DNA repair factor APLF acts as a H2A-H2B histone chaperone through binding its DNA interaction surface

Abstract

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.

A three layered histone epigenetics in breast cancer metastasis

“…Aprataxin and PNK (Polynucleotide Kinase) like factor (APLF) is a histone chaperone [37]. Apart from histone chaperone activity, APLF is associated with the non-homologous end joining DNA repair pathway [33,38].…”

“…Apart from histone chaperone activity, APLF is associated with the non-homologous end joining DNA repair pathway [33,38]. The acidic C-terminal domain (AD) of APLF is responsible for histone chaperone activity [37]. APLF AD domain is found to be structurally homologous to NAP1 protein (Nucleosome Assembly protein 1), characterized as a histone chaperone [39].…”

“…(2) Second layer of histone epigenetics is histone variants, which plays an important role of maintaining chromatin switch [ON/OFF state]. (3) Final layer of histone epigenetics is Histone Modifications which further modifies the chromatin state by addition of functional groups into the tails of histones [37]. The Cancer Genome Atlas (TCGA) database analysis for invasive ductal breast cancer patient sample comprising of a cohort of 815 patient samples, showed significant increase of APLF in triple negative breast cancer (TNBC) sample and invasive breast cancer of the basal type [40].…”

Molecular Structure, Binding Affinity, and Biological Activity in the Epigenome

“…It was found that both histone systems bind the APLF reader protein with micromolar affinity, both are enthalpically favorable interactions, yet the binding of (H3-H4)2 is entropically unfavorable, which might explain the difference in their Kd values. ITC did not detect additional lower affinity binding modes, resulting in a stoichiometry of n = 1 in both cases [141].…”

“…ITC is also applicable for the thermodynamic analysis of large complexes of the epigenome. For example, binding of aprataxin and polynucleotide kinase like factor (APLF) to histone dimers and tetramers H2A-H2B and (H3-H4)2 was investigated in a study [141]. It was found that both histone systems bind the APLF reader protein with micromolar affinity, both are enthalpically favorable interactions, yet the binding of (H3-H4)2 is entropically unfavorable, which might explain the difference in their Kd values.…”