Abstract
Chromatin Assembly Factor 1 (CAF-1) is a histone chaperone that assembles acetylated histones H3/H4 onto newly synthesized DNA, allowing the de novo assembly of nucleosomes during replication. CAF-1 is an evolutionary conserved heterotrimeric protein complex. In Arabidopsis, the three CAF-1 subunits are encoded by FAS1, FAS2 and MSI1. Atfas1-4 mutants have reduced fertility due to a decrease in the number of cells that enter meiosis. Interestingly, the number of DNA double-strand breaks (DSBs), measured by scoring the presence of γH2AX, AtRAD51 and AtDMC1 foci, is higher than in wild-type (WT) plants, and meiotic recombination genes such AtCOM1/SAE2, AtBRCA1, AtRAD51 and AtDMC1 are overexpressed. An increase in DSBs in this mutant does not have a significant effect in the mean chiasma frequency at metaphase I, nor a different number of AtMLH1 nor AtMUS81 foci per cell compared to WT at pachytene. Nevertheless, this mutant does show a higher gene conversion (GC) frequency. To examine how an increase in DSBs influences meiotic recombination and synaptonemal complex (SC) formation, we analyzed double mutants defective for AtFAS1 and different homologous recombination (HR) proteins. Most showed significant increases in both the mean number of synapsis initiation points (SIPs) and the total length of AtZYP1 stretches in comparison with the corresponding single mutants. These experiments also provide new insight into the relationships between the recombinases in Arabidopsis, suggesting a prominent role for AtDMC1 versus AtRAD51 in establishing interhomolog interactions. In Arabidopsis an increase in the number of DSBs does not translate to an increase in the number of crossovers (COs) but instead in a higher GC frequency. We discuss different mechanisms to explain these results including the possible existence of CO homeostasis in plants.
Highlights
Histone chaperones are a family of proteins that facilitate appropriate interactions between histones and DNA by regulating the assembly and disassembly of chromatin in response to cellular requirements [1,2,3]
These results indicate that Atfas1-4 plants have reduced fertility
Our results show that the number of COs can be constrained in plant species even when the number of double-strand breaks (DSBs) increases during meiosis
Summary
Histone chaperones are a family of proteins that facilitate appropriate interactions between histones and DNA by regulating the assembly and disassembly of chromatin in response to cellular requirements [1,2,3]. CAF-1 is a heterotrimeric histone chaperone complex that mediates nucleosome assembly on newly replicated DNA in fungi, animals and plants [4]. The AtFAS2 subunit enables protein-protein interactions within CAF-1 and with Anti-Silencing Function 1 (ASF1), another major evolutionarily conserved H3/H4 histone chaperone, which participates in replication-independent nucleosome assembly [8,9,10,11]. Depletion of either subunit increased the frequency of somatic homologous recombination (HR) ~40-fold, as well as increased T-DNA integration [19]. These findings suggest that the loss of CAF-1 activity produces defects in chromatin assembly that lead to genomic instability. Double mutants show reduced growth and defects in organ development, and overexpression of genes involved in S-phase checkpoints and DNA repair by HR [11]
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