A Single Residue Unique to DinB-Like Proteins Limits Formation of the Polymerase IV Multiprotein Complex in Escherichia coli
A Single Residue Unique to DinB-Like Proteins Limits Formation of the Polymerase IV Multiprotein Complex in Escherichia coli
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DOI：
10.1128/jb.01349-12
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发表时间：
2013-03-01
影响因子：
3.2
通讯作者：
Godoy, Veronica G.
中科院分区：
文献类型：
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作者：
Cafarelli, Tiziana M.;Rands, Thomas J.;Godoy, Veronica G.
DinB的活性由与RecA和UmuD形成的多蛋白复合物（MPC）控制。我们在DinB中鉴定了两个高度保守的表面残基，半胱氨酸66（C66）和脯氨酸67（P67）。DinB三级结构的映射表明这些是非催化的，多序列比对表明它们在DinB样蛋白中是独特的。为了研究含C66的表面在MPC形成中的作用，我们构建了dinB（C66A）衍生物。我们发现，DinB（C66A）与其相互作用的合作伙伴，RecA和UmuD，在更大程度上比DinB。值得注意的是，RecA与DinB的共纯化在不存在UmuD的情况下有所增强，并且对于DinB（C66A）进一步增加。体外拉下测定也表明DinB（C66A）比DinB更好地结合RecA和UmuD。我们注意到DinB（C66A）对UmuD的增加的亲和力是RecA依赖性的。因此，含有C66的结合表面似乎对调节与UmuD的相互作用，特别是与RecA的相互作用至关重要。dinB（C66A）从染色体上的表达导致dinB依赖性病变旁路保真度和同源重组的可检测差异。对这种DinB衍生物的研究揭示了DinB上的关键表面，其似乎调节MPC结合的强度，并提出了RecA和UmuD与DinB的结合顺序。这些发现最终将允许操纵这些酶来阻止细菌抗生素耐药性的获得，并深入了解人类癌症的发展。
The activity of DinB is governed by the formation of a multiprotein complex (MPC) with RecA and UmuD. We identified two highly conserved surface residues in DinB, cysteine 66 (C66) and proline 67 (P67). Mapping on the DinB tertiary structure suggests these are noncatalytic, and multiple-sequence alignments indicate that they are unique among DinB-like proteins. To investigate the role of the C66-containing surface in MPC formation, we constructed the dinB(C66A) derivative. We found that DinB(C66A) copurifies with its interacting partners, RecA and UmuD, to a greater extent than DinB. Notably, copurification of RecA with DinB is somewhat enhanced in the absence of UmuD and is further increased for DinB(C66A). In vitro pulldown assays also indicate that DinB(C66A) binds RecA and UmuD better than DinB. We note that the increased affinity of DinB(C66A) for UmuD is RecA dependent. Thus, the C66-containing binding surface appears to be critical to modulate interaction with UmuD, and particularly with RecA. Expression of dinB(C66A) from the chromosome resulted in detectable differences in dinB-dependent lesion bypass fidelity and homologous recombination. Study of this DinB derivative has revealed a key surface on DinB, which appears to modulate the strength of MPC binding, and has suggested a binding order of RecA and UmuD to DinB. These findings will ultimately permit the manipulation of these enzymes to deter bacterial antibiotic resistance acquisition and to gain insights into cancer development in humans.