The Functional Analysis Of DNA Replication Factor RFC1in Meiotic Recombination In Arabidopsis

Meiosis is essential for eukaryotic sexual reproduction. In this process, the recombination of homologous chromosomes enables the exchange of genetic material between, which is the basis for the formation of biodiversity and genetic breeding. Meiotic recombination includes DNA double strand break, processing, synthesis and ligation. DNA synthesis is very important for the development and growth of eukaryotes and is greatly needed in the process of meiotic recombination, The null mutant of DNA synthesis factor always causes embryo lethality, but few DNA synthesis factors have been studied for possible meiotic functions so far. Current meiotic recombination model mainly focuses on the leading strand DNA synthesis, while the lagging strand DNA synthesis is much neglected. In this study, DNA replication factor C subunit1(RFC1) was observed to examine its function in DNA lagging strand synthesis. To address whether meiotic recombination needs the DNA lagging strand synthesis, a T-DNA insertional weak mutant allele Atrfcl-2and meiotic specific RNAi line DMC1-RFC1RNAi were analyzed by molecular genetic and cell biology methods. The main results are as follows:RFC complexes are composed of a large subunit (RFC1) and four small sub units (RPC2, RFC3, RFC4, RFC5). RFC1, the largest subunit of RFC complex, contains an ATPase domain. Phylogenetic tree of RFC1showed that it is a highly conserved single copy gene. Real-time PCR analysis of RFClexression showed that it is widely expressed and further in-situ hybridization analysis showed that RFC1was expressed much higher at pollen mother cell than in surrounding somatic cells, suggesting that RFC1may has a role in meiosis. Phenotypic analysis revealed that the Atrfcl-2mutant was sterile and had multivalents, non-homologs chromosome interaction, abnormal chromosome segregation, and chromosome fragmentation. DMC1-RFC1RNAi and trans-heterozygous Atrfcl-2+/-Atrfcl-3+/-shared the similar meiotic chromosome behavior with Atrfcl-2mutant. Genetic analyses of Atrfcl-2+mutant demonstrated that the meiotic defects were caused by single copy gene mutation. Overexpression of RFC1gene in Atrfcl-2-/-background can rescue the mutant fertility and meiotic defects. These results demonstrated that the mutation of RFC1causes the meiotic and fertility defects. The key event of meiosis is the interaction between homologs, including pairing, synapsis and recombination. Telomere clustering in early leptotene is one phenomenon to indicate the early pairing. FISH were used to analyze the chromosome behavior. The signals of telomere in Atrfcl-2mutants showed no obvious difference compared with the wild type, suggesting that the early pairing is normal in Atrfcl-2mutant. However, non-homologs interactions in Atrfcl-2mutant were detected by specific probe with BAC clone. Immunolocalization of ASY1and ZYP1protein foci were similar in wild type and Atrfcl-2from leptotene to pachytene stage, except that the Atrfcl-2 pachytene chromosomes sometimes had a "bubble" lacking the ZYP1signal, suggesting a partial defect in synapsis. Meiotic recombination initiates the formation of double strand break catalyzed by SPOIL To investigate whether the meiotic defects in rfcl-2mutant were caused by SPO11, the Atrfcl-2spoll-1double mutant were generated. Chromosome behavior analysis by DAPI found that the chromosome fragments in Atrfcl-2were depended on SPO11, indicating that RFC1acts in meiotic recombination. RAD51is a single strand associated protein and promotes the single strand invasion following the double strand break formation. Further genetic analysis of Atrfcl-2rad51double mutant and Atrfcl-2mutant found that RFC1acted downstream of RAD51. Immunofluorescence showed that RAD51foci number were similar in wild type and Atrfcl-2at zygotene, but with persisted signals at pachytene stage, suggesting that the repair of double strand break (DSB) is delayed in the mutant. During meiotic recombination, the programmed DSBs are processed into MSH4-dependent interference sensitive crossovers (COs), MUS81-dependent interference insensitive COs and non-COs (NCOs). Genetic analyses of the mutations of Atrfcl-2, Atrfcl-2msh4-l, Atrfcl-2ptd-1and Atrfcl-2mus81-1supported that RFC1functioned in the MSH4-dependent interference-sensitive CO formation pathway. The formation of multivalent and residual viable pollens in Atrfcl-2was dependent on MUS81. The meiotic recombination rate of Atrfcl-2mutant and WT were detected by fluorescent tagged lines (FTLs). The result showed that the distribution of meiotic crossover had recombinational hot regions and cool regions. The remaining COs in Atrfcl-2were interference insensitive.In conclusion, our results provide strong evidence to support the hypothesis that DNA lagging strand synthesis is also needed in meiotic recombination. Because RFC1is very conserved in eukaryote and important for lagging strand synthesis, meiotic recombination requires lagging strand synthesis could be very conserved in eukaryote. Therefore, our results not only provide a theoretical basis for genetic manipulation of crop genetic exchange by molecular breeding, but also have important implications for understanding the molecular genetic basis of human reproductive health.