Fudrini Olivencia Begonia, Müller Andreas U, Roschitzki Bernd, Burger Sibylle, Weber-Ban Eilika, Imkamp Frank
University of Zurich, Institute of Medical Microbiology, Zurich, Switzerland.
ETH Zurich, Institute of Molecular Biology and Biophysics, Zurich, Switzerland.
Sci Rep. 2017 Oct 25;7(1):13987. doi: 10.1038/s41598-017-14410-z.
Two genes, pafB and pafC, are organized in an operon with the Pup-ligase gene pafA, which is part of the Pup-proteasome system (PPS) present in mycobacteria and other actinobacteria. The PPS is crucial for Mycobacterium tuberculosis resistance towards reactive nitrogen intermediates (RNI). However, pafB and pafC apparently play only a minor role in RNI resistance. To characterize their function, we generated a pafBC deletion in Mycobacterium smegmatis (Msm). Proteome analysis of the mutant strain revealed decreased cellular levels of various proteins involved in DNA damage repair, including recombinase A (RecA). In agreement with this finding, Msm ΔpafBC displayed increased sensitivity to DNA damaging agents. In mycobacteria two pathways regulate DNA repair genes: the LexA/RecA-dependent SOS response and a predominant pathway that controls gene expression via a LexA/RecA-independent promoter, termed P1. PafB and PafC feature winged helix-turn-helix DNA binding motifs and we demonstrate that together they form a stable heterodimer in vitro, implying a function as a heterodimeric transcriptional regulator. Indeed, P1-driven transcription of recA was decreased in Msm ΔpafBC under standard conditions and induction of recA expression upon DNA damage was strongly impaired. Taken together, our data indicate an important regulatory function of PafBC in the mycobacterial DNA damage response.
两个基因pafB和pafC与Pup连接酶基因pafA一起组成一个操纵子，pafA是分枝杆菌和其他放线菌中存在的Pup蛋白酶体系统（PPS）的一部分。PPS对于结核分枝杆菌抵抗活性氮中间体（RNI）至关重要。然而，pafB和pafC在抵抗RNI方面显然只起次要作用。为了表征它们的功能，我们在耻垢分枝杆菌（Msm）中产生了pafBC缺失。对突变菌株的蛋白质组分析显示，参与DNA损伤修复的各种蛋白质的细胞水平降低，包括重组酶A（RecA）。与这一发现一致，Msm ΔpafBC对DNA损伤剂的敏感性增加。在分枝杆菌中，有两条途径调节DNA修复基因：LexA/RecA依赖性SOS反应和一条主要途径，该途径通过一个不依赖LexA/RecA的启动子P1控制基因表达。PafB和PafC具有带翼的螺旋-转角-螺旋DNA结合基序，我们证明它们在体外一起形成稳定的异二聚体，这意味着它们作为异二聚体转录调节因子发挥作用。事实上，在标准条件下，Msm ΔpafBC中recA的P1驱动转录降低，DNA损伤后recA表达的诱导受到严重损害。综上所述，我们的数据表明PafBC在分枝杆菌DNA损伤反应中具有重要的调节功能。
