Probing the Flexibility of the Catalytic Nucleophile in the Lyase Catalytic Pocket of Human DNA Polymerase β with Unnatural Lysine Analogues,Biochemistry

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Probing the Flexibility of the Catalytic Nucleophile in the Lyase Catalytic Pocket of Human DNA Polymerase β with Unnatural Lysine Analogues
Biochemistry ( IF 2.9 ) Pub Date : 2017-01-12 00:00:00 , DOI: 10.1021/acs.biochem.6b00807
Sasha M. Daskalova ₁ , Chandrabali Bhattacharya ₁ , Larisa M. Dedkova ₁ , Sidney M. Hecht ₁

Affiliation

DNA polymerase β (Pol β) is a key enzyme in mammalian base excision repair (BER), contributing stepwise 5′-deoxyribose phosphate (dRP) lyase and “gap-filling” DNA polymerase activities. The lyase reaction is believed to occur via a β-elimination reaction following the formation of a Schiff base between the dRP group at the pre-incised apurinic/apyrimidinic site and the ε-amino group of Lys72. To probe the steric constraints on the formation and subsequent resolution of the putative Schiff base intermediate within the lyase catalytic pocket, Lys72 was replaced with each of several nonproteinogenic lysine analogues. The modified Pol β enzymes were produced by coupled in vitro transcription and translation from a modified DNA template containing a TAG codon at the position corresponding to Lys72. In the presence of a misacylated tRNA_CUA transcript, suppression of the UAG codon in the transcribed mRNA led to elaboration of full length Pol β having a lysine analogue at position 72. Replacement of the primary nucleophilic amine with a secondary amine in the form of N-methyllysine (4) affected mainly the stability of the Schiff base intermediate and resulted in relatively moderate inhibition of lyase activity and BER. Elongation of the side chain of the catalytic residue by one methylene group, achieved by introduction of homolysine (6) at position 72, apparently shifted the amino group to a position less favorable for Schiff base formation. Interestingly, this effect was attenuated when the side chain was elongated by replacing one side-chain methylene group with a bridging S atom (thialysine, 2). In comparison, replacement of lysine 72 with an analogue having a guanidine moiety in lieu of an ε-amino group (homoarginine, 5) or a sterically constrained secondary amine (piperidinylalanine, 3) led to almost complete suppression of dRP excision activity and the ability of Pol β to support BER. These results help to define the tolerance of Pol β to subtle local structural and functional alterations.

中文翻译：

用非天然赖氨酸类似物探索人类DNA聚合酶β的裂解酶催化口袋中催化亲核试剂的灵活性

DNA聚合酶β（Polβ）是哺乳动物碱基切除修复（BER）中的关键酶，可逐步促进5'-脱氧核糖磷酸（dRP）裂解酶和“填补缺口”的DNA聚合酶活性。认为裂解酶反应是通过β消除反应而发生的，该反应是在预先增加的嘌呤/嘧啶基位点的dRP基团与Lys72的ε-氨基之间形成席夫碱之后形成的。为了探查在裂解酶催化口袋中推定的席夫碱中间体的形成和后续拆分的空间位阻，将Lys72替换为几种非蛋白原性赖氨酸类似物。修饰的Polβ酶是通过体外偶联产生的修饰的DNA模板的转录和翻译，该模板在与Lys72对应的位置包含TAG密码子。在存在错误酰化的tRNA _CUA转录本的情况下，转录的mRNA中UAG密码子的抑制导致形成在72位具有赖氨酸类似物的全长Polβ 。用N形式的仲胺取代伯亲核胺-甲基赖氨酸（4）主要影响Schiff碱中间体的稳定性，并导致相对适度的裂解酶活性和BER抑制。通过引入高赖氨酸实现催化残基侧链的一个亚甲基的延长（6）在72位上，显然将氨基转移到不利于席夫碱形成的位置上。有趣的是，当通过用桥连的S原子（硫代赖氨酸，2）取代一个侧链亚甲基而延长了侧链时，这种作用减弱了。相比之下，用具有胍基部分代替ε-氨基（高精氨酸，5）或空间受限的仲胺（哌啶基丙氨酸，3）的类似物替代赖氨酸72几乎完全抑制了dRP切除活性和能力。 Polβ支持BER。这些结果有助于确定Polβ对细微的局部结构和功能变化的耐受性。

更新日期：2017-01-12

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