tailieunhanh - Purification of Saccharomyces cerevisiae recombinant Crp1

A complex Mus81-Mm4 is a DNA structure–specific endonuclease in Saccharomyces cerevisiae. Mus81-Mms4 functions in processing of recombination intermediates that could arise during the repair of stalled and blocked replication forks and double stranded breaks. Mus81-Mms4 works with many proteins involved in DNA repair, replication fork stability, and joint molecule formation/resolution during homologous recombination repair. | 22 Tạp chí Khoa học Công nghệ Số 4 Purification of Saccharomyces cerevisiae recombinant Crpl Huong Thi Thu Phung Diem Hong Tran Nguyen Tat Thanh Hi-Tech Institute Nguyen Tat Thanh University ptthuong@ Abstract A complex Mus81-Mm4 is a DNA structure-specific endonuclease in Saccharomyces cerevisiae. Mus81-Mms4 functions in processing of recombination intermediates that could arise during the repair of stalled and blocked replication forks and double stranded breaks. Mus81-Mms4 works with many proteins involved in DNA repair replication fork stability and joint molecule formation resolution during homologous recombination repair. A biochemical screening of protein s that enhances the Mus81-Mms4 endonuclease activity on its preferable substrates in vitro revealed that Crp1 a cruciform DNA-recognizing protein which can specifically bind to DNA four-way junction structures like Holliday junctions could be the potential factor. To further demonstrate that Crp1 interacts functionally with Mus81-Mms4 in vitro we carried out the purification of recombinant Crp1 using Escherichia coli system. Our results showed that the purified Crp1 was highly homogenous and active that is ready for biochemical use. 2018 Journal of Science and Technology - NTTU Nhận Được duyệt Công bố Keywords Crp1 DNA binding Mus81 purification. 1 Introduction Mus81 is a conserved DNA structure-specific endonuclease which belongs to the XPF Rad1 family of proteins involved in DNA nucleotide excision repair 1 . XPF Rad1 family members typically contain a pair of helix-hairpin-helix HhH motifs and a conserved catalytic domain in their C-terminal region with an ERKX3D active site motif 1 . In Mus81 the HhH motifs are positioned at both ends whereas in XPF they are positioned at the C-terminal end. The HhH motifs may play a role in DNA binding and dimer formation and are also required for nuclease activity 2 3 . Mus81 was shown to be active as a structure-specific .