Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10065, USA.
J Biol Chem. 2010 Jan 22;285(4):2632-41. doi: 10.1074/jbc.M109.076133. Epub 2009 Nov 17.
Mycobacterial AdnAB exemplifies a family of heterodimeric motor-nucleases involved in processing DNA double strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N-terminal UvrD-like motor domain and a C-terminal RecB-like nuclease module. Here we conducted a biochemical characterization of the AdnAB motor, using a nuclease-inactivated heterodimer. AdnAB is a vigorous single strand DNA (ssDNA)-dependent ATPase (k(cat) 415 s(-1)), and the affinity of the motor for the ssDNA cofactor increases 140-fold as DNA length is extended from 12 to 44 nucleotides. Using a streptavidin displacement assay, we demonstrate that AdnAB is a 3' --> 5' translocase on ssDNA. AdnAB binds stably to DSB ends. In the presence of ATP, the motor unwinds the DNA duplex without requiring an ssDNA loading strand. We integrate these findings into a model of DSB unwinding in which the "leading" AdnB and "lagging" AdnA motor domains track in tandem, 3' to 5', along the same DNA single strand. This contrasts with RecBCD, in which the RecB and RecD motors track in parallel along the two separated DNA single strands. The effects of 5' and 3' terminal obstacles on ssDNA cleavage by wild-type AdnAB suggest that the AdnA nuclease receives and processes the displaced 5' strand, while the AdnB nuclease cleaves the displaced 3' strand. We present evidence that the distinctive "molecular ruler" function of the ATP-dependent single strand DNase, whereby AdnAB measures the distance from the 5'-end to the sites of incision, reflects directional pumping of the ssDNA through the AdnAB motor into the AdnB nuclease. These and other findings suggest a scenario for the descent of the RecBCD- and AddAB-type DSB-processing machines from an ancestral AdnAB-like enzyme.
分枝杆菌 AdnAB 是参与加工 DNA 双链断裂 (DSB) 的异二聚体运动-核酸酶家族的典范。AdnA 和 AdnB 亚基各由一个 N 端 UvrD 样运动结构域和一个 C 端 RecB 样核酸酶模块组成。在这里，我们使用失活的异二聚体对 AdnAB 运动进行了生化特性分析。AdnAB 是一种强有力的单链 DNA (ssDNA) 依赖性 ATP 酶 (k(cat) 415 s(-1))，当 DNA 长度从 12 个核苷酸延伸至 44 个核苷酸时，马达对 ssDNA 辅助因子的亲和力增加 140 倍。使用链霉亲和素置换测定法，我们证明 AdnAB 是 ssDNA 上的 3' --> 5' 转位酶。AdnAB 稳定地结合 DSB 末端。在 ATP 存在下，该马达无需 ssDNA 加载链即可解开 DNA 双链。我们将这些发现整合到 DSB 解开模型中，其中“先导”AdnB 和“滞后”AdnA 运动结构域沿着同一 DNA 单链 3' 至 5' 串联跟踪。这与 RecBCD 形成对比，在 RecBCD 中，RecB 和 RecD 马达沿着两条分开的 DNA 单链平行跟踪。5' 和 3' 末端障碍物对野生型 AdnAB 切割 ssDNA 的影响表明，AdnA 核酸酶接收并处理取代的 5' 链，而 AdnB 核酸酶切割取代的 3' 链。我们提供的证据表明，ATP 依赖性单链 DNA 核酸酶独特的“分子标尺”功能，即 AdnAB 测量从 5'-末端到切口位点的距离，反映了 ssDNA 通过 AdnAB 马达进入 AdnB 核酸酶的定向泵送。这些和其他发现表明，RecBCD 和 AddAB 型 DSB 加工机器从祖先的 AdnAB 样酶下降的情况。
