experiments. Single-molecule fluorescence methods are powerful tools to investigate DNA-protein interaction dynamics in real time. In this chapter, we focus on using single-molecule Förster Resonance Energy Transfer (smFRET) to probe the binding dynamics of individual proteins on single DNA molecules. We provide a detailed discussion of total internal reflection fluorescence (TIRF) instrument design, nucleic acid labeling with fluorophores, flow cell surface passivation, and data analysis methods.


Subject(s)
DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Fluorescence Resonance Energy Transfer/methods , Nanotechnology/methods , Nucleic Acids/chemistry , Nucleic Acids/metabolism , Protein Binding
2.
Nat Commun ; 6: 10209, 2015 Dec 18.
Article in English | MEDLINE | ID: mdl-26681117

ABSTRACT

Activation-induced deoxycytidine deaminase (AID) generates antibody diversity in B cells by initiating somatic hypermutation (SHM) and class-switch recombination (CSR) during transcription of immunoglobulin variable (IgV) and switch region (IgS) DNA. Using single-molecule FRET, we show that AID binds to transcribed dsDNA and translocates unidirectionally in concert with RNA polymerase (RNAP) on moving transcription bubbles, while increasing the fraction of stalled bubbles. AID scans randomly when constrained in an 8 nt model bubble. When unconstrained on single-stranded (ss) DNA, AID moves in random bidirectional short slides/hops over the entire molecule while remaining bound for ∼ 5 min. Our analysis distinguishes dynamic scanning from static ssDNA creasing. That AID alone can track along with RNAP during transcription and scan within stalled transcription bubbles suggests a mechanism by which AID can initiate SHM and CSR when properly regulated, yet when unregulated can access non-Ig genes and cause cancer.


Subject(s)
Antibody Diversity/genetics , B-Lymphocytes/metabolism , Cytidine Deaminase/metabolism , DNA, Single-Stranded/metabolism , DNA-Directed RNA Polymerases/metabolism , DNA/metabolism , Viral Proteins/metabolism , Animals , Antibody Diversity/immunology , B-Lymphocytes/immunology , Cytidine Deaminase/immunology , Escherichia coli , Fluorescence Resonance Energy Transfer , Immunoglobulin Class Switching/genetics , Immunoglobulin Class Switching/immunology , Sf9 Cells , Somatic Hypermutation, Immunoglobulin/genetics , Somatic Hypermutation, Immunoglobulin/immunology , Spodoptera , Transcription, Genetic/genetics , Transcription, Genetic/immunology
3.
Cell ; 162(2): 271-286, 2015 Jul 16.
Article in English | MEDLINE | ID: mdl-26186187

ABSTRACT

Repair of DNA double strand breaks by homologous recombination (HR) is initiated by Rad51 filament nucleation on single-stranded DNA (ssDNA), which catalyzes strand exchange with homologous duplex DNA. BRCA2 and the Rad51 paralogs are tumor suppressors and critical mediators of Rad51. To gain insight into Rad51 paralog function, we investigated a heterodimeric Rad51 paralog complex, RFS-1/RIP-1, and uncovered the molecular basis by which Rad51 paralogs promote HR. Unlike BRCA2, which nucleates RAD-51-ssDNA filaments, RFS-1/RIP-1 binds and remodels pre-synaptic filaments to a stabilized, "open," and flexible conformation, in which the ssDNA is more accessible to nuclease digestion and RAD-51 dissociation rate is reduced. Walker box mutations in RFS-1, which abolish filament remodeling, fail to stimulate RAD-51 strand exchange activity, demonstrating that remodeling is essential for RFS-1/RIP-1 function. We propose that Rad51 paralogs stimulate HR by remodeling the Rad51 filament, priming it for strand exchange with the template duplex.


