Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island , Kingston, Rhode Island 02881, United States.
Biochemistry. 2014 Jun 24;53(24):4059-71. doi: 10.1021/bi5003212. Epub 2014 Jun 10.
Adduct-induced DNA damage can affect transcription efficiency and DNA replication and repair. We previously investigated the effects of the 3'-next flanking base (GCT vs GCA; G*, FABP, N-(2'-deoxyguanosin-8-yl)-4'-fluoro-4-aminobiphenyl; FAF, N-(2'-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene) on the conformation of arylamine-DNA lesions in relation to E. coli nucleotide excision repair ( Jain , V. , Hilton , B. , Lin , B. , Patnaik , S. , Liang , F. , Darian , E. , Zou , Y. , Mackerell , A. D. , Jr. , and Cho , B. P. ( 2013 ) Nucleic Acids Res. , 41 , 869 - 880 ). Here, we report the differential effects of the same pair of sequences on DNA replication in vitro by the polymerases exofree Klenow fragment (Kf-exo(-)) and Dpo4. We obtained dynamic (19)F NMR spectra for two 19-mer modified templates during primer elongation: GCA [d(5'-CTTACCATCGCAACCATTC-3')] and GCT [d(5'-CTTACCATCGCTACCATTC-3')]. We found that lesion stacking is favored in the GCT sequence compared to the GCA counterpart. Surface plasmon resonance binding results showed consistently weaker affinities for the modified DNA with the binding strength in the order of FABP > FAF and GCA > GCT. Primer extension was stalled at (n) and near (n - 1 and n + 1) the lesion site, and the extent of blockage and the extension rates across the lesion were influenced by not only the DNA sequences but also the nature of the adduct's chemical structure (FAF vs FABP) and the polymerase employed (Kf-exo(-) vs Dpo4). Steady-state kinetics analysis with Kf-exo(-) revealed the most dramatic sequence and lesion effects at the lesion (n) and postinsertion (n + 1) sites, respectively. Taken together, these results provide insights into the important role of lesion-induced conformational heterogeneity in modulating translesion DNA synthesis.
加合物诱导的 DNA 损伤会影响转录效率和 DNA 复制和修复。我们之前研究了 3'-下一个侧翼碱基（GCT 与 GCA；G*，FABP，N-(2'-脱氧鸟嘌呤-8-基)-4'-氟-4-氨基联苯；FAF，N-(2'-脱氧鸟嘌呤-8-基)-7-氟-2-氨基芴）对大肠杆菌核苷酸切除修复（Jain，V.，Hilton，B.，Lin，B.，Patnaik，S.，Liang，F.，Darian，E.，Zou，Y.，Mackerell，A.D.，Jr.，和 Cho，B.P.（2013）Nucleic Acids Res.，41，869-880）中芳基胺-DNA 损伤构象的影响。在这里，我们报告了同一对序列对体外聚合酶 exofree Klenow 片段（Kf-exo(-)）和 Dpo4 复制 DNA 的不同影响。我们在引物延伸过程中为两个 19 -mer 修饰模板获得了动态（19）F NMR 光谱：GCA [d(5'-CTTACCATCGCAACCATTC-3')]和 GCT [d(5'-CTTACCATCGCTACCATTC-3')]。我们发现与 GCA 对应物相比，加合物堆积在 GCT 序列中更有利。表面等离子体共振结合结果一致显示，修饰 DNA 的亲和力较弱，结合强度顺序为 FABP > FAF 和 GCA > GCT。引物延伸在损伤位点（n）及其附近（n-1 和 n+1）处停滞，阻断程度和跨损伤的延伸率不仅受 DNA 序列的影响，还受加合物化学结构的性质（FAF 与 FABP）和使用的聚合酶（Kf-exo(-)与 Dpo4）的影响。使用 Kf-exo(-)进行稳态动力学分析显示，在损伤（n）和插入后（n+1）位点，序列和损伤的影响最为显著。总之，这些结果提供了关于损伤诱导的构象异质性在调节跨损伤 DNA 合成中的重要作用的见解。
