The Absence of the Arabidopsis Chaperone Complex CAF-1 Produces Mitotic Chromosome Abnormalities and Changes in the Expression Profiles of Genes Involved in DNA Repair

Abstract

Chromatin Assembly Factor 1 (CAF-1) is an evolutionary conserved heterotrimeric chaperone complex that facilitates the incorporation of histones H3 and H4 onto newly synthesized DNA. We demonstrate here that the mutant deficient for the large subunit of the complex, fas1-4, and in minor extent, the mutant deficient for the middle subunit, fas2-1, display chromosome abnormalities throughout Arabidopsis mitosis. Among them, we observed multicentromeric chromosomes at metaphase, and chromatid bridges and acentric fragments at anaphase-telophase. 45S rDNA and telomeric sequences were frequently involved in bridges and fragments. Gene expression analysis by real-time qPCR has revealed that several genes related to homologous recombination (HR) and alternative non-homologous end-joining (aNHEJ) are overexpressed in fas1-4. These results concur with previous studies which have indicated that HR may be involved in the progressive loss of 45S rDNA and telomeres displayed by fas mutants. However, increased expression of PARP1, PARP2, and LIG6 in fas1-4, and the phenotype shown by the double mutant fas1 rad51 suggest that aNHEJ should also be responsible for the chromosomal aberrations observed. The activity of different DNA repair pathways in absence of CAF-1 is discussed.

Disruption ofNAP1genes supresses thefas1mutant phenotype and enhances genome stability

“…H3.3 or H2A.Z (Jin et al, 2009; Chereji et al, 2017). The increased digestion rate in fas1 may reflect the lower density of nucleosomes and the different H3.1/H3.3 balance, as previously observed in fas1 (Kirik et al, 2006; Otero et al, 2016; Varas et al, 2017), thus the higher protection of chromatin in fas1m132-2 possibly suggests changes in chromatin structure in the quadruple mutant (Figure 10). The total abundance of H3 in fas1 or fas1m123-2 did not differ (Figure 8), indicating that the observed changes were not caused by altered levels of H3.…”

“…To discover a possible relationship between FAS1 and NAP1;1-3 histone chaperones, and elucidate their roles in chromatin maintenance, we crossed the fas1-4 single mutant (G1) with the nap1;1nap1;2nap1;3-2 triple mutant ( m123-2 , adopted from (Liu et al, 2009)), thereby creating the fas1-4nap1;1nap1;2nap1;3 ( fas1m123-2 ) quadruple mutant. Parental A. thaliana plants carrying a T-DNA insertion in all three NAP1;1-3 genes (m123-2 ) grow relatively well under standard laboratory conditions without any apparent phenotypic changes (Liu et al, 2009), while dysfunction of the FAS1 gene causes growth and developmental problems and reduced fertility (Kaya et al, 2001; Kirik et al, 2006; Mozgova et al, 2010; Hisanaga et al, 2013; Varas et al, 2017).…”

Meiotic DNA Repair in the Nucleolus Employs a Nonhomologous End-Joining Mechanism

“…fas1 Arabidopsis mutants, deficient for histone replacement, are characterized by enrichment of gH2Ax at the rDNA, mitotic anaphase bridges between the rDNA loci, and loss of rDNA copy number. rDNA loss in fas1 2/2 is alleviated in a rad51 2/2 background, indicating that it is a consequence of HR (Muchová et al, 2015;Varas et al, 2017). In Saccharomyces cerevisiae, meiotic DSBs are suppressed within the rDNA array by the histone deacetylase Sir2 and the AAA1 ATPases Pch2 and Orc1 (Gottlieb and Esposito, 1989;Ozenberger and Roeder, 1991;Mieczkowski et al, 2007;Vader et al, 2011).…”

Meiotic DNA repair in the nucleolus employs a non-homologous end joining mechanism

“…Arabidopsis plants with dysfunctional FAS1, a subunit of the Chromatin assembly factor complex (CAF-1) involved in histone replacement, are enriched in γH2Ax (a histone variant present at DNA DSBs) at the rDNA, show rDNA bridges in mitotic anaphase and suffer from rDNA loss. The rDNA loss phenotype is alleviated in a rad51 -/background, indicating that it is a consequence of HR (Muchova et al 2015;Varas et al 2017) DSB formation is an essential part of meiosis. In the yeast Saccharomyces cerevisiae meiotic DSBs, and therefore potential non-allelic recombination events, are suppressed within the rDNA array by the histone deacetylase Sir2, the AAA+ ATPases Pch2 and Orc1 (also part of the origin of replication complex) (Gottlieb and Esposito 1989;Ozenberger and Roeder 1991;Mieczkowski et al 2007;Vader et al 2011).…”