Subject(s)
Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/metabolism , Carrier Proteins/metabolism , DNA, Single-Stranded/metabolism , DNA-Binding Proteins/metabolism , Homologous Recombination , Rad51 Recombinase/metabolism , Animals , Caenorhabditis elegans Proteins/genetics , DNA-Binding Proteins/genetics , HEK293 Cells , Humans , Mutation , Nuclear Pore Complex Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism
4.
Nat Chem ; 6(1): 28-33, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24345943

ABSTRACT

The human APOBEC3 proteins are a family of DNA-editing enzymes that play an important role in the innate immune response against retroviruses and retrotransposons. APOBEC3G is a member of this family that inhibits HIV-1 replication in the absence of the viral infectivity factor Vif. Inhibition of HIV replication occurs by both deamination of viral single-stranded DNA and a deamination-independent mechanism. Efficient deamination requires rapid binding to and dissociation from ssDNA. However, a relatively slow dissociation rate is required for the proposed deaminase-independent roadblock mechanism in which APOBEC3G binds the viral template strand and blocks reverse transcriptase-catalysed DNA elongation. Here, we show that APOBEC3G initially binds ssDNA with rapid on-off rates and subsequently converts to a slowly dissociating mode. In contrast, an oligomerization-deficient APOBEC3G mutant did not exhibit a slow off rate. We propose that catalytically active monomers or dimers slowly oligomerize on the viral genome and inhibit reverse transcription.


Subject(s)
Biopolymers/chemistry , Cytidine Deaminase/metabolism , APOBEC-3G Deaminase , Cytidine Deaminase/chemistry , Deamination , HIV-1/physiology , Humans , RNA-Directed DNA Polymerase/metabolism , Virus Replication
5.
Nucleic Acids Res ; 41(19): 8959-68, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23901012

ABSTRACT

Replication by Escherichia coli DNA polymerase III is disrupted on encountering DNA damage. Consequently, specialized Y-family DNA polymerases are used to bypass DNA damage. The protein UmuD is extensively involved in modulating cellular responses to DNA damage and may play a role in DNA polymerase exchange for damage tolerance. In the absence of DNA, UmuD interacts with the α subunit of DNA polymerase III at two distinct binding sites, one of which is adjacent to the single-stranded DNA-binding site of α. Here, we use single molecule DNA stretching experiments to demonstrate that UmuD specifically inhibits binding of α to ssDNA. We predict using molecular modeling that UmuD residues D91 and G92 are involved in this interaction and demonstrate that mutation of these residues disrupts the interaction. Our results suggest that competition between UmuD and ssDNA for α binding is a new mechanism for polymerase exchange.


Subject(s)
DNA Polymerase III/metabolism , DNA, Single-Stranded/metabolism , DNA-Directed DNA Polymerase/metabolism , Escherichia coli Proteins/metabolism , Binding, Competitive , DNA Polymerase III/chemistry , DNA-Directed DNA Polymerase/chemistry , DNA-Directed DNA Polymerase/genetics , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Mutation
6.
Nucleic Acids Res ; 40(2): 751-60, 2012 Jan.
Article in English | MEDLINE | ID: mdl-21917850

ABSTRACT

Reverse transcription in retroviruses and retrotransposons requires nucleic acid chaperones, which drive the rearrangement of nucleic acid conformation. The nucleic acid chaperone properties of the human immunodeficiency virus type-1 (HIV-1) nucleocapsid (NC) protein have been extensively studied, and nucleic acid aggregation, duplex destabilization and rapid binding kinetics have been identified as major components of its activity. However, the properties of other nucleic acid chaperone proteins, such as retrotransposon Ty3 NC, a likely ancestor of HIV-1 NC, are not well understood. In addition, it is unclear whether a single zinc finger is sufficient to optimize the properties characteristic of HIV-1 NC. We used single-molecule DNA stretching as a method for detailed characterization of Ty3 NC chaperone activity. We found that wild type Ty3 NC aggregates single- and double-stranded DNA, weakly stabilizes dsDNA, and exhibits rapid binding kinetics. Single-molecule studies in the presence of Ty3 NC mutants show that the N-terminal basic residues and the unique zinc finger at the C-terminus are required for optimum chaperone activity in this system. While the single zinc finger is capable of optimizing Ty3 NC's DNA interaction kinetics, two zinc fingers may be necessary in order to facilitate the DNA destabilization exhibited by HIV-1 NC.


