The Bacillus subtilis PriA Winged Helix Domain Is Critical for Surviving DNA Damage.
The Bacillus subtilis PriA Winged Helix Domain Is Critical for Surviving DNA Damage.
复制标题
枯草芽孢杆菌 PriA 有翼螺旋结构域对于 DNA 损伤的存活至关重要。
DOI：
10.1128/jb.00539-21
复制
发表时间：
2022
影响因子：
3.2
通讯作者：
Simmons,LyleA
中科院分区：
文献类型：
--
作者：
Matthews,LindsayA;Simmons,LyleA
DNA复制叉经常遇到病变或其他障碍，导致叉进展受阻。PriA是几乎所有真细菌用来在导致复制叉运动受阻的条件下生存的关键蛋白质之一。PriA直接绑定停滞的复制叉，并启动叉重新启动，允许染色体在压力条件下完全复制。我们使用CRISPR-Cas基因编辑方法来定位对于B中几种抗生素诱导的DNA损伤存活至关重要的PriA残基。枯草杆菌。我们发现B中的翼螺旋（WH）结构域。枯草杆菌PriA对DNA损伤的存活至关重要，并参与DNA结合。WH结构域的重要功能定位于不同的表面，这些表面在几种革兰氏阳性人类病原体中也是保守的。此外，我们鉴定了邻近WH结构域的氨基酸接头，其在B中大大延伸。由于插入而导致的枯草杆菌。缩短这个接头诱导了对DNA损伤的超敏表型，表明其延长的长度对于体内有效的复制叉重启是至关重要的。由于E. coli PriA中，我们的研究结果证明了在B中分叉重启期间WH结构域的贡献的重要差异。枯草杆菌。此外，我们的研究结果表明，这个高度可变的区域在PriA可以提供不同的功能在不同的细菌organism.IMPORTANCEPriA是一个重要的蛋白质，发现在几乎所有的细菌，识别停滞的复制叉编排叉重新启动。PriA同源物含有翼螺旋（WH）结构域。急诊coliPriA WH结构域是一个保守的结构域，在大肠杆菌外不保守的叉重启动途径中起作用。大肠杆菌和密切相关的变形菌。我们分析了WH结构域和相关接头在B中的重要性。枯草杆菌，并发现两者都是生存的DNA损伤的关键。该功能映射到WH结构域C-末端的小基序，其在致病细菌中也是保守的。该基序不是DNA结合所必需的，因此可能在复制叉重启途径中执行新的功能。
DNA replication forks regularly encounter lesions or other impediments that result in a blockage to fork progression. PriA is one of the key proteins used by virtually all eubacteria to survive conditions that result in a blockage to replication fork movement. PriA directly binds stalled replication forks and initiates fork restart allowing for chromosomes to be fully duplicated under stressful conditions. We used a CRISPR-Cas gene editing approach to map PriA residues critical for surviving DNA damage induced by several antibiotics in B. subtilis. We find that the winged helix (WH) domain in B. subtilis PriA is critical for surviving DNA damage and participates in DNA binding. The importantin vivofunction of the WH domain mapped to distinct surfaces that were also conserved among several Gram-positive human pathogens. In addition, we identified an amino acid linker neighboring the WH domain that is greatly extended in B. subtilis due to an insertion. Shortening this linker induced a hypersensitive phenotype to DNA damage, suggesting that its extended length is critical for efficient replication fork restartin vivo. Because the WH domain is dispensable in E. coli PriA, our findings demonstrate an important difference in the contribution of the WH domain during fork restart in B. subtilis. Furthermore, with our results we suggest that this highly variable region in PriA could provide different functions across diverse bacterial organisms.IMPORTANCEPriA is an important protein found in virtually all bacteria that recognizes stalled replication forks orchestrating fork restart. PriA homologs contain a winged helix (WH) domain. The E. coli PriA WH domain is dispensable and functions in a fork restart pathway that is not conserved outside of E. coli and closely related proteobacteria. We analyzed the importance of the WH domain and an associated linker in B. subtilis and found that both are critical for surviving DNA damage. This function mapped to a small motif at the C-terminal end of the WH domain, which is also conserved in pathogenic bacteria. The motif was not required for DNA binding and therefore may perform a novel function in the replication fork restart pathway.