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Science advances 8(30), eabo0517 (2022). doi:10.1126/sciadv.abo0517, Nucleosome assembly requires the coordinated deposition of histone complexes H3-H4 and H2A-H2B to form a histone octamer on DNA. In the current paradigm, specific histone chaperones guide the deposition of first H3-H4 and then H2A-H2B. Here, we show that the acidic domain of DNA repair factor APLF (APLF$^{AD}$) can assemble the histone octamer in a single step and deposit it on DNA to form nucleosomes. The crystal structure of the APLF$^{AD}$-histone octamer complex shows that APLF$^{AD}$ tethers the histones in their nucleosomal conformation. Mutations of key aromatic anchor residues in APLF$^{AD}$ affect chaperone activity in vitro and in cells. Together, we propose that chaperoning of the histone octamer is a mechanism for histone chaperone function at sites where chromatin is temporarily disrupted., Published by Assoc., Washington, DC [u.a.]

At any time in the nucleus of a eukaryotic cell, chromatin, the protein-DNA supercomplex that harbors the genome, undergoes various processes to meet the needs of the cell during its life cycle. These processes serve to regulate the replication, transcription, or repair of the DNA and are as such dependent on the dynamics of the nucleosome, the fundamental repeating unit of chromatin. A nucleosome is assembled from a protein core of histone proteins around which DNA is wrapped. However, for nuclear processes in which the DNA serves as template for proteins, DNA has to be (partially) unwrapped in a process called nucleosome disassembly. Nucleosome assembly and disassembly are dependent on a family of proteins called histone chaperones. These chaperones bind and manage the histone proteins for all chromatin processes. One of these processes is repair of DNA double-strand breaks (DSBs), a common type of DNA damage in eukaryotic cells. The major pathway for DSB repair in human cells is non-homologous end joining (NHEJ). The NHEJ DNA repair factor Aprataxin and Polynucleotide kinase Like Factor (APLF) was shown to harbor histone chaperone function in its C-terminal acidic domain (APLFAD). The topic of this dissertation is the characterization of the histone chaperone function of APLFAD. A multidisciplinary approach is used to better understand how this histone chaperone functions, from histone binding to nucleosome assembly. The aim is to provide a better understanding of NHEJ DNA repair and the mechanism of nucleosome assembly.

Genome replication, transcription and repair require the assembly/disassembly of the nucleosome. Histone chaperones are regulators of this process by preventing formation of non-nucleosomal histone-DNA complexes. Aprataxin and polynucleotide kinase like factor (APLF) is a non-homologous end-joining (NHEJ) DNA repair factor that possesses histone chaperone activity in its acidic domain (APLFAD). Here, we studied the molecular basis of this activity using biochemical and structural methods. We find that APLFAD is intrinsically disordered and binds histone complexes (H3-H4)2 and H2A-H2B specifically and with high affinity. APLFAD prevents unspecific complex formation between H2A-H2B and DNA in a chaperone assay, establishing for the first time its specific histone chaperone function for H2A-H2B. On the basis of a series of nuclear magnetic resonance studies, supported by mutational analysis, we show that the APLFAD histone binding domain uses two aromatic side chains to anchor to the α1-α2 patches on both H2A and H2B, thereby covering most of their DNA-interaction surface. An additional binding site on both APLFAD and H2A-H2B may be involved in the handoff between APLF and DNA or other chaperones. Together, our data support the view that APLF provides not only a scaffold but also generic histone chaperone activity for the NHEJ-complex.

Architectural DNA-binding proteins are key to the organization and compaction of genomic DNA inside cells. The activity of architectural proteins is often subject to further modulation and regulation through the interaction with a diverse array of other protein factors. Detailed knowledge on the binding modes involved is crucial for our understanding of how these protein-protein and protein-DNA interactions shape the functional landscape of chromatin in all kingdoms of life: bacteria, archaea, and eukarya. Microscale thermophoresis (MST) is a biophysical technique that has seen increasing application in the study of biomolecular interactions thanks to its solution-based nature, its rapid application, modest sample demand, and the sensitivity of the thermophoresis effect to binding events. Here, we describe the use of MST in the study of chromatin interactions, with emphasis on the wide range of ways in which these experiments are set up and the diverse types of information they reveal. These aspects are illustrated with four very different systems: the sequence-dependent DNA compaction by architectural protein HMfB; the sequential binding of core histone complexes to histone chaperone APLF; the impact of the nucleosomal context on the recognition of histone modifications; and the binding of a LANA-derived peptide to nucleosome core. Special emphasis is given to the key steps in the design, execution, and analysis of MST experiments in the context of the provided examples.