DNA strand exchange activity of rice recombinase OsDmc1 monitored by fluorescence resonance energy transfer and the role of ATP hydrolysis

Spliceosomal small nuclear ribonucleoprotein particles (snRNPs) are required for pre-mRNA splicing throughout the nucleoplasm, yet snRNPs also concentrate in Cajal bodies (CBs). To address a proposed role of CBs in snRNP assembly, we have used fluorescence resonance energy transfer (FRET) microscopy to investigate the subnuclear distribution of specific snRNP intermediates. Two distinct complexes containing the protein SART3 (p110), required for U4/U6 snRNP assembly, were localized: SART3•U6 snRNP and SART3•U4/U6 snRNP. These complexes segregated to different nuclear compartments, with SART3•U6 snRNPs exclusively in the nucleoplasm and SART3•U4/U6 snRNPs preferentially in CBs. Mutant cells lacking the CB-specific protein coilin and consequently lacking CBs exhibited increased nucleoplasmic levels of SART3•U4/U6 snRNP complexes. Reconstitution of CBs in these cells by expression of exogenous coilin restored accumulation of SART3•U4/U6 snRNP in CBs. Thus, while some U4/U6 snRNP assembly can occur in the nucleoplasm, these data provide evidence that SART3•U6 snRNPs form in the nucleoplasm and translocate to CBs where U4/U6 snRNP assembly occurs.

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Human Rad54 Protein Stimulates DNA Strand Exchange Activity of hRad51 Protein in the Presence of Ca2+

Journal of Biological Chemistry ◽

10.1074/jbc.m410244200 ◽

2004 ◽

Vol 279 (50) ◽

pp. 52042-52051 ◽

Cited By ~ 56

Author(s):

Olga M. Mazina ◽

Alexander V. Mazin

Keyword(s):

Homologous Recombination ◽

Atp Hydrolysis ◽

Specific Interaction ◽

Strand Exchange ◽

Rad51 Protein ◽

Recombination Proteins ◽

Dna Strand Exchange ◽

Dna Strand ◽

Exchange Activity

Rad51 and Rad54 proteins play a key role in homologous recombination in eukaryotes. Recently, we reported that Ca2+is requiredin vitrofor human Rad51 protein to form an active nucleoprotein filament that is important for the search of homologous DNA and for DNA strand exchange, two critical steps of homologous recombination. Here we find that Ca2+is also required for hRad54 protein to effectively stimulate DNA strand exchange activity of hRad51 protein. This finding identifies Ca2+as a universal cofactor of DNA strand exchange promoted by mammalian homologous recombination proteinsin vitro. We further investigated the hRad54-dependent stimulation of DNA strand exchange. The mechanism of stimulation appeared to include specific interaction of hRad54 protein with the hRad51 nucleoprotein filament. Our results show that hRad54 protein significantly stimulates homology-independent coaggregation of dsDNA with the filament, which represents an essential step of the search for homologous DNA. The results obtained indicate that hRad54 protein serves as a dsDNA gateway for the hRad51-ssDNA filament, promoting binding and an ATP hydrolysis-dependent translocation of dsDNA during the search for homologous sequences.

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