Supplementary MaterialsS1 Fig: plants display a reduction in the number of ovules per gynoecium. loop intermediate mediated by AtRAD51 and AtDMC1 and other proteins. If the second end is captured and the broken DNA strands are ligated, a dHJ is formed. This intermediate Marimastat manufacturer is resolved as CO1, sensitive to interference, or NCO upon appropriate resolution of the two HJs. On the other hand, this dHJ can be dissolved as a NCO. Alternatively, the D loop can be processed to generate a CO2 (insensitive to interference). When the D-loop is dissociated before the second end capture SDSA pathway occurs, Marimastat manufacturer the invading strand dissociates after DNA synthesis. This strand then re-anneals to the original parent, resulting in repair of the DSB and a heteroduplex DNA. This pathway is always resolved as a NCO.(TIF) pgen.1005301.s004.tif (902K) GUID:?5BACA6AD-A1AE-4543-9AC0-C71E83975927 S5 Fig: DSBs are not produced in nuclei. (A) AtASY1 (green) and H2AX (red). (B) AtASY1 (green) Marimastat manufacturer and AtRAD51 (red). (C) AtASY1 (green) and AtDMC1 (red). Bars = 5m.(TIF) pgen.1005301.s005.tif (690K) GUID:?E6143F40-4DC4-443C-A2F0-DA38E34A2208 S6 Fig: Chromosomal localization of the different NFTL alleles. (TIF) pgen.1005301.s006.tif (110K) GUID:?4544D582-95A0-4C65-90AC-895C3BF3669F S1 Table: Comparisons of H2AX, AtRAD51, AtDMC1, AtMSH4 and AtMLH1 foci between WT and assembly of nucleosomes during replication. CAF-1 is an evolutionary conserved heterotrimeric protein complex. In Arabidopsis, the three CAF-1 subunits are encoded by and 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 H2AX, AtRAD51 and AtDMC1 foci, is higher than in wild-type (WT) plants, and meiotic recombination genes such and 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 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. Author Summary Meiosis is a special cell division common in all sexually reproducing organisms. It consists of two successive rounds of chromosome segregation, preceded BMP13 by a single DNA replication event. Homologous recombination is a key process that occurs during the first meiotic division. It guarantees the association of the homologous chromosomes by chiasmata, the cytological manifestations of reciprocal interchanges (crossovers, COs). The formation of COs during meiosis is fine-tuned by several mechanisms. One of them, reported in some model organisms, is CO homeostasis, which ensures a consistent number of COs despite variability in early recombination events. Here we described the analysis of has a higher gene conversion (GC) frequency. These results demonstrate that Arabidopsis meiocytes are able to maintain WT levels of COs even when DSBs numbers are increased. Furthermore, we provide evidence for a prominent role for AtDMC1 in establishing interhomolog interactions in Arabidopsis. Introduction 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 [1C3]. CAF-1 is a heterotrimeric histone chaperone complex that mediates nucleosome assembly on newly replicated DNA in fungi, animals and plants [4]. The CAF-1 complex is composed of: FASCIATA 1 (AtFAS1), AtFAS2, and the MULTICOPY SUPPRESOR OF IRA1 (AtMSI1) [5]. The large subunit AtFAS1 binds acetylated histones H3/H4 and interacts with Proliferating Cell Antigen (PCNA) [6, 7]. The AtFAS2 subunit enables protein-protein interactions within CAF-1 and with Anti-Silencing Function 1 (ASF1), another major evolutionarily conserved H3/H4 histone.