Assembly of ribosomal subunits into active ribosomal complexes is integral to protein synthesis. Release of eIF6 from the 60S ribosomal subunit primes 60S to associate with the 40S subunit and engage in translation. The dynamics of eIF6 interaction with the uL14 (RPL23) interface of 60S and its perturbation by somatic mutations acquired in Shwachman-Diamond Syndrome (SDS) is yet to be clearly understood. Here, by using a modified strategy to obtain high yields of recombinant human eIF6 we have uncovered the critical interface entailing eight key residues in the C-tail of uL14 that is essential for physical interactions between 60S and eIF6. Disruption of the complementary binding interface by conformational changes in eIF6 disease variants provide a mechanism for weakened interactions of variants with the 60S. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) analyses uncovered dynamic configurational rearrangements in eIF6 induced by binding to uL14 and exposed an allosteric interface regulated by the C-tail of eIF6. Disrupting key residues in the eIF6-60S binding interface markedly limits proliferation of cancer cells, which highlights the significance of therapeutically targeting this interface. Establishing these key interfaces thus provide a therapeutic framework for targeting eIF6 in cancers and SDS.

Replication Protein A (RPA) is a heterotrimeric complex that binds to single-stranded DNA (ssDNA) and recruits over three dozen RPA-interacting proteins to coordinate multiple aspects of DNA metabolism including DNA replication, repair, and recombination. Rtt105 is a molecular chaperone that regulates nuclear localization of RPA. Here, we show that Rtt105 binds to multiple DNA binding and protein-interaction domains of RPA and configurationally staples the complex. In the absence of ssDNA, Rtt105 inhibits RPA binding to Rad52, thus preventing spurious binding to RPA-interacting proteins. When ssDNA is available, Rtt105 promotes formation of high-density RPA nucleoprotein filaments and dissociates during this process. Free Rtt105 further stabilizes the RPA-ssDNA filaments by inhibiting the facilitated exchange activity of RPA. Collectively, our data suggest that Rtt105 sequesters free RPA in the nucleus to prevent untimely binding to RPA-interacting proteins, while stabilizing RPA-ssDNA filaments at DNA lesion sites.

Human DNA helicase B (HELB) is a poorly characterized helicase suggested to play both positive and negative regulatory roles in DNA replication and recombination. In this work, we used bulk and single-molecule approaches to characterize the biochemical activities of HELB protein with a particular focus on its interactions with Replication Protein A (RPA) and RPA–single-stranded DNA (ssDNA) filaments. HELB is a monomeric protein that binds tightly to ssDNA with a site size of ∼20 nucleotides. It couples ATP hydrolysis to translocation along ssDNA in the 5′ to 3′ direction accompanied by the formation of DNA loops. HELB also displays classical helicase activity, but this is very weak in the absence of an assisting force. HELB binds specifically to human RPA, which enhances its ATPase and ssDNA translocase activities but inhibits DNA unwinding. Direct observation of HELB on RPA nucleoprotein filaments shows that translocating HELB concomitantly clears RPA from ssDNA. This activity, which can allow other proteins access to ssDNA intermediates despite their shielding by RPA, may underpin the diverse roles of HELB in cellular DNA transactions.

The ability to site-specifically modify proteins at multiple sites will enable the study of protein function in its native environment with unprecedented levels of detail. Here, we present a versatile two-step strategy to meet this goal involving site-specific encoding of two distinct noncanonical amino acids bearing bioorthogonal handles into proteins followed by mutually orthogonal labeling. This general approach, that we call ual ncoding nd abeling (DEAL), allowed us to efficiently encode tetrazine- and azide-bearing amino acids into a protein and demonstrate for the first time that the bioorthogonal labeling reactions with strained alkene and alkyne labels can function simultaneously and intracellularly with high yields when site-specifically encoded in a single protein. Using our DEAL system, we were able to perform topologically defined protein-protein cross-linking, intramolecular stapling, and site-specific installation of fluorophores all inside living cells, as well as study the DNA-binding properties of yeast Replication Protein A . By enabling the efficient dual modification of proteins , this DEAL approach provides a tool for the characterization and engineering of proteins .

Lamin A/C provides a nuclear scaffold for compartmentalization of genome function that is important for genome integrity. Lamin A/C dysfunction is associated with cancer, aging, and degenerative diseases. The mechanisms whereby lamin A/C regulates genome stability remain poorly understood. We demonstrate a crucial role for lamin A/C in DNA replication. Lamin A/C binds to nascent DNA, especially during replication stress (RS), ensuring the recruitment of replication fork protective factors RPA and RAD51. These ssDNA-binding proteins, considered the first and second responders to RS respectively, function in the stabilization, remodeling, and repair of the stalled fork to ensure proper restart and genome stability. Reduced recruitment of RPA and RAD51 upon lamin A/C depletion elicits replication fork instability (RFI) characterized by MRE11 nuclease-mediated degradation of nascent DNA, RS-induced DNA damage, and sensitivity to replication inhibitors. Importantly, unlike homologous recombination-deficient cells, RFI in lamin A/C-depleted cells is not linked to replication fork reversal. Thus, the point of entry of nucleases is not the reversed fork but regions of ssDNA generated during RS that are not protected by RPA and RAD51. Consistently, RFI in lamin A/C-depleted cells is rescued by exogenous overexpression of RPA or RAD51. These data unveil involvement of structural nuclear proteins in the protection of ssDNA from nucleases during RS by promoting recruitment of RPA and RAD51 to stalled forks. Supporting this model, we show physical interaction between RPA and lamin A/C. We suggest that RS is a major source of genomic instability in laminopathies and lamin A/C-deficient tumors.