Howard Hughes Medical Institute, The Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02454, USA.
Proc Natl Acad Sci U S A. 2012 Feb 28;109(9):3353-8. doi: 10.1073/pnas.1115105109. Epub 2012 Feb 13.
Transcription-coupled DNA repair targets DNA lesions that block progression of elongating RNA polymerases. In bacteria, the transcription-repair coupling factor (TRCF; also known as Mfd) SF2 ATPase recognizes RNA polymerase stalled at a site of DNA damage, removes the enzyme from the DNA, and recruits the Uvr(A)BC nucleotide excision repair machinery via UvrA binding. Previous studies of TRCF revealed a molecular architecture incompatible with UvrA binding, leaving its recruitment mechanism unclear. Here, we examine the UvrA recognition determinants of TRCF using X-ray crystallography of a core TRCF-UvrA complex and probe the conformational flexibility of TRCF in the absence and presence of nucleotides using small-angle X-ray scattering. We demonstrate that the C-terminal domain of TRCF is inhibitory for UvrA binding, but not RNA polymerase release, and show that nucleotide binding induces concerted multidomain motions. Our studies suggest that autoinhibition of UvrA binding in TRCF may be relieved only upon engaging the DNA damage.
转录偶联 DNA 修复靶向那些阻碍延伸 RNA 聚合酶前进的 DNA 损伤。在细菌中，转录修复偶联因子（TRCF；也称为 Mfd）SF2 ATP 酶识别在 DNA 损伤部位停滞的 RNA 聚合酶，将酶从 DNA 上脱离，并通过 UvrA 结合招募 Uvr（A）BC 核苷酸切除修复机制。先前对 TRCF 的研究揭示了一种与 UvrA 结合不兼容的分子结构，使其招募机制尚不清楚。在这里，我们使用核心 TRCF-UvrA 复合物的 X 射线晶体学研究来检查 TRCF 的 UvrA 识别决定因素，并使用小角度 X 射线散射研究在没有和存在核苷酸的情况下探测 TRCF 的构象灵活性。我们证明 TRCF 的 C 末端结构域对 UvrA 结合具有抑制作用，但对 RNA 聚合酶释放没有抑制作用，并表明核苷酸结合诱导协同的多结构域运动。我们的研究表明，TRCF 中 UvrA 结合的自动抑制可能仅在与 DNA 损伤结合时才会解除。
