Nigam, Richa and Roy, Anindya
(2019)
Functional characterization of DNA dealkylation repair
protein AlkB from Escherichia coli
and use of indenone derivatives as inhibitors of AlkB and
human AlkB homologue 3 (AlkBH3).
PhD thesis, Indian institute of technology Hyderabad.
Abstract
DNA alkylation damage, emanating from the exposure to environmental alkylating
agents or produced by certain endogenous metabolic processes, affects cell viability
and genomic stability. Fe(II)/2-oxoglutarate-dependent dioxygenase enzymes, such
as Escherichia coli (E coli) AlkB is involved in protecting DNA from alkylation
damage. Previous studies have shown that AlkB preferentially removes alkyl DNA
adducts from single-stranded DNA (ssDNA). 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. Therefore, AlkB-mediated DNA repair using SSBbound DNA as substrate is studied in the present thesis. By using in vitro repair
reaction, it is found that AlkB can efficiently remove N3-methyl Cytosine (N3meC)
adducts inasmuch as DNA length is shorter than 20 nucleotides. However, when
longer N3meC-containing oligonuleotides were used as the substrate, efficiency of
AlkB catalyzed reaction is found to be abated compared to SSB-bound DNA
substrate of identical length. It is also observed here that truncated SSB containing
only the DNA binding domain can support the stimulation of AlkB activity,
suggesting the importance of SSB-DNA interaction for AlkB function. Using 70-
mer oligonucleotide containing single N3meC, it is demonstrated that SSB-AlkB
interaction promotes faster repair of the methyl DNA adducts.
Intrinsically disordered regions (IDRs) of proteins often regulate function
through interactions with folded domains. E. coli single-stranded DNA binding
protein SSB binds and stabilizes ssDNA. The N-terminal of SSB contains
characteristic oligonucleotide/oligosaccharide-binding (OB) fold which binds
ssDNA tightly but non-specifically. SSB also forms complexes with large number
proteins via the C-terminal interaction domain consisting mostly of acidic amino
xi
acid residues. The amino acid residues located between the OB-fold and C-terminal
acidic domain are known to constitute an intrinsically disordered region (IDR) and
no functional significance has been attributed to this region. Although SSB is known
to bind many DNA repair protein, it is not known whether it binds to DNA
dealkylation repair protein AlkB. Therefore, AlkB-SSB interaction is characterized
in detail in this thesis. It is demonstrated by in vitro pull-down and yeast two-hybrid
analysis that SSB binds to AlkB via the IDR. The site of contact is found to be the
residues 152-169 of SSB. The present study also reveals that unlike most of the
SSB-binding proteins which utilize C-terminal acidic domain for interaction, IDR of
SSB is necessary and sufficient for AlkB interaction.
The human homologue of AlkB, AlkB homologue 3 (AlkBH3), is also a
member of the dioxygenase family of enzymes involved in DNA dealkylation repair.
Because of its role in promoting tumor cell proliferation and metastasis of cancer,
extensive efforts are being directed in developing selective inhibitors for AlkBH3.
The present thesis also reports screening and evaluation of panel of arylated
indenone derivatives as new class of inhibitors of AlkB and AlkBH3 DNA repair
activity. The indenone-derived AlkB and AlkBH3 inhibitors are found to display
specific binding and competitive mode of inhibition. It is demonstrated that AlkB
inhibitor has the ability to sensitize cells to methyl methane sulfonate (MMS) that
mainly produce DNA alkylation damage and AlkBH3 inhibitor can prevent the
proliferation of lung cancer cell line and enhance sensitivity to MMS.
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