Dissociation Dynamics of XPC-RAD23B from Damaged DNA Is a Determining Factor of NER Efficiency

XPC-RAD23B (XPC) plays a critical role in human nucleotide excision repair (hNER) as this complex recognizes DNA adducts to initiate NER. To determine the mutagenic potential of structurally different bulky DNA damages, various studies have been conducted to define the correlation of XPC-DNA damage equilibrium binding affinity with NER efficiency. However, little is known about the effects of XPC-DNA damage recognition kinetics on hNER. Although association of XPC is important, our current work shows that the XPC-DNA dissociation rate also plays a pivotal role in achieving NER efficiency.We characterized for the first time the binding of XPC to mono- versus di-AAF-modified sequences by using the real time monitoring surface plasmon resonance technique. Strikingly, the half-life (t1/2 or the retention time of XPC in association with damaged DNA) shares an inverse relationship with NER efficiency. This is particularly true when XPC remained bound to clustered adducts for a much longer period of time as compared to mono-adducts. Our results suggest that XPC dissociation from the damage site could become a rate-limiting step in NER of certain types of DNA adducts, leading to repression of NER

Dissociation Dynamics of XPC-RAD23B From Damaged Dna Is a Determining Factor of NER Efficiency

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. XPC-RAD23B (XPC) plays a critical role in human nucleotide excision repair (hNER) as this complex recognizes DNA adducts to initiate NER. To determine the mutagenic potential of structurally different bulky DNA damages, various studies have been conducted to define the correlation of XPC-DNA damage equilibrium binding affinity with NER efficiