Singh Vandana, Kumari Bhavini, Maity Banibrata, Seth Debabrata, Das Prolay
Department of Chemistry, Indian Institute of Technology Patna, Govt. Polytechnic Campus, Patliputra Colony, Patna 800013, Bihar, India.
Department of Chemistry, Indian Institute of Technology Patna, Govt. Polytechnic Campus, Patliputra Colony, Patna 800013, Bihar, India.
Mutat Res. 2014 Aug-Sep;766-767:56-65. doi: 10.1016/j.mrfmmm.2014.06.002. Epub 2014 Jun 21.
Sequences like the core element of TATA box and CpG island are frequently encountered in the genome and related to transcription. The fate of repair of clustered abasic sites in such sequences of genomic importance is largely unknown. This prompted us to investigate the sequence dependence of cleavage efficiency of APE1 enzyme at abasic sites within the core sequences of TATA box and CpG island using fluorescence dynamics and reaction kinetics. Simultaneous molecular dynamics study through steady state and time resolved fluorescence spectroscopy using unique ethidium bromide dye release assay confirmed an elevated amount of abasic site cleavage of the TATA box sequence as compared to the core CpG island. Reaction kinetics showed that catalytic efficiency of APE1 for abasic site cleavage of core CpG island sequence was ∼4 times lower as compared to that of the TATA box. Higher value of Km was obtained from the core CpG island sequence than the TATA box sequence. This suggests a greater binding effect of APE1 enzyme on TATA sequence that signifies a prominent role of the sequence context of the DNA substrate. Evidently, a faster response from APE1 was obtained for clustered abasic damage repair of TATA box core sequences than CpG island consensus sequences. The neighboring bases of the abasic sites in the complementary DNA strand were found to have significant contribution in addition to the flanking bases in modulating APE1 activity. The repair refractivity of the bistranded clustered abasic sites arise from the slow processing of the second abasic site, consequently resulting in decreased overall production of potentially lethal double strand breaks.
诸如TATA盒核心元件和CpG岛等序列在基因组中经常出现且与转录相关。在这些具有基因组重要性的序列中，成簇无碱基位点的修复命运在很大程度上尚不清楚。这促使我们利用荧光动力学和反应动力学研究APE1酶在TATA盒和CpG岛核心序列内无碱基位点的切割效率的序列依赖性。通过使用独特的溴化乙锭染料释放测定法的稳态和时间分辨荧光光谱进行的同步分子动力学研究证实，与核心CpG岛相比，TATA盒序列的无碱基位点切割量有所增加。反应动力学表明，与TATA盒相比，APE1对核心CpG岛序列无碱基位点切割的催化效率低约4倍。从核心CpG岛序列获得的Km值高于TATA盒序列。这表明APE1酶对TATA序列具有更大的结合作用，这意味着DNA底物的序列背景起着重要作用。显然，与CpG岛共有序列相比，APE1对TATA盒核心序列的成簇无碱基损伤修复反应更快。除了侧翼碱基外，互补DNA链中无碱基位点的相邻碱基在调节APE1活性方面也有显著贡献。双链成簇无碱基位点的修复抗性源于第二个无碱基位点的缓慢处理，从而导致潜在致死性双链断裂的总体产生减少。
