Highlights d Repeat expansions in transcription factors (TFs) alter their phase separation capacity d Repeat expansions in TFs perturb the composition of TFcontaining condensates d Hoxd13 repeat expansions alter the transcriptional program in a synpolydactyly model d Features of intrinsically disordered regions in TFs are linked to condensation behavior

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. Atomic force microscopy experiments revealed that A and C capsids were mechanically indistinguishable, indicating that the presence of DNA does not account for changes in mechanical properties during capsid maturation. Despite having the same rigidity, the scaffold-containing B capsids broke at significantly lower forces than A and C capsids. An extraction of pentons with guanidine hydrochloride (GuHCl) increased the flexibility of all capsids. Surprisingly, the breaking forces of the modified A and C capsids dropped to similar values as those of the GuHCl-treated B capsids, indicating that mechanical reinforcement occurs at the vertices. Nonetheless, it also showed that HSV1 capsids possess a remarkable structural integrity that was preserved after removal of pentons. We suggest that HSV1 capsids are stabilized after removal of the scaffold proteins, and that this stabilization is triggered by the packaging of DNA, but independent of the actual presence of DNA.atomic force microscopy ͉ nanoindentation ͉ penton ͉ viral structure ͉ virus mechanics H erpes simplex virus type 1 (HSV1) virions encapsidate their 152 kbp double-stranded DNA genome in an icosahedral capsid that is surrounded by an amorphous protein layer, called the tegument, and a lipid-containing envelope. Assembly of herpesviruses is initiated in the nucleus where procapsids selfassemble around a protein scaffold and subsequently mature (1-4). This transformation is characterized by massive conformational changes of the Ϸ200 MDa shell, resulting in stable, mature capsids, and is in many aspects analogous to maturation processes in bacteriophages (5, 6). The scaffold is proteolytically cleaved and removed, whereas the outer shell transforms from a spherical into an icosahedral shape (7, 8). The mature capsid has an outer diameter of 125 nm with an overall shell thickness of Ϸ15 nm (9). Capsomeres converge at their proximal ends, forming a contiguous shell of Ϸ4 nm thickness that is only interrupted by channels passing through all of the 162 capsomeres (10, 11). The capsomeres are hexamers (hexons) and pentamers (pentons) of the major capsid protein VP5 that form a shell with a triangulation number of T ϭ 16. In total, 320 triplexes, each formed by a heterotrimer of 1 VP19c and 2 VP23 molecules, connect these capsomeres (10). One of the 12 capsid vertices is occupied by the pUL6 portal (12). Three particle types can be isolated from the nuclei of infected cells due to their different sedimentation behavior: B capsids, which still have the scaffold inside; the lighter A capsids, which are empty; and the denser C capsids, which contain the DN...

The human APOBEC3 proteins are a family of DNA-editing enzymes that play an important role in the innate immune response and have broad activity 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-catalyzed 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.

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. Although many of their interactions have been characterized, it remains poorly understood how replication proteins enter and leave the replisome. In this study, we visualize fluorescently labeled bacteriophage T7 DNA polymerases within the replisome while we simultaneously observe the kinetics of the replication process. This combination of observables allows us to monitor both the activity and dynamics of individual polymerases during coordinated leading-and lagging-strand synthesis. Our data suggest that lagging-strand polymerases are exchanged at a frequency similar to that of Okazaki fragment synthesis and that two or more polymerases are present in the replisome during DNA replication. Our studies imply a highly dynamic picture of the replisome with lagging-strand DNA polymerases residing at the fork for the synthesis of only a few Okazaki fragments. Further, new lagging-strand polymerases are readily recruited from a pool of polymerases that are proximally bound to the replisome and continuously replenished from solution.polymerase exchange | single molecule | fluorescence microscopy T he organization of replisomes is highly conserved among various organisms (1), underlining the evolutionary importance of the replication machinery architecture. The bacteriophage T7 replication system offers an attractive model system to study the interplay between replication proteins because its replication machinery is relatively simple; a functional replisome can be reconstituted by just four purified proteins. Three of these proteins are encoded by the phage itself: helicase-primase (gp4), DNA polymerase (gp5), and single-stranded DNA (ssDNA) binding protein (gp2.5). A processivity factor for the gp5 polymerase, thioredoxin (trx), is provided by the host Escherichia coli.