title: Direct observation of preferential processing of clustered abasic DNA damages with APE1 in TATA box and CpG island by reaction kinetics and fluorescence dynamics.
abstract: 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.
authors.affiliation: Department of Chemistry, Indian Institute of Technology Patna, Govt. Polytechnic Campus, Patliputra Colony, Patna 800013, Bihar, India.
authors.forename: Vandana
authors.initials: V
authors.lastname: Singh
authors.affiliation: Department of Chemistry, Indian Institute of Technology Patna, Govt. Polytechnic Campus, Patliputra Colony, Patna 800013, Bihar, India.
authors.forename: Bhavini
authors.initials: B
authors.lastname: Kumari
authors.affiliation: Department of Chemistry, Indian Institute of Technology Patna, Govt. Polytechnic Campus, Patliputra Colony, Patna 800013, Bihar, India.
authors.forename: Banibrata
authors.initials: B
authors.lastname: Maity
authors.affiliation: Department of Chemistry, Indian Institute of Technology Patna, Govt. Polytechnic Campus, Patliputra Colony, Patna 800013, Bihar, India.
authors.forename: Debabrata
authors.initials: D
authors.lastname: Seth
authors.affiliation: Department of Chemistry, Indian Institute of Technology Patna, Govt. Polytechnic Campus, Patliputra Colony, Patna 800013, Bihar, India. Electronic address: prolay@iitp.ac.in.
authors.forename: Prolay
authors.initials: P
authors.lastname: Das
mesh_terms: D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D018899:CpG Islands; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D016385:TATA Box
chemical_list: D004247:DNA; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase
keywords: APE1; Abasic site; CpG island; DNA damage; TATA box; Time resolved fluorescence spectroscopy