Partial Depletion of Histone H4 Increases Homologous Recombination-Mediated Genetic Instability

Abstract

DNA replication can be a source of genetic instability. Given the tight connection between DNA replication and nucleosome assembly, we analyzed the effect of a partial depletion of histone H4 on genetic instability mediated by homologous recombination. A Saccharomyces cerevisiae strain was constructed in which the expression of histone H4 was driven by the regulated tet promoter. In agreement with defective nucleosome assembly, partial depletion of histone H4 led to subtle changes in plasmid superhelical density and chromatin sensitivity to micrococcal nuclease. Under these conditions, homologous recombination between ectopic DNA sequences was increased 20-fold above the wild-type levels. This hyperrecombination was not associated with either defective repair or transcription but with an accumulation of recombinogenic DNA lesions during the S and G₂/M phases, as determined by an increase in the proportion of budded cells containing Rad52-yellow fluorescent protein foci. Consistently, partial depletion of histone H4 caused a delay during the S and G₂/M phases. Our results suggest that histone deposition defects lead to the formation of recombinogenic DNA structures during replication that increase genomic instability.

ACKNOWLEDGMENTS

We thank R. Rothstein for providing the Rad52-YFP construct, G. Vicent for the anti-H4 antibodies, M. Nieto for excellent technical assistance, M. Fidalgo for help with confocal microscopy and flow cytometry, R. E. Wellinger and M. C. Muñoz-Centeno for critical reading of the manuscript, and Diane Haun for style supervision.

The research was funded by the Spanish Ministry of Science and Technology (BMC2000-0439 and SAF2003-00204 grants).