Strand Invasion

Single-strand DNA tails created by Spoll and the MRN complex search for and invade a homologous sequence. Rad5l and Dmcl, which are homologues of the bacterial recombinase RecA, catalyze this step. Rad5l functions in both mitotic and meiotic recombination, whereas Dmcl is meiosis-specific (Bishop et al. l992; Shinohara et al. l992). Both proteins have been identified in Arabidopsis, and the corresponding mutants have been characterized (Klimyuk and Jones l997; Couteau et al. l999; Li et al. 2004).

The atrad51-1 mutant shows a completely sterile phenotype and displays defects in synapsis and chromosome fragmentations during meiosis. These chromosome fragmentations observed in the atrad51-1 mutant depend on AtSpoll-l (Li et al. 2004). These results indicate that AtRad5l acts downstream of AtSpoll-l during meiotic recombination. In contrast, atdmc1 mutants show reduced fertility, but nevertheless produce a small number of seeds. In addition, atdmc1 mutants are unable to form chiasmata (Couteau et al. l999). These results indicate that AtDmcl is required for bivalent formation of chromosome and chromosome segregation during meiotic recombination in Arabidopsis. In rice, biochemical analyses of two Dmcl proteins (DmclA and DmclB) have been completed (Sakane et al. 2008).

Interestingly, rice DmclA and DmclB proteins could promote strand exchange in the absence of RPA with long DNA substrates containing several thousand base pairs. In contrast, the human Dmcl protein strictly required RPA to promote strand exchange with these long DNA substrates. The DNA binding activity of the rice DmclA protein is greater than that of the rice DmclB protein. The biochemical difference between the two rice Dmc1 proteins may indicate that these proteins have functional differences during meiosis in rice (Sakane et al. 2008).

In addition to AtRad5l and AtDmcl, the five Rad5l paralogues (Rad5lB, Rad5lC, Rad5lD, XRCC2 and XRCC3) identified in vertebrates are also present in the Arabidopsis genome, while yeast has only two Rad5l paralogues (Rad55 and Rad57; Osakabe et al. 2002; Bleuyard et al. 2005; Durrant et al. 2007). AtRad5lC and AtXRCC3 function in both meiotic and mitotic recombination, whereas AtRad5lB, AtRad5lD and AtXRCC2 are mitosis-specific. atrad51C and atxrcc3 mutants show a completely sterile phenotype and display chromosome fragmentation during meiosis (Bleuyard and White 2004; Abe et al. 2005; Bleuyard et al. 2005; Li et al. 2005). The chromosome fragmentation observed in atrad51C and atxrcc3 mutants depends on AtSpoll-l (Bleuyard et al. 2004b; Li et al. 2005). The meiotic phenotypes of atrad51C and atxrcc3 mutants are similar to those of atrad51-1 mutants. Moreover, AtRad5lC interacts with AtXRCC3 in yeast two-hybrid assays (Osakabe et al. 2002). These results indicate that AtRad5lC and AtXRCC3 proteins form a heterodimer complex and cooperate with AtRad5l downstream of AtSpoll-l during meiotic recombination.

Several proteins also cooperate with Dmcl/Rad5l in strand invasion. Homologues of BRCA2, Mndl and AHP2 were identified recently as key players in meiotic recombination in Arabidopsis (Schommer et al. 2003; Siaud et al. 2004; Kerzendorfer et al. 2006). Despite its absence in yeast, the BRCA2 protein appears to be conserved amongst eukaryotes, including Arabidopsis. In humans, mutations of the BRAC2 gene are associated with susceptibility to breast cancer. BRCA2 facilitates the loading of Rad5l on single-stranded DNA. Arabidopsis has two closely related BRCA2 homologues. In Arabidopsis, the silencing of the two AtBRCA2 genes by RNA interference (RNAi) leads to a sterile phenotype and chromosome fragmentation. The chromosome fragmentations observed in AtBRCA2-RNAi plants depend on AtSpoll-l (Siaud et al. 2004). These results indicate that AtBRCA2 acts downstream of AtSpoll-l during meiosis. In addition, AtBRCA2 interacts with both AtDmcl and AtRad5l in yeast two-hybrid assays (Siaud et al. 2004). This result indicates that the BRCA2 function of loading Rad5l and Dmcl on single-stranded DNA is conserved in plants.

In yeast, Mndl and Hop2/AHP2 form a complex and function in the early stages of meiotic chromosome pairing and DSB repair. Both proteins have been identified in Arabidopsis, and the corresponding mutants have been characterized. atmnd1 and atahp2 mutants show a sterile phenotype and display defects in synapsis and chromosome fragmentation during meiosis (Schommer et al. 2003; Kerzendorfer et al. 2006). The chromosome fragmentation observed in atmnd1 mutants depends on AtSpoll-l. These results indicate that AtMndl and AtAHP2 act downstream of AtSpoll-l during meiotic recombination. In addition, AtMndl interacts with

AtAHP2 in yeast two-hybrid assays (Kerzendorfer et al. 2006). These data suggest that the function of the Mnd1-Hop2/AHP2 heterodimer is conserved from yeast to plants.

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