Subject(s)
DNA/metabolism , Nucleocapsid Proteins/chemistry , Nucleocapsid Proteins/metabolism , Retroelements , Zinc Fingers , Amino Acid Sequence , Kinetics , Molecular Sequence Data , Nucleocapsid Proteins/genetics , Sequence Deletion , Yeasts/genetics
7.
Nucleic Acids Res ; 39(13): 5611-21, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21441536

ABSTRACT

Retrotransposition amplifies LINE-1 (L1) to high copy number in mammalian genomes. The L1 protein encoded by ORF1 (ORF1p) is required for retrotransposition. This dependence on ORF1p was investigated by mutating three highly conserved residues, R238, R284 and Y318 to alanine, thereby inactivating retrotransposition. R284A and Y318A were rescued by further substituting the alanine with the appropriate conservative amino acid, e.g. lysine or phenylalanine, respectively, whereas R238K remained inactive. Quantification of the steady-state levels of L1 RNA and ORF1p failed to discriminate active from inactive variants, indicating loss of L1 retrotransposition resulted from loss of function rather than reduced expression. The two biochemical properties known for ORF1p are high-affinity RNA binding and nucleic acid chaperone activity. Only R238A/K exhibited significantly reduced RNA affinities. The nucleic acid chaperone activities of the remaining paired mutants were assessed by single-molecule DNA stretching and found to mirror retrotransposition activity. To further examine ORF1p chaperone function, their energetic barriers to DNA annealing and melting were derived from kinetic work. When plotted against each other, the ratio of these two activities distinguished functional from non-functional ORF1p variants. These findings enhance our understanding of the requirements for ORF1p in LINE-1 retrotransposition and, more generally, nucleic acid chaperone function.


Subject(s)
Long Interspersed Nucleotide Elements , Retroelements/genetics , Amino Acid Substitution , Animals , DNA/chemistry , Mice , Mutation , Nucleic Acid Denaturation , RNA/metabolism
9.
Phys Life Rev ; 7(3): 299-341, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20576476

ABSTRACT

Single molecule force spectroscopy is a powerful method that uses the mechanical properties of DNA to explore DNA interactions. Here we describe how DNA stretching experiments quantitatively characterize the DNA binding of small molecules and proteins. Small molecules exhibit diverse DNA binding modes, including binding into the major and minor grooves and intercalation between base pairs of double-stranded DNA (dsDNA). Histones bind and package dsDNA, while other nuclear proteins such as high mobility group proteins bind to the backbone and bend dsDNA. Single-stranded DNA (ssDNA) binding proteins slide along dsDNA to locate and stabilize ssDNA during replication. Other proteins exhibit binding to both dsDNA and ssDNA. Nucleic acid chaperone proteins can switch rapidly between dsDNA and ssDNA binding modes, while DNA polymerases bind both forms of DNA with high affinity at distinct binding sites at the replication fork. Single molecule force measurements quantitatively characterize these DNA binding mechanisms, elucidating small molecule interactions and protein function.


Subject(s)
Biophysics/methods , DNA-Binding Proteins/metabolism , DNA/metabolism , Microscopy, Atomic Force , DNA/chemistry , DNA/ultrastructure , DNA, Single-Stranded/chemistry , DNA, Single-Stranded/metabolism , DNA-Binding Proteins/chemistry , DNA-Directed DNA Polymerase/chemistry , DNA-Directed DNA Polymerase/metabolism , Molecular Chaperones/chemistry , Molecular Chaperones/metabolism
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