ASF1A Human

What is the molecular function of ASF1A in human cells?

ASF1A functions as a major histone chaperone that regulates the supply of histone H3-H4 and facilitates nucleosome assembly to maintain chromatin structure. It plays an essential role in DNA-dependent processes including replication, repair, and transcription . The protein operates by binding to the H3-H4 dimer and transferring these histones to downstream chaperones like CAF-1 (Chromatin Assembly Factor 1) for incorporation into nucleosomes . This histone transfer function is critical for maintaining genomic integrity during cellular processes that require nucleosome disassembly and reassembly .

Experimentally, ASF1A function can be studied using cell-free systems that support processes like nucleotide excision repair (NER) on damaged plasmids. Supercoiling assays can be employed to monitor nucleosome formation, where the accumulation of supercoils is proportional to the number of nucleosomes formed on circular DNA .

How does ASF1A interact with CAF-1 in nucleosome assembly pathways?

ASF1A interacts directly with the p60 subunit of CAF-1 to facilitate nucleosome assembly. This interaction has been demonstrated through multiple experimental approaches:

• Pull-down assays using GST-ASF1 fusion proteins show that GST-ASF1A can efficiently pull down the CAF-1 subunits p150 and p60 from human cell-free extracts .

• Reciprocal immunoprecipitation experiments confirm that ASF1A co-immunoprecipitates with native p60, with interactions stable even in 1M NaCl conditions .

• Direct binding studies with purified components demonstrate that ASF1A interacts specifically with the p60 subunit of CAF-1, not with p150 or p48 .

The functional significance of this interaction is evidenced by synergistic activity in nucleosome assembly coupled to DNA repair. When ASF1A is added together with CAF-1 and purified histones to a cell-free system supporting nucleotide excision repair, there is a marked increase in nucleosome assembly specifically on repaired DNA compared to either protein alone .

What experimental methods are most effective for studying ASF1A-mediated nucleosome assembly?

Several complementary experimental approaches have proven effective for investigating ASF1A function:

For nucleosome assembly studies specifically, the supercoiling assay provides a quantitative measure where the accumulation of supercoils is proportional to the number of nucleosomes formed on circular DNA. Partial micrococcal nuclease (MNase) digestion of assembled DNA can confirm that the observed supercoiling is due to the deposition of regularly spaced nucleosomes .

How does CODANIN-1 negatively regulate ASF1A function at the molecular level?

CODANIN-1 acts as a negative regulator of ASF1A through a sophisticated molecular mechanism revealed by cryo-EM structural studies. The 3.75 Å resolution structure of the human CODANIN-1_ASF1A complex has shown that:

• CODANIN-1 forms a dimer where each monomer can hold two ASF1A molecules .

• The binding occurs through two distinct modules in CODANIN-1: a B-domain and a histone H3 mimic helix (HMH) .

• The interaction effectively hijacks ASF1A by occupying its binding sites for both histones and downstream chaperones .

Specifically, CODANIN-1 has two ASF1A-binding modules:

• A distal ASF1A-binding module (BD-N and HMH-N)

• A proximal ASF1A-binding module (BD-C and HMH-C)

Binding kinetics analysis through Bio-Layer Interferometry (BLI) has revealed that wild-type CODANIN-1 has a 12.7 × 10^4 kon (M^-1 s^-1) for ASF1A binding, with no measurable dissociation (koff) . Among the individual binding domains, BD-N shows the strongest binding to ASF1A, resulting in a 10-fold reduction in kon when mutated .

Through this sequestration mechanism, CODANIN-1 inhibits the formation of the ASF1A/H3-H4 complex and retains ASF1A in the cytoplasm, preventing it from participating in nucleosome assembly .

What is the structural basis for the interaction between ASF1A and histone H3-H4?

ASF1A interacts with the histone H3-H4 dimer through specific structural elements. CODANIN-1's ability to inhibit this interaction provides valuable insights into the ASF1A-histone binding interface. The histone H3 mimic helix (HMH) of CODANIN-1 mimics the structure of histone H3's α3 helix, which is critical for ASF1A binding .

Key findings about this structural interaction include:

• The HMH domains of CODANIN-1 (HMH-N and HMH-C) mimic the histone H3 binding surface to occupy the histone-binding pocket of ASF1A .

