Measuring DNA mechanics on the genome scale.

Mechanical deformations of DNA such as bending are ubiquitous and have been implicated in diverse cellular functions. However, the lack of high-throughput tools to measure the mechanical properties of DNA has limited our understanding of how DNA mechanics influence chromatin transactions across the genome. Here we develop 'loop-seq'-a high-throughput assay to measure the propensity for DNA looping-and determine the intrinsic cyclizabilities of 270,806 50-base-pair DNA fragments that span Saccharomyces cerevisiae chromosome V, other genomic regions, and random sequences.

FACT and Ubp10 collaborate to modulate H2B deubiquitination and nucleosome dynamics.

Monoubiquitination of histone H2B (H2B-Ub) plays a role in transcription and DNA replication, and is required for normal localization of the histone chaperone, FACT. In yeast, H2B-Ub is deubiquitinated by Ubp8, a subunit of SAGA, and Ubp10. Although they target the same substrate, loss of Ubp8 and Ubp10 cause different phenotypes and alter the transcription of different genes.

Competition Assay for Measuring Deubiquitinating Enzyme Substrate Affinity.

Assays of the affinity of a deubiquitinating enzyme for substrate, either through binding studies or determination of the Michaelis constant, K, can shed light on substrate selectivity and the effects of mutations on substrate interactions. The difficulty in generating sufficient quantities of ubiquitinated substrate frequently presents a barrier to these studies. We describe here an alternative approach that can be used in cases where non-hydrolyzable, chemically ubiquitinated substrate analogs can be more readily generated.

Active site alanine mutations convert deubiquitinases into high-affinity ubiquitin-binding proteins.

A common strategy for exploring the biological roles of deubiquitinating enzymes (DUBs) in different pathways is to study the effects of replacing the wild-type DUB with a catalytically inactive mutant in cells. We report here that a commonly studied DUB mutation, in which the catalytic cysteine is replaced with alanine, can dramatically increase the affinity of some DUBs for ubiquitin. Overexpression of these tight-binding mutants thus has the potential to sequester cellular pools of monoubiquitin and ubiquitin chains.

Hydrazide Mimics for Protein Lysine Acylation To Assess Nucleosome Dynamics and Deubiquitinase Action.

A range of acyl-lysine (acyl-Lys) modifications on histones and other proteins have been mapped over the past decade but for most, their functional and structural significance remains poorly characterized. One limitation in the study of acyl-Lys containing proteins is the challenge of producing them or their mimics in site-specifically modified forms. We describe a cysteine alkylation-based method to install hydrazide mimics of acyl-Lys post-translational modifications (PTMs) on proteins.

Recognition of ubiquitinated nucleosomes.

Histone ubiquitination plays a non-degradative role in regulating transcription and the DNA damage response. A mechanistic understanding of this chromatin modification has lagged that of small histone modifications because of the technical challenges in preparing ubiquitinated nucleosomes. The recent structure of the DUB module of the SAGA coactivator complex bound to a nucleosome containing monoubiquitinated H2B has provided the first view of how specialized subunits target this enzyme to its substrate.

Structural basis for histone H2B deubiquitination by the SAGA DUB module.

Monoubiquitinated histone H2B plays multiple roles in transcription activation. H2B is deubiquitinated by the Spt-Ada-Gcn5 acetyltransferase (SAGA) coactivator, which contains a four-protein subcomplex known as the deubiquitinating (DUB) module. The crystal structure of the Ubp8/Sgf11/Sus1/Sgf73 DUB module bound to a ubiquitinated nucleosome reveals that the DUB module primarily contacts H2A/H2B, with an arginine cluster on the Sgf11 zinc finger domain docking on the conserved H2A/H2B acidic patch.

Responsiveness of self-report measures in individuals with vertigo, dizziness, and unsteadiness.

Objective: The responsiveness (sensitivity to change) of many self-report measures commonly used with individuals who have balance and vestibular dysfunction has not been assessed. The purpose of this study was to determine the responsiveness of 4 self-report measures including the Activities-Specific Balance Confidence (ABC) scale, Dizziness Handicap Inventory (DHI), Falls Efficacy Scale-International (FES-I), and Vestibular Activities and Participation (VAP) scale in people seeking treatment for vertigo, dizziness, and unsteadiness.

Study Design: A prospective descriptive study.

Reliability and validity of the Falls Efficacy Scale-International (FES-I) in individuals with dizziness and imbalance.

Hypothesis: The purpose of this research is to establish the test-retest reliability and convergent validity of the Falls Efficacy Scale-International (FES-I) in people with vestibular disorders.

Background: Individuals with vestibular dysfunction have an increased risk of falling. The FES-I is a measure used to quantify an individual's concern of falling during different tasks.

Stoichiometry and affinity for thymine DNA glycosylase binding to specific and nonspecific DNA.

Deamination of 5-methylcytosine to thymine creates mutagenic G · T mispairs, contributing to cancer and genetic disease. Thymine DNA glycosylase (TDG) removes thymine from these G · T lesions, and follow-on base excision repair yields a G · C pair. A previous crystal structure revealed TDG (catalytic domain) bound to abasic DNA product in a 2:1 complex, one subunit at the abasic site and the other bound to undamaged DNA.

Role of two strictly conserved residues in nucleotide flipping and N-glycosylic bond cleavage by human thymine DNA glycosylase.

Thymine DNA glycosylase (TDG) promotes genomic integrity by excising thymine from mutagenic G.T mismatches arising by deamination of 5-methylcytosine, and follow-on base excision repair enzymes restore a G.C pair.

Solution structure of S100A1 bound to the CapZ peptide (TRTK12).

As is typical for S100-target protein interactions, a Ca 2+-dependent conformational change in S100A1 is required to bind to a 12-residue peptide (TRTK12) derived from the actin-capping protein CapZ. In addition, the Ca 2+-binding affinity of S100A1 is found to be tightened (greater than threefold) when TRTK12 is bound. To examine the biophysical basis for these observations, we determined the solution NMR structure of TRTK12 in a complex with Ca 2+-loaded S100A1.

Crystal structure of human thymine DNA glycosylase bound to DNA elucidates sequence-specific mismatch recognition.

Cytosine methylation at CpG dinucleotides produces m(5)CpG, an epigenetic modification that is important for transcriptional regulation and genomic stability in vertebrate cells. However, m(5)C deamination yields mutagenic G.T mispairs, which are implicated in genetic disease, cancer, and aging.

Excision of 5-halogenated uracils by human thymine DNA glycosylase. Robust activity for DNA contexts other than CpG.

Thymine DNA glycosylase (TDG) excises thymine from G.T mispairs and removes a variety of damaged bases (X) with a preference for lesions in a CpG.X context.