Eggington Julie Malia, Haruta Nami, Wood Elizabeth Anne, Cox Michael Matthew
Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
BMC Microbiol. 2004 Jan 12;4:2. doi: 10.1186/1471-2180-4-2.
Deinococcus radiodurans R1 is one of the most radiation-resistant organisms known and is able to repair an unusually large amount of DNA damage without induced mutation. Single-stranded DNA-binding (SSB) protein is an essential protein in all organisms and is involved in DNA replication, recombination and repair. The published genomic sequence from Deinococcus radiodurans includes a putative single-stranded DNA-binding protein gene (ssb; DR0100) requiring a translational frameshift for synthesis of a complete SSB protein. The apparently tripartite gene has inspired considerable speculation in the literature about potentially novel frameshifting or RNA editing mechanisms. Immediately upstream of the ssb gene is another gene (DR0099) given an ssb-like annotation, but left unexplored.
A segment of the Deinococcus radiodurans strain R1 genome encompassing the ssb gene has been re-sequenced, and two errors involving omitted guanine nucleotides have been documented. The corrected sequence incorporates both of the open reading frames designated DR0099 and DR0100 into one contiguous ssb open reading frame (ORF). The corrected gene requires no translational frameshifts and contains two predicted oligonucleotide/oligosaccharide-binding (OB) folds. The protein has been purified and its sequence is closely related to the Thermus thermophilus and Thermus aquaticus SSB proteins. Like the Thermus SSB proteins, the SSBDr functions as a homodimer. The Deinococcus radiodurans SSB homodimer stimulates Deinococcus radiodurans RecA protein and Escherichia coli RecA protein-promoted DNA three-strand exchange reactions with at least the same efficiency as the Escherichia coli SSB homotetramer.
The correct Deinococcus radiodurans ssb gene is a contiguous open reading frame that codes for the largest bacterial SSB monomer identified to date. The Deinococcus radiodurans SSB protein includes two OB folds per monomer and functions as a homodimer. The Deinococcus radiodurans SSB protein efficiently stimulates Deinococcus radiodurans RecA and also Escherichia coli RecA protein-promoted DNA strand exchange reactions. The identification and purification of Deinococcus radiodurans SSB protein not only allows for greater understanding of the SSB protein family but provides an essential yet previously missing player in the current efforts to understand the extraordinary DNA repair capacity of Deinococcus radiodurans.
耐辐射球菌R1是已知的最耐辐射的生物体之一，能够修复大量DNA损伤而不发生诱导突变。单链DNA结合（SSB）蛋白是所有生物体中的一种必需蛋白，参与DNA复制、重组和修复。已公布的耐辐射球菌基因组序列包含一个推定的单链DNA结合蛋白基因（ssb；DR0100），该基因需要通过翻译移码来合成完整的SSB蛋白。这个明显的三联体基因引发了文献中关于潜在的新型移码或RNA编辑机制的大量推测。在ssb基因的紧邻上游是另一个基因（DR0099），其注释为类ssb，但尚未进行研究。
对包含ssb基因的耐辐射球菌R1菌株基因组片段进行了重新测序，记录了两个涉及鸟嘌呤核苷酸缺失的错误。校正后的序列将指定为DR0099和DR0100的两个开放阅读框合并为一个连续的ssb开放阅读框（ORF）。校正后的基因不需要翻译移码，并且包含两个预测的寡核苷酸/寡糖结合（OB）折叠。该蛋白已被纯化，其序列与嗜热栖热菌和嗜热水生栖热菌的SSB蛋白密切相关。与嗜热栖热菌的SSB蛋白一样，SSBDr以同二聚体形式发挥作用。耐辐射球菌SSB同二聚体刺激耐辐射球菌RecA蛋白和大肠杆菌RecA蛋白促进的DNA三链交换反应，其效率至少与大肠杆菌SSB同四聚体相同。
正确的耐辐射球菌ssb基因是一个连续的开放阅读框，编码迄今为止鉴定出的最大的细菌SSB单体。耐辐射球菌SSB蛋白每个单体包含两个OB折叠，并以同二聚体形式发挥作用。耐辐射球菌SSB蛋白有效地刺激耐辐射球菌RecA以及大肠杆菌RecA蛋白促进的DNA链交换反应。耐辐射球菌SSB蛋白的鉴定和纯化不仅有助于更深入地了解SSB蛋白家族，而且为当前理解耐辐射球菌非凡的DNA修复能力的努力提供了一个必不可少但以前缺失的因素。
