Unblending of Transcriptional Condensates in Human Repeat Expansion Disease.

Expansions of amino acid repeats occur in >20 inherited human disorders, and many occur in intrinsically disordered regions (IDRs) of transcription factors (TFs). Such diseases are associated with protein aggregation, but the contribution of aggregates to pathology has been controversial. Here, we report that alanine repeat expansions in the HOXD13 TF, which cause hereditary synpolydactyly in humans, alter its phase separation capacity and its capacity to co-condense with transcriptional co-activators.

Simultaneous Real-Time Imaging of Leading and Lagging Strand Synthesis Reveals the Coordination Dynamics of Single Replisomes.

The molecular machinery responsible for DNA replication, the replisome, must efficiently coordinate DNA unwinding with priming and synthesis to complete duplication of both strands. Due to the anti-parallel nature of DNA, the leading strand is copied continuously, while the lagging strand is produced by repeated cycles of priming, DNA looping, and Okazaki-fragment synthesis. Here, we report a multidimensional single-molecule approach to visualize this coordination in the bacteriophage T7 replisome by simultaneously monitoring the kinetics of loop growth and leading-strand synthesis.

Single-molecule observation of prokaryotic DNA replication.

Replication of DNA requires the coordinated activity of a number of proteins within a multiprotein complex, the replisome. Recent advances in single-molecule techniques have enabled the observation of dynamic behavior of individual replisome components and of the replisome as a whole, aspects that previously often have been obscured by ensemble averaging in more classical solution-phase biochemical experiments. To improve robustness and reproducibility of single-molecule assays of replication and allow objective analysis and comparison of results obtained from such assays, common practices should be established.

Single-molecule imaging at high fluorophore concentrations by local activation of dye.

Single-molecule fluorescence microscopy is a powerful tool for observing biomolecular interactions with high spatial and temporal resolution. Detecting fluorescent signals from individual labeled proteins above high levels of background fluorescence remains challenging, however. For this reason, the concentrations of labeled proteins in in vitro assays are often kept low compared to their in vivo concentrations.

Single-molecule studies of polymerase dynamics and stoichiometry at the bacteriophage T7 replication machinery.

Replication of DNA plays a central role in transmitting hereditary information from cell to cell. To achieve reliable DNA replication, multiple proteins form a stable complex, known as the replisome, enabling them to act together in a highly coordinated fashion. Over the past decade, the roles of the various proteins within the replisome have been determined.

Oligomerization transforms human APOBEC3G from an efficient enzyme to a slowly dissociating nucleic acid-binding protein.

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.

A single-molecule view of DNA replication: the dynamic nature of multi-protein complexes revealed.

Recent advances in the development of single-molecule approaches have made it possible to study the dynamics of biomolecular systems in great detail. More recently, such tools have been applied to study the dynamic nature of large multi-protein complexes that support multiple enzymatic activities. In this review, we will discuss single-molecule studies of the replisome, the protein complex responsible for the coordinated replication of double-stranded DNA.

A single zinc finger optimizes the DNA interactions of the nucleocapsid protein of the yeast retrotransposon Ty3.

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.

Scaffold expulsion and genome packaging trigger stabilization of herpes simplex virus capsids.

Herpes simplex virus type 1 (HSV1) capsids undergo extensive structural changes during maturation and DNA packaging. As a result, they become more stable and competent for nuclear egress. To further elucidate this stabilization process, we used biochemical and nanoindentation approaches to analyze the structural and mechanical properties of scaffold-containing (B), empty (A), and DNA-containing (C) nuclear capsids.