Department of Biochemistry, Indian Institute of Science, Bengaluru, India.
Department of Biochemistry, Indian Institute of Science, Bengaluru, India.
J Biol Chem. 2024 Oct;300(10):107732. doi: 10.1016/j.jbc.2024.107732. Epub 2024 Sep 1.
In eubacteria, Holliday junction (HJ) resolvases (HJRs) are crucial for faithful segregation of newly replicated chromosomes, homologous recombination, and repair of stalled/collapsed DNA replication forks. However, compared with the Escherichia coli HJRs, little is known about their orthologs in mycobacterial species. A genome-wide analysis of Mycobacterium smegmatis identified two genes encoding putative HJRs, namely RuvC (MsRuvC) and RuvX (MsRuvX); but whether they play redundant, overlapping, or distinct roles remains unknown. Here, we reveal that MsRuvC exists as a homodimer while MsRuvX as a monomer in solution, and both showed high-binding affinity for branched DNAs compared with unbranched DNA species. Interestingly, the DNA cleavage specificities of MsRuvC and MsRuvX were found to be mutually exclusive: the former efficiently promotes HJ resolution, in a manner analogous to the Escherichia coli RuvC, but does not cleave other branched DNA species; whereas the latter is a versatile DNase capable of cleaving a variety of branched DNA structures, including 3' and 5' flap DNA, splayed-arm DNA and dsDNA with 3' and 5' overhangs but lacks the HJ resolution activity. Point mutations in the RNase H-like domains of MsRuvC and MsRuvX pinpointed critical residues required for their DNA cleavage activities and also demonstrated uncoupling between DNA-binding and DNA cleavage activities. Unexpectedly, we found robust evidence that MsRuvX possesses a double-strand/single-strand junction-specific endonuclease and ssDNA exonucleolytic activities. Combined, our findings highlight that the RuvC and RuvX DNases play distinct complementary, and not redundant, roles in the processing of branched DNA structures in M. smegmatis.
在真细菌中，霍利迪连接点（HJ）解旋酶（HJRs）对于新复制染色体的准确分离、同源重组以及停滞/崩溃的 DNA 复制叉的修复至关重要。然而，与大肠杆菌 HJR 相比，人们对分枝杆菌种的同源物知之甚少。对耻垢分枝杆菌的全基因组分析鉴定出两个编码假定 HJR 的基因，即 RuvC（MsRuvC）和 RuvX（MsRuvX）；但它们是否发挥冗余、重叠或独特的作用仍不清楚。在这里，我们揭示 MsRuvC 以同源二聚体的形式存在，而 MsRuvX 以单体的形式存在于溶液中，并且与未分枝的 DNA 相比，它们都对分枝 DNA 具有高结合亲和力。有趣的是，发现 MsRuvC 和 MsRuvX 的 DNA 切割特异性是相互排斥的：前者有效地促进 HJ 分辨率，类似于大肠杆菌 RuvC，但不切割其他分枝 DNA ；而后者是一种多功能的核酸内切酶，能够切割多种分枝 DNA 结构，包括 3' 和 5' flap DNA、张开臂 DNA 和带有 3' 和 5' 突出端的 dsDNA，但缺乏 HJ 分辨率活性。在 MsRuvC 和 MsRuvX 的 RNase H 样结构域中的点突变确定了其 DNA 切割活性所需的关键残基，并也证明了 DNA 结合和 DNA 切割活性之间的解耦。出乎意料的是，我们发现了强有力的证据表明 MsRuvX 具有双链/单链连接特异性内切核酸酶和 ssDNA 外切核酸酶活性。综合来看，我们的发现强调了 RuvC 和 RuvX 核酸酶在分枝 DNA 结构的加工中发挥着不同但互补的作用，而不是冗余的作用。
