Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, Massachusetts, USA.
PLoS One. 2013;8(3):e58015. doi: 10.1371/journal.pone.0058015. Epub 2013 Mar 12.
Non homologous end joining (NHEJ) is an important process that repairs double strand DNA breaks (DSBs) in eukaryotic cells. Cells defective in NHEJ are unable to join chromosomal breaks. Two different NHEJ assays are typically used to determine the efficiency of NHEJ. One requires NHEJ of linearized plasmid DNA transformed into the test organism; the other requires NHEJ of a single chromosomal break induced either by HO endonuclease or the I-SceI restriction enzyme. These two assays are generally considered equivalent and rely on the same set of NHEJ genes. PC4 is an abundant DNA binding protein that has been suggested to stimulate NHEJ. Here we tested the role of PC4's yeast homolog SUB1 in repair of DNA double strand breaks using different assays. We found SUB1 is required for NHEJ repair of DSBs in plasmid DNA, but not in chromosomal DNA. Our results suggest that these two assays, while similar are not equivalent and that repair of plasmid DNA requires additional factor(s) that are not required for NHEJ repair of chromosomal double-strand DNA breaks. Possible roles for Sub1 proteins in NHEJ of plasmid DNA are discussed.
非同源末端连接（NHEJ）是修复真核细胞双链 DNA 断裂（DSBs）的重要过程。NHEJ 缺陷的细胞无法连接染色体断裂。通常使用两种不同的 NHEJ 测定法来确定 NHEJ 的效率。一种需要线性化质粒 DNA 的 NHEJ 转化为测试生物；另一种需要由 HO 内切酶或 I-SceI 限制酶诱导的单个染色体断裂的 NHEJ。这两种测定通常被认为是等效的，并依赖于相同的 NHEJ 基因集。PC4 是一种丰富的 DNA 结合蛋白，已被提议刺激 NHEJ。在这里，我们使用不同的测定法测试了酵母同源物 SUB1 在修复质粒 DNA 中的 DNA 双链断裂中的作用。我们发现 SUB1 是质粒 DNA 中 DSBs 的 NHEJ 修复所必需的，但在染色体 DNA 中不是必需的。我们的结果表明，这两种测定虽然相似但并不等效，并且质粒 DNA 的修复需要额外的因子（而不是修复染色体双链 DNA 断裂所必需的）。讨论了 Sub1 蛋白在质粒 DNA 的 NHEJ 中的可能作用。
