Posttranslational modification of proteins expands their structural and functional capabilities beyond those directly specified by the genetic code. However, the vast diversity of chemically plausible (including unnatural but functionally relevant) side chains is not readily accessible. We describe C (sp)-C (sp) bond-forming reactions on proteins under biocompatible conditions, which exploit unusual carbon free-radical chemistry, and use them to form Cβ-Cγ bonds with altered side chains. We demonstrate how these transformations enable a wide diversity of natural, unnatural, posttranslationally modified (methylated, glycosylated, phosphorylated, hydroxylated), and labeled (fluorinated, isotopically labeled) side chains to be added to a common, readily accessible dehydroalanine precursor in a range of representative protein types and scaffolds. This approach, outside of the rigid constraints of the ribosome and enzymatic processing, may be modified more generally for access to diverse proteins.

The linker histone H1 is the most abundant group of eukaryotic chromatin-binding proteins. The mechanism underlying the diverse physiological functions of H1 remains unclear. Here we used singlemolecule fluorescence and force microscopy to observe the behavior of H1 on DNA under different tensions. Unexpectedly, we found that H1 coalesces around nascent ssDNA. Molecular dynamics simulations revealed that multivalent and transient interactions between H1 and ssDNA mediate their phase separation. We further showed that longer and unpaired nucleic acids result in more viscous, gellike H1 droplets. Finally, we imaged H1 puncta in cells under normal and stressed conditions and observed that RPA and H1 occupy separate nuclear regions. Overall, our results provide a new perspective to understanding the role of H1 in genome organization and maintenance.

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A wealth of in vivo evidence demonstrates the physiological importance of histone H3 trimethylation at lysine 36 (H3K36me3), to which chromodomain-containing proteins, such as MRG15, bind preferentially compared to their dimethyl (H3K36me2) counterparts.However, in vitro studies using isolated H3 peptides have failed to recapitulate a causal interaction. Here, we show that MRG15 can clearly discriminate between synthetic, fully intact model nucleosomes containing H3K36me2 and H3K36me3. MRG15 docking studies, along with experimental observations and nucleosome structure analysis suggest a model where the H3K36 side chain is sequestered in intact nucleosomes via a hydrogen bonding interaction with the DNA backbone, which is abrogated when the third methyl group is added to form H3K36me3. Hence, this mechanism provides a 'methyl-switch' for contextdependent reader selectivity. These results highlight the importance of such intra-chromatin interactions in understanding epigenetic regulation, a feature which is absent in commonlyused peptide or histone-only models.