Eukaryotic chromatin is highly dynamic and turns over rapidly
even in the absence of DNA replication. In vitro, the acidic
histone chaperone ‘nucleosome assembly protein 1 (NAP-1)
reversibly removes H2A-H2B or histone variant dimers from
assembled nucleosomes, resulting in active histone exchange.
Transient removal of H2A-H2B dimers facilitates nucleosome
sliding along the DNA to a thermodynamically favorable position.
We have determined the structure of yNAP-1 which reveals a novel
fold that suggests mechanisms by which histones are bound and
shuttled into the nucleus.
NAP-1 dependent histone exchange as well as nucleosome
sliding is independent of ATP and relies on the presence of the
C-terminal acidic domain of yeast NAP-1, even though this region
is not required for histone binding and chromatin assembly. Our
results suggest a novel role for NAP-1 (and perhaps for other
histone chaperones) in mediating chromatin fluidity by
incorporating histone variants and assisting nucleosome sliding.
The 3.0 Å crystal structure of yeast NAP-1 reveals a homodimer
with a novel fold. A long
α-helix is responsible for dimerization via a novel antiparallel non-coiled coil, and an
α/β domain is implicated in protein – protein interactions. The
four-stranded anti-parallel β-sheet that characterizes the
α/β
domain is found in all histone chaperones, despite absence of
homology in sequence, structural context, or quarternery
structure. This is the first structure of a member of the large
NAP family of proteins, and suggests a mechanism by which
histones are bound, and by which the shuttling of histones to
and from the nucleus is regulated.
We have an ongoing interest in elucidating the effect of
mitomycin – based anti-cancer drugs on nucleosomal DNA, in
collaboration with the Williams laboratory at Colorado State
University. These drugs (which are currently in clinical trials)
are known to cause DNA crosslinks and mono-alkylation on free
DNA, however, their effect in a nucleosomal context has not been
studied to date. We find that nucleosomal DNA is a favored
substrates for drug-mediated monoalkylation, but not a very
efficient substrate for DNA crosslinking. Intriguingly,
crosslinking of free DNA has an inhibitory effect on chromatin
assembly in vitro. In vivo, this drug-mediated inhibition of
chromatin assembly can result in apoptosis in rapidly dividing
cancer cells, whereas in cells that are not dividing at a fast
rate the mechanism of inhibition may be at the transcriptional
level. This may in part explain the effects of the drug on the
post-replicative processes of cancerous cells.
