Li Fuyang, Dong Junchao, Pan Xuewen, Oum Ji-Hyun, Boeke Jef D, Lee Sang Eun
Department of Molecular Medicine and Institute of Biotechnology, University of Texas Health Science Center at San Antonio, 15355 Lambda Drive, San Antonio, TX 78245, USA.
Mol Cell. 2008 May 9;30(3):325-35. doi: 10.1016/j.molcel.2008.02.028.
Elimination of a double-strand break (DSB) flanked by direct repeat sequences is mediated by single-strand annealing (SSA), which relies on a distinct set of gene products involving recombination, mismatch repair, and nucleotide excision repair. Here, we screened for yeast mutants defective in SSA with a plasmid-based SSA assay coupled to a barcode microarray readout. The screen identified Yal027Wp/Saw1 (single-strand annealing weakened 1) and Slx4 besides other known SSA proteins. Saw1 interacts physically with Rad1/Rad10, Msh2/Msh3, and Rad52 proteins, and cells lacking SLX4 or SAW1 accumulate recombination intermediates blocked at the Rad1/Rad10-dependent 3' flap cleavage step. Slx4 and Saw1 also contribute to the integrity of ribosomal DNA arrays. Saw1 mutants that fail to interact with Rad1, but retain interaction with Rad52 and Msh2, are defective in 3' flap removal and SSA repair. Deletion of SAW1 abolished association of Rad1 at SSA intermediates in vivo. We propose that Saw1 targets Rad1/Rad10 to Rad52-coated recombination intermediates.
由单链退火（SSA）介导的、两侧带有同向重复序列的双链断裂（DSB）的消除，依赖于一组独特的基因产物，这些产物涉及重组、错配修复和核苷酸切除修复。在这里，我们通过基于质粒的SSA检测与条形码微阵列读数相结合的方法，筛选了在SSA中存在缺陷的酵母突变体。该筛选除了鉴定出其他已知的SSA蛋白外，还鉴定出了Yal027Wp/Saw1（单链退火减弱1）和Slx4。Saw1与Rad1/Rad10、Msh2/Msh3和Rad52蛋白发生物理相互作用，缺乏SLX4或SAW1的细胞会积累在Rad1/Rad10依赖性3' 瓣切割步骤受阻的重组中间体。Slx4和Saw1也有助于核糖体DNA阵列的完整性。无法与Rad1相互作用，但仍与Rad52和Msh2相互作用的Saw1突变体，在3' 瓣切除和SSA修复方面存在缺陷。在体内，SAW1的缺失消除了Rad1在SSA中间体处的结合。我们提出，Saw1将Rad1/Rad10靶向到由Rad52包被的重组中间体上。
