Liriano Melissa L, Berger Madison B, Nguyen Kenny, Korzhnev Dmitry M, Cisneros G Andrés, Beuning Penny J
Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts 02115, United States.
Department of Chemistry and Biochemistry, University of Texas at Dallas, Richardson, Texas 75080, United States.
Biochemistry. 2025 Jul 15;64(14):3126-3136. doi: 10.1021/acs.biochem.5c00135. Epub 2025 Jun 27.
The bacterial processivity factor, the β sliding clamp, endows Pol III DNA polymerase α with efficiency and processivity during DNA replication by tethering α to DNA. The dimeric β clamp is loaded onto primer:template DNA junctions by a five-subunit clamp loader complex. We previously showed that tuning the stability of the β dimer interface affected clamp opening dynamics and function. The L82D clamp mutation destabilized the dimer interface due to altered hydrophobic interactions and β L82D exhibited lower thermostability, a change in dimerization state, and a decrease in loading activity. Molecular modeling revealed a rearrangement of the local hydrogen-bond network near L82D in Domain 1, leading to new, non-native interactions involving residues Q265 and R269 in Domain 3 of the partner protomer. We hypothesize that altering this newly formed network in the context of the β L82D variant may stabilize the dimer interface and rescue clamp activity. To test these hypotheses, Q265 and R269 were mutated in WT β and in the L82D variant. The β R269 residue is essential for maintaining a stable dimer interface, functional oligomeric state, and efficient DNA loading, while Q265 is not, as Q265A is tolerated. Disrupting native interactions of R269 with the formation of an alternate bonding network in L82D seems to be the primary driver for the lower thermostability, altered dimerization state, and loss of clamp activity previously observed in β L82D. Our observations show that a complex balance of noncovalent interactions contributes to the stability of the β clamp dimer interface.
细菌持续性因子β滑动夹通过将α固定在DNA上，在DNA复制过程中赋予DNA聚合酶Ⅲα效率和持续性。二聚体β夹通过一个五亚基夹装载复合物加载到引物：模板DNA连接处。我们之前表明，调节β二聚体界面的稳定性会影响夹打开的动力学和功能。L82D夹突变由于疏水相互作用改变而使二聚体界面不稳定，β L82D表现出较低的热稳定性、二聚化状态改变和加载活性降低。分子模拟显示，结构域1中L82D附近的局部氢键网络发生重排，导致与伙伴原体结构域3中的残基Q265和R269形成新的非天然相互作用。我们假设，在β L82D变体的背景下改变这个新形成的网络可能会稳定二聚体界面并挽救夹活性。为了验证这些假设，在野生型β和L82D变体中对Q265和R269进行了突变。β R269残基对于维持稳定的二聚体界面、功能性寡聚状态和有效的DNA加载至关重要，而Q265则不然，因为Q265A是可耐受的。在L82D中破坏R269的天然相互作用并形成替代键合网络似乎是先前在β L82D中观察到的较低热稳定性、二聚化状态改变和夹活性丧失的主要驱动因素。我们的观察结果表明，非共价相互作用的复杂平衡有助于β夹二聚体界面的稳定性。