• Mutational studies of the HMH domains reveal that specific residues are critical for this interaction:

  • Mutations L545A/L549A in HMH-C significantly reduce ASF1A binding .

  • The hydrophobic residues in the HMH are particularly important for mimicking histone H3 binding .

When mutations are introduced to both the BD-N (R195A/R196A) and HMH-C (L545A/L549A) domains simultaneously, ASF1A binding is completely abolished, confirming the critical nature of these interaction surfaces .

How do mutations in ASF1A binding domains affect its function in DNA repair?

Mutations in ASF1A's binding domains can significantly impact its function in DNA repair processes. The synergistic activity between ASF1A and CAF-1 in nucleosome assembly coupled to nucleotide excision repair (NER) depends on their specific interaction .

Experimental evidence shows:

• ASF1A functions synergistically with CAF-1 to assemble nucleosomes during NER, but this activity requires an intact interaction interface .

• B-domains contribute more critically to ASF1A binding than HMH domains, as demonstrated by BLI experiments where mutations of both BD-N and BD-C (Mut8) strongly abrogated binding to ASF1A .

The critical nature of these interactions is highlighted by the observation that disrupting the ASF1A-CAF-1 interaction can impair nucleosome assembly during DNA repair. In yeast, defects in Asf1p significantly increase the sensitivity of cac-1 mutants to UV irradiation, and asf1 mutants alone are hypersensitive to a range of DNA-damaging agents, suggesting that proper ASF1-CAF-1 interaction is essential for the DNA damage response .

What methodological approaches are optimal for characterizing ASF1A interactions with different binding partners?

Characterizing ASF1A's interactions with various binding partners requires a multi-faceted methodological approach:

For studying ASF1A interactions specifically, bio-layer interferometry (BLI) has proven particularly valuable. This technique allowed researchers to determine that wild-type CODANIN-1 has a 12.7 × 10^4 kon (M^-1 s^-1) for ASF1A binding with no measurable dissociation, while various mutations in the binding domains showed different effects on binding kinetics .

Combining structural studies with functional assays provides the most comprehensive understanding of ASF1A interactions. For example, the supercoiling assay coupled with partial MNase digestion can confirm the functional relevance of interactions observed in binding studies .

How does phosphorylation affect ASF1A function in cellular processes?

Phosphorylation represents an important regulatory mechanism for ASF1A activity. Unlike yeast Asf1p, human ASF1A (and ASF1B) are phosphorylated in a replication-dependent manner by Tousled-like kinases (Tlks) .

Key findings regarding ASF1A phosphorylation include:

• Human ASF1A is phosphorylated by Tousled-like kinases in a manner dependent on DNA replication .

• The C-terminal acidic stretch present in yeast Asf1p is absent in human ASF1A; it has been suggested that phosphorylation of human ASF1A by Tlk might provide the structural equivalent to this acidic region .

• Initial experiments suggest that phosphorylation status does not significantly affect the direct interaction between ASF1A and CAF-1 p60 in pulldown assays .

What are the unresolved questions regarding ASF1A functional differences from ASF1B?

A significant question in the field concerns the functional distinction between the two human ASF1 variants (ASF1A and ASF1B), which share 71% homology . While both variants can function synergistically with CAF-1 to assemble nucleosomes during nucleotide excision repair, there are hints of functional specialization:

• A Triton-resistant ASF1A fraction has been detected by western blotting, representing the first hint of a distinction between the two proteins .

• Both variants are phosphorylated in a replication-dependent manner by Tousled-like kinases, but the functional consequences of this phosphorylation may differ .

Future research should focus on determining:

• The specific cellular contexts where ASF1A versus ASF1B may be preferentially utilized

• Distinct binding partners unique to each variant

• Differential regulation mechanisms that may exist for the two proteins

• Potential specialization in different DNA-dependent processes (replication, repair, transcription)

How can advanced structural analysis further our understanding of ASF1A regulation?

• High-resolution structures of ASF1A in complex with other regulatory partners beyond CODANIN-1

• Conformational changes in ASF1A during the histone transfer process

• Structural basis for potential ASF1A-ASF1B functional differences

• Impact of post-translational modifications on ASF1A structure and interactions

Addressing these questions will require integrating multiple structural approaches:

• Cryo-EM for large complexes

• X-ray crystallography for high-resolution details of specific domains

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) for conformational dynamics

• Integrative modeling approaches that combine multiple data types