Chromatin: Keep it level

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Research has unveiled a Rad53 kinase-dependent surveillance mechanism that regulates histone protein levels in yeast.

During DNA replication, histone metabolism and DNA synthesis must be carefully controlled, because an excess of histones is toxic to the cell. And, scientists have now identified Rad53 kinase as a key controller of the delicate balance between these processes.

Rad53 functions in the DNA-damage response and is essential for deoxyribonucleotide triphosphate (dNTP) production. To study the role of Rad53 in histone metabolism, Akash Gunjan and Alain Verreault overexpressed histones in wild-type and rad53 Delta budding yeast strains and found that excess histones were far more toxic to rad53 Delta mutant cells than to wild-type cells.

To understand why rad53 Delta mutants were sensitive to histone overexpression, the authors followed the fate of the overexpressed histones. In cells that were arrested in G1 phase, overexpressed histone H3 was rapidly degraded in wild-type cells, but not in rad53 Delta cells. When histone overexpression was induced in S-phase cells, a fraction of those histones was incorporated into chromatin. Both wild-type and rad53 Delta cells incorporated similar amounts of histone H3 into chromatin, whereas unincorporated histones were quickly degraded in wild-type cells but not in the rad53 Delta strain. Rad53 is therefore specifically needed to trigger degradation of the excess histones that are not packaged into chromatin.

Newly synthesized histones normally associate with histone chaperones during nucleosome assembly. The amounts of histone H3 associated with the histone chaperones CAF-1 and Hir2 were much higher in rad53 Delta mutant cells compared with wild-type cells, which confirmed that rad53 Delta mutant cells accumulated newly synthesized histones. The treatment of cells with genotoxic agents that slow or block DNA replication also led to an increase in histones that were bound to histone chaperones.

The authors showed that a reduction in histone H3/H4 gene dosage suppressed the slow-growth phenotype of rad53 Delta mutant cells. Other damaging effects of histone overexpression — including mitotic chromosome loss and DNA damage sensitivity — were also reduced, which shows that excess histones interfere with several distinct processes that require access to genetic information.

But how does Rad53 sense excess histones and target them for degradation? Gunjan and Verreault found that overexpressed histones associated transiently with Rad53. By contrast, higher levels of histones were associated with a kinase-defective mutant. This indicates that the Rad53 kinase activity is needed to avoid the accumulation of histones in a Rad53-containing complex, by triggering their degradation. Whether histone phosphorylation has a role in their degradation is not clear. Also, whether Rad53 binds histones directly or through histone chaperones remains to be determined. To elucidate the precise mechanism of this Rad53-dependent surveillance system for histone levels, researchers will undoubtedly focus on proteins that function downstream of Rad53.

Arianne Heinrichs

References

Gunjan, A. & Verreault, A. A Rad53 kinase-dependent surveillance mechanism that regulates histone protein levels in S. cerevisiae. Cell 115, 537–549 (2003) | PubMed |