Research Interest
DNA alkylation affects important biological processes and linked to increased risk of cancer. The research interests of the laboratory reside in the understanding the DNA alkylation damage and its repair mechanisms. Particularly, we strive to know the details of the enzymes engaged in removing the alkyl groups from the modified DNA bases. These enzymes are family of iron-dependent dioxygenases and the most well-characterized member of this family is AlkB in Escherichia coli. There are nine known AlkB homologues in humans, known as AlkBH1-9, which share structural homology to AlkB. The goal of our research is to gain deeper understanding of the functions of the ALKBH1-9 enzymes. We think this line of research has potential to lead to efficient methods of treating cancer.
Some of the research highlight from our lab in the past few years are:
E coli AlkB functions in collaboration with SSB and RecA:
· Research Question: AlkB was reported to repair single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and RNA. However, most of the ssDNA is wrapped by SSB (single-stranded DNA binding) proteins. We were interested to know whether AlkB can repair methyl adduct when the ssDNA remains bound to SSB protein.
· Result: Our results revealed that AlkB could efficiently remove alkyl adducts from shorter length DNA length (<20 nucleotides). However, when longer ssDNA were used as the substrate, efficiency of AlkB-catalyzed reaction was poor with ‘naked’ ssDNA compared to SSB-bound DNA substrate of identical length. Our results suggest that SSB-bound ssDNA could support AlkB mediated repair. We have also mapped the segment of SSB involved in interaction with AlkB.
· Publication details: See publication [1-3].
E coli AlkB is recruited by RecA:
· Research Question: AlkB was reported to repair single-stranded DNA. Single-stranded DNA is created during homologous recombination and this single-stranded DNA is susceptible to alkylation damage. How AlkB is recruited to the ssDNA generated during homologous recombination?
· Result: We studied the mechanism of AlkB recruitment and shown that E coli AlkB interacts with homologous recombination protein RecA and this interaction helps AlkB recruitment to ssDNA. Our results have also showed that RecA-AlkB interaction results in enhanced rate of repair of alkylation damage [4]. Our results could explain how RecA-AlkB interaction might help promote DNA repair by ALkB and facilitate restart of DNA polymerase stalled at alkyl-adducts.
· Publication details: See publication [4].
E coli AlkB is regulated by iron and oxygen availability
· Research Question: AlkB family of enzymes being iron-dependent dioxygenases. Are they regulated by availability of intracellular iron and oxygen?
· Result: We have proved that intracellular iron concentration is crucial in keeping the DNA free of alkylation damage.
· Publication details: [5] and [6].
Human AlkBH3 is recruited by Rad51C:
· Research Question: Just like E coli AlkB and RecA, single-stranded DNA is created during homologous recombination in human cells [7]. Human homologous recombination is mediated by Rad51 and Rad51 orthologues. We asked which AlkBH protein is is recruited to the ssDNA generated during homologous recombination?
· Result: our result with human cells revealed that human AlkB homologue AlkBH3 interacts with human Rad51 paralogue Rad51C and this interaction facilitates damage removal. We observed that AlkBH3-Rad51C interaction particularly facilitates repair of alkyl adducts present on a 3’-overhang substrate [8].
· Publication details: See publication [5], [6].
Development of quantitative assays for AlkBH activity:
· Research Question: When we started working with AlkB, there were no simple assay for AlkB activity.
· Result: We developed three simple strategies for AlkB enzyme assays and characterized the substrate specificity
· Publication details: See publication [6, 9-11].
New small molecule inhibitors of AlkBH enzymes:
· Research Question: Small molecule inhibitor of AlkBH protein might render cancer cells more succeptible to DNA damage by conventional DNA-damaging drugs.
· Result: Using the high-throughput assays that we developed, we are trying to develop of new inhibitors DNA repair enzymes AlkB, AlkBH2 and AlkBH3. We have developed natural product indenone-based small molecule inhibitor of AlkBH3
· Publication details: See publication [12].
Presently our lab is pursuing two major research interests:
(A) Understanding the mechanism of DNA alkylation repair in human ALKBH enzymes
(B) Increasing efficacy of alkylating cancer drugs by inhibition of ALKBH enzymes
Important Publications
[1] R. Nigam, R. Anindya, Escherichia coli single-stranded DNA binding protein SSB promotes AlkB-mediated DNA dealkylation repair, Biochemical and biophysical research communications, 496 (2018) 274-279.
[2] M. Mohan, V. Pandya, R. Anindya, Escherichia coli AlkB and single-stranded DNA binding protein SSB interaction explored by Molecular Dynamics Simulation, J Mol Graph Model, 84 (2018) 29-35.
[3] R. Nigam, M. Mohan, G. Shivange, P.K. Dewangan, R. Anindya, Escherichia coli AlkB interacts with single-stranded DNA binding protein SSB by an intrinsically disordered region of SSB, Molecular biology reports, 45 (2018) 865-870.
[4] G. Shivange, M. Monisha, R. Nigam, N. Kodipelli, R. Anindya, RecA stimulates AlkB-mediated direct repair of DNA adducts, Nucleic acids research, 44 (2016) 8754-8763.
[5] R. Anindya, Non-heme dioxygenases in tumor hypoxia: They're all bound with the same fate, DNA Repair (Amst), 49 (2017) 21-25.
[6] A. Deepa, K. Naveena, R. Anindya, DNA repair activity of Fe(II)/2OG-dependent dioxygenases affected by low iron level in Saccharomyces cerevisiae, FEMS Yeast Res, 18 (2018).
[7] R. Anindya, Single-stranded DNA damage: Protecting the single-stranded DNA from chemical attack, DNA Repair (Amst), 87 (2020) 102804.
[8] M. Mohan, D. Akula, A. Dhillon, A. Goyal, R. Anindya, Human RAD51 paralogue RAD51C fosters repair of alkylated DNA by interacting with the ALKBH3 demethylase, Nucleic acids research, 47 (2019) 11729-11745.
[9] G. Shivange, N. Kodipelli, R. Anindya, A nonradioactive restriction enzyme-mediated assay to detect DNA repair by Fe(II)/2-oxoglutarate-dependent dioxygenase, Anal Biochem, 465 (2014) 35-37.
[10] G. Shivange, N. Kodipelli, M. Monisha, R. Anindya, A role for Saccharomyces cerevisiae Tpa1 protein in direct alkylation repair, The Journal of biological chemistry, 289 (2014) 35939-35952.
[11] G. Shivange, N. Kodipelli, R. Anindya, 2-Hydrazinobenzothiazole-based etheno-adduct repair protocol (HERP): a method for quantitative determination of direct repair of etheno-bases, DNA Repair (Amst), 28 (2015) 8-13.
[12] R. Nigam, K.R. Babu, T. Ghosh, B. Kumari, D. Akula, S.N. Rath, P. Das, R. Anindya, F.A. Khan, Indenone derivatives as inhibitor of human DNA dealkylation repair enzyme AlkBH3, Bioorg Med Chem, 26 (2018) 4100-4112.