Mascarenhas Judita, Sanchez Humberto, Tadesse Serkalem, Kidane Dawit, Krisnamurthy Mahalakshmi, Alonso Juan C, Graumann Peter L
Institut für Mikrobiologie, Albert-Ludwigs Universität Freiburg, Stefan Meier Str, 19, 79104 Freiburg, Germany.
BMC Mol Biol. 2006 Jun 16;7:20. doi: 10.1186/1471-2199-7-20.
Several distinct pathways for the repair of damaged DNA exist in all cells. DNA modifications are repaired by base excision or nucleotide excision repair, while DNA double strand breaks (DSBs) can be repaired through direct joining of broken ends (non homologous end joining, NHEJ) or through recombination with the non broken sister chromosome (homologous recombination, HR). Rad50 protein plays an important role in repair of DNA damage in eukaryotic cells, and forms a complex with the Mre11 nuclease. The prokaryotic ortholog of Rad50, SbcC, also forms a complex with a nuclease, SbcD, in Escherichia coli, and has been implicated in the removal of hairpin structures that can arise during DNA replication. Ku protein is a component of the NHEJ pathway in pro- and eukaryotic cells.
A deletion of the sbcC gene rendered Bacillus subtilis cells sensitive to DNA damage caused by Mitomycin C (MMC) or by gamma irradiation. The deletion of the sbcC gene in a recN mutant background increased the sensitivity of the single recN mutant strain. SbcC was also non-epistatic with AddAB (analog of Escherichia coli RecBCD), but epistatic with RecA. A deletion of the ykoV gene encoding the B. subtilis Ku protein in a sbcC mutant strain did not resulted in an increase in sensitivity towards MMC and gamma irradiation, but exacerbated the phenotype of a recN or a recA mutant strain. In exponentially growing cells, SbcC-GFP was present throughout the cells, or as a central focus in rare cases. Upon induction of DNA damage, SbcC formed 1, rarely 2, foci on the nucleoids. Different to RecN protein, which forms repair centers at any location on the nucleoids, SbcC foci mostly co-localized with the DNA polymerase complex. In contrast to this, AddA-GFP or AddB-GFP did not form detectable foci upon addition of MMC.
Our experiments show that SbcC plays an important role in the repair of DNA inter-strand cross-links (induced by MMC), most likely through HR, and suggest that NHEJ via Ku serves as a backup DNA repair system. The cell biological experiments show that SbcC functions in close proximity to the replication machinery, suggesting that SbcC may act on stalled or collapsed replication forks. Our results show that different patterns of localization exist for DNA repair proteins, and that the B. subtilis SMC proteins RecN and SbcC play distinct roles in the repair of DNA damage.
所有细胞中都存在几种不同的受损DNA修复途径。DNA修饰通过碱基切除或核苷酸切除修复进行修复，而DNA双链断裂（DSB）可通过断裂末端的直接连接（非同源末端连接，NHEJ）或与未断裂的姐妹染色体进行重组（同源重组，HR）来修复。Rad50蛋白在真核细胞的DNA损伤修复中起重要作用，并与Mre11核酸酶形成复合物。Rad50的原核直系同源物SbcC在大肠杆菌中也与核酸酶SbcD形成复合物，并与去除DNA复制过程中可能出现的发夹结构有关。Ku蛋白是原核和真核细胞中NHEJ途径的一个组成部分。
sbcC基因的缺失使枯草芽孢杆菌细胞对丝裂霉素C（MMC）或γ射线引起的DNA损伤敏感。在recN突变体背景中缺失sbcC基因增加了单recN突变体菌株的敏感性。SbcC与AddAB（大肠杆菌RecBCD的类似物）也不存在上位性，但与RecA存在上位性。在sbcC突变体菌株中缺失编码枯草芽孢杆菌Ku蛋白的ykoV基因，不会导致对MMC和γ射线的敏感性增加，但会加剧recN或recA突变体菌株的表型。在指数生长的细胞中，SbcC-GFP在整个细胞中都有分布，在极少数情况下呈中央聚集。在诱导DNA损伤后，SbcC在类核上形成1个，很少形成2个聚集点。与在类核上任何位置形成修复中心的RecN蛋白不同，SbcC聚集点大多与DNA聚合酶复合物共定位。与此相反，添加MMC后，AddA-GFP或AddB-GFP不会形成可检测到的聚集点。
我们的实验表明，SbcC在DNA链间交联（由MMC诱导）的修复中起重要作用，最有可能是通过HR，并表明通过Ku的NHEJ作为备用的DNA修复系统。细胞生物学实验表明，SbcC在靠近复制机制的位置发挥作用，这表明SbcC可能作用于停滞或崩溃的复制叉。我们的结果表明，DNA修复蛋白存在不同的定位模式，并且枯草芽孢杆菌SMC蛋白RecN和SbcC在DNA损伤修复中发挥不同的作用。
