TY  - JOUR
A1  - Tessmer, Ingrid
A1  - Melikishvili, Manana
A1  - Fried, Michael G.
T1  - Cooperative cluster formation, DNA bending and base-flipping by O\(^6\)-alkylguanine-DNA alkyltransferase
JF  - Nucleic Acids Research
N2  - O\(^6\)-Alkylguanine-DNA alkyltransferase (AGT) repairs mutagenic O\(^6\)-alkylguanine and O\(^4\)-alkylthymine adducts in DNA, protecting the genome and also contributing to the resistance of tumors to chemotherapeutic alkylating agents. AGT binds DNA cooperatively, and cooperative interactions are likely to be important in lesion search and repair. We examined morphologies of complexes on long, unmodified DNAs, using analytical ultracentrifugation and atomic force microscopy. AGT formed clusters of 11 proteins. Longer clusters, predicted by the McGhee-von Hippel model, were not seen even at high [protein]. Interestingly, torsional stress due to DNA unwinding has the potential to limit cluster size to the observed range. DNA at cluster sites showed bend angles (similar to 0, similar to 30 and similar to 60 degrees) that are consistent with models in which each protein induces a bend of similar to 30 degrees. Distributions of complexes along the DNA are incompatible with sequence specificity but suggest modest preference for DNA ends. These properties tell us about environments in which AGT may function. Small cooperative clusters and the ability to accommodate a range of DNA bends allow function where DNA topology is constrained, such as near DNA-replication complexes. The low sequence specificity allows efficient and unbiased lesion search across the entire genome.
KW  - inactivation
KW  - nucleotide excision-repair
KW  - atomic-force microscopy
KW  - noncooperative binding
KW  - restricition enzymes
KW  - complex stability
KW  - stranded DNAs
KW  - protein
KW  - chemotherapy
KW  - AGT
Y1  - 2012
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-133949
VL  - 40
IS  - 17
ER  - 
TY  - JOUR
A1  - Khan, Irfan
A1  - Suhasini, Avvaru N.
A1  - Banerjee, Taraswi
A1  - Sommers, Joshua A.
A1  - Kaplan, Daniel L.
A1  - Kuper, Jochen
A1  - Kisker, Caroline
A1  - Brosh, Jr., Robert M.
T1  - Impact of Age-Associated Cyclopurine Lesions on DNA Repair Helicases
JF  - PLOS ONE
N2  - 8,5' cyclopurine deoxynucleosides (cPu) are locally distorting DNA base lesions corrected by nucleotide excision repair (NER) and proposed to play a role in neurodegeneration prevalent in genetically defined Xeroderma pigmentosum (XP) patients. In the current study, purified recombinant helicases from different classifications based on sequence homology were examined for their ability to unwind partial duplex DNA substrates harboring a single site-specific cPu adduct. Superfamily (SF) 2 RecQ helicases (RECQ1, BLM, WRN, RecQ) were inhibited by cPu in the helicase translocating strand, whereas helicases from SF1 (UvrD) and SF4 (DnaB) tolerated cPu in either strand. SF2 Fe-S helicases (FANCJ, DDX11 (ChlR1), DinG, XPD) displayed marked differences in their ability to unwind the cPu DNA substrates. Archaeal Thermoplasma acidophilum XPD (taXPD), homologue to the human XPD helicase involved in NER DNA damage verification, was impeded by cPu in the non-translocating strand, while FANCJ was uniquely inhibited by the cPu in the translocating strand. Sequestration experiments demonstrated that FANCJ became trapped by the translocating strand cPu whereas RECQ1 was not, suggesting the two SF2 helicases interact with the cPu lesion by distinct mechanisms despite strand-specific inhibition for both. Using a protein trap to simulate single-turnover conditions, the rate of FANCJ or RECQ1 helicase activity was reduced 10-fold and 4.5-fold, respectively, by cPu in the translocating strand. In contrast, single-turnover rates of DNA unwinding by DDX11 and UvrD helicases were only modestly affected by the cPu lesion in the translocating strand. The marked difference in effect of the translocating strand cPu on rate of DNA unwinding between DDX11 and FANCJ helicase suggests the two Fe-S cluster helicases unwind damaged DNA by distinct mechanisms. The apparent complexity of helicase encounters with an unusual form of oxidative damage is likely to have important consequences in the cellular response to DNA damage and DNA repair.
KW  - nucleotide excision-repair
KW  - replication fork
KW  - substrate specificity
KW  - translesion synthesis
KW  - genomic stability
KW  - Warsaw breakage syndrome
KW  - escherichia coli
KW  - xeroderma-pigmentosum
KW  - human cells
KW  - biochemical characterization
Y1  - 2014
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-114635
VL  - 9
IS  - 11
ER  -