Manthei Kelly A, Munson Lia M, Nandakumar Jayakrishnan, Simmons Lyle A
Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan, USA.
bioRxiv. 2024 Feb 17:2024.02.15.580553. doi: 10.1101/2024.02.15.580553.
Mitomycin C (MMC) repair factor A () and factor B (), encode a conserved helicase and exonuclease that repair DNA damage in the soil-dwelling bacterium . Here we have focused on the characterization of MrfB, a DEDDh exonuclease in the DnaQ superfamily. We solved the structure of the exonuclease core of MrfB to a resolution of 2.1 Å, in what appears to be an inactive state. In this conformation, a predicted α-helix containing the catalytic DEDDh residue Asp172 adopts a random coil, which moves Asp172 away from the active site and results in the occupancy of only one of the two catalytic Mg ions. We propose that MrfB resides in this inactive state until it interacts with DNA to become activated. By comparing our structure to an AlphaFold prediction as well as other DnaQ-family structures, we located residues hypothesized to be important for exonuclease function. Using exonuclease assays we show that MrfB is a Mg-dependent 3'-5' DNA exonuclease. We show that Leu113 aids in coordinating the 3' end of the DNA substrate, and that a basic loop is important for substrate binding. This work provides insight into the function of a recently discovered bacterial exonuclease important for the repair of MMC-induced DNA adducts.
丝裂霉素C（MMC）修复因子A（）和因子B（）编码一种保守的解旋酶和核酸外切酶，可修复土壤细菌中的DNA损伤。在这里，我们重点研究了MrfB的特性，它是DnaQ超家族中的一种DEDDh核酸外切酶。我们解析了MrfB核酸外切酶核心的结构，分辨率为2.1 Å，其状态似乎是无活性的。在这种构象中，一个包含催化性DEDDh残基Asp172的预测α螺旋呈无规卷曲状态，这使得Asp172远离活性位点，导致两个催化性镁离子中只有一个被占据。我们提出，MrfB处于这种无活性状态，直到它与DNA相互作用而被激活。通过将我们的结构与AlphaFold预测结果以及其他DnaQ家族结构进行比较，我们确定了据推测对核酸外切酶功能很重要的残基。使用核酸外切酶测定法，我们表明MrfB是一种依赖镁的3'-5' DNA核酸外切酶。我们表明Leu113有助于协调DNA底物的3'末端，并且一个碱性环对底物结合很重要。这项工作为一种最近发现的对MMC诱导的DNA加合物修复很重要的细菌核酸外切酶的功能提供了见解。
