Functional attributes of the Saccharomyces cerevisiae meiotic recombinase Dmc1

• Published in: DNA repair 2013-09, Vol.12 (9), p.707-712
• Main Authors: Busygina, Valeria, Gaines, William A., Xu, Yuanyuan, Kwon, Youngho, Williams, Gareth J., Lin, Sheng-Wei, Chang, Hao-Yen, Chi, Peter, Wang, Hong-Wei, Sung, Patrick
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - chemistry; Calcium - chemistry; Cell Cycle Proteins - chemistry; Cell Cycle Proteins - isolation & purification; Cell Cycle Proteins - physiology; Chromatography, Gel; Dmc1 recombinase; DNA Helicases - chemistry; DNA Repair Enzymes - chemistry; DNA Topoisomerases - chemistry; DNA, Fungal - chemistry; DNA, Fungal - ultrastructure; DNA, Single-Stranded - chemistry; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - isolation & purification; DNA-Binding Proteins - physiology; Homologous Recombination; Humans; Hydrolysis; Meiosis; Protein Binding; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - isolation & purification; Saccharomyces cerevisiae Proteins - physiology
• ISSN: 1568-7864; 1568-7856
• DOI: 10.1016/j.dnarep.2013.05.004

•Inclusion of ATP and Mg2+ during purificationprevents ScDmc1 aggregation.•ScDmc1 so purified is active as recombinase and responsive to Rad54 and Rdh54.•3D reconstruction of ScDmc1 on ssDNA illustrateslikeness with other recombinases. The role of Dmc1 as a meiosis-specific general recombinase was first demonstrated in Saccharomyces cerevisiae. Progress in understanding the biochemical mechanism of ScDmc1 has been hampered by its tendency to form inactive aggregates. We have found that the inclusion of ATP during protein purification prevents Dmc1 aggregation. ScDmc1 so prepared is capable of forming D-loops and responsive to its accessory factors Rad54 and Rdh54. Negative staining electron microscopy and iterative helical real-space reconstruction revealed that the ScDmc1-ssDNA nucleoprotein filament harbors 6.5 protomers per turn with a pitch of ∼106Å. The ScDmc1 purification procedure and companion molecular analyses should facilitate future studies on this recombinase.