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Identification of the Dimer Exchange Interface of the Bacterial DNA Damage Response Protein UmuD
Biochemistry ( IF 2.9 ) Pub Date : 2017-08-29 00:00:00 , DOI: 10.1021/acs.biochem.7b00560 David A. Murison 1 , Rebecca C. Timson 1 , Bilyana N. Koleva 1 , Michael Ordazzo 1 , Penny J. Beuning 1
Biochemistry ( IF 2.9 ) Pub Date : 2017-08-29 00:00:00 , DOI: 10.1021/acs.biochem.7b00560 David A. Murison 1 , Rebecca C. Timson 1 , Bilyana N. Koleva 1 , Michael Ordazzo 1 , Penny J. Beuning 1
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The Escherichia coli SOS response, an induced DNA damage response pathway, confers survival on bacterial cells by providing accurate repair mechanisms as well as the potentially mutagenic pathway translesion synthesis (TLS). The umuD gene products are upregulated after DNA damage and play roles in both nonmutagenic and mutagenic aspects of the SOS response. Full-length UmuD is expressed as a homodimer of 139-amino-acid subunits, which eventually cleaves its N-terminal 24 amino acids to form UmuD′. The cleavage product UmuD′ and UmuC form the Y-family polymerase DNA Pol V (UmuD′2C) capable of performing TLS. UmuD and UmuD′ exist as homodimers, but their subunits can readily exchange to form UmuDD′ heterodimers preferentially. Heterodimer formation is an essential step in the degradation pathway of UmuD′. The recognition sequence for ClpXP protease is located within the first 24 amino acids of full-length UmuD, and the partner of full-length UmuD, whether UmuD or UmuD′, is degraded by ClpXP. To better understand the mechanism by which UmuD subunits exchange, we measured the kinetics of exchange of a number of fluorescently labeled single-cysteine UmuD variants as detected by Förster resonance energy transfer. Labeling sites near the dimer interface correlate with increased rates of exchange, indicating that weakening the dimer interface facilitates exchange, whereas labeling sites on the exterior decrease the rate of exchange. In most but not all cases, homodimer and heterodimer exchange exhibit similar rates, indicating that somewhat different molecular surfaces mediate homodimer exchange and heterodimer formation.
中文翻译：
细菌DNA损伤反应蛋白UmuD的二聚体交换界面的鉴定。
的大肠杆菌SOS反应，诱导DNA损伤应答途径，赋予存活细菌细胞通过提供准确修复机制以及潜在的诱变途径跨损伤合成（TLS）。DNA损伤后，umuD基因产物被上调，并且在SOS反应的非诱变和诱变方面均发挥作用。全长UmuD被表达为139个氨基酸亚基的同型二聚体，其最终裂解其N末端的24个氨基酸以形成UmuD'。裂解产物UmuD'和UMUC形成Y-家族聚合酶DNA聚合酶V（UmuD' 2C）能够执行TLS。UmuD和UmuD'以同源二聚体存在，但是它们的亚基可以容易地交换以优先形成UmuDD'异二聚体。异二聚体的形成是UmuD'降解途径中必不可少的步骤。ClpXP蛋白酶的识别序列位于全长UmuD的前24个氨基酸内，并且全长UmuD的伴侣，无论是UmuD还是UmuD'，都被ClpXP降解。为了更好地了解UmuD亚基交换的机制，我们通过Förster共振能量转移检测了许多荧光标记的单半胱氨酸UmuD变体的交换动力学。二聚体界面附近的标记位点与交换速率增加相关，表明减弱二聚体界面有助于交换，而在外部贴标签的位置会降低交换速率。在大多数但不是全部情况下，同型二聚体和异型二聚体的交换速率相似，这表明有些不同的分子表面介导了同型二聚体的交换和异质二聚体的形成。
更新日期：2017-08-30
中文翻译：
细菌DNA损伤反应蛋白UmuD的二聚体交换界面的鉴定。
的大肠杆菌SOS反应，诱导DNA损伤应答途径，赋予存活细菌细胞通过提供准确修复机制以及潜在的诱变途径跨损伤合成（TLS）。DNA损伤后，umuD基因产物被上调，并且在SOS反应的非诱变和诱变方面均发挥作用。全长UmuD被表达为139个氨基酸亚基的同型二聚体，其最终裂解其N末端的24个氨基酸以形成UmuD'。裂解产物UmuD'和UMUC形成Y-家族聚合酶DNA聚合酶V（UmuD' 2C）能够执行TLS。UmuD和UmuD'以同源二聚体存在，但是它们的亚基可以容易地交换以优先形成UmuDD'异二聚体。异二聚体的形成是UmuD'降解途径中必不可少的步骤。ClpXP蛋白酶的识别序列位于全长UmuD的前24个氨基酸内，并且全长UmuD的伴侣，无论是UmuD还是UmuD'，都被ClpXP降解。为了更好地了解UmuD亚基交换的机制，我们通过Förster共振能量转移检测了许多荧光标记的单半胱氨酸UmuD变体的交换动力学。二聚体界面附近的标记位点与交换速率增加相关，表明减弱二聚体界面有助于交换，而在外部贴标签的位置会降低交换速率。在大多数但不是全部情况下，同型二聚体和异型二聚体的交换速率相似，这表明有些不同的分子表面介导了同型二聚体的交换和异质二聚体的形成。
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