Escherichia coli single-stranded DNA binding protein SSB promotes AlkB-mediated DNA dealkylation repair
- Department of Biotechnology, Indian Institute of Technology Hyderabad, Kandi, Sangareddy, Hyderabad, Telangana, 502285 (India)
Highlights: • E. coli single stranded DNA binding protein (SSB) wraps single-stranded DNA (ssDNA). • SSB is known to be involved in base excision repair and recombination repair but not in DNA dealkylation repair. • The alkyl-adduct positioned at the 5′ of ssDNA is more accessible to AlkB compared to the internally located alkyl-adducts. • The presence of SSB lowers the K{sub M} of AlkB-catalyzed reaction and promote repair of the longer substrate. • AlkB-mediated repair of methyl adducts from very short ssDNA is rather efficient and SSB-independent. Repair of alkylation damage in DNA is essential for maintaining genome integrity. Escherichia coli (E.coli) protein AlkB removes various alkyl DNA adducts including N1-methyladenine (N{sup 1}meA) and N3-methylcytosine (N{sup 3}meC) by oxidative demethylation. Previous studies showed that AlkB preferentially removes N{sup 1}meA and N{sup 3}meC from single-stranded DNA (ssDNA). It can also remove N{sup 1}meA and N{sup 3}meC from double-stranded DNA by base-flipping. Notably, ssDNA produced during DNA replication and recombination, remains bound to E. coli single-stranded DNA binding protein SSB and it is not known whether AlkB can repair methyl adduct present in SSB-coated DNA. Here we have studied AlkB-mediated DNA repair using SSB-bound DNA as substrate. In vitro repair reaction revealed that AlkB could efficiently remove N{sup 3}meC adducts inasmuch as DNA length is shorter than 20 nucleotides. However, when longer N{sup 3}meC-containing oligonuleotides were used as the substrate, efficiency of AlkB catalyzed reaction was abated compared to SSB-bound DNA substrate of identical length. Truncated SSB containing only the DNA binding domain could also support the stimulation of AlkB activity, suggesting the importance of SSB-DNA interaction for AlkB function. Using 70-mer oligonucleotide containing single N{sup 3}meC we demonstrate that SSB-AlkB interaction promotes faster repair of the methyl DNA adducts.
- OSTI ID:
- 23127425
- Journal Information:
- Biochemical and Biophysical Research Communications, Vol. 496, Issue 2; Other Information: Copyright (c) 2018 Elsevier Inc. All rights reserved.; Country of input: International Atomic Energy Agency (IAEA); ISSN 0006-291X
- Country of Publication:
- United States
- Language:
- English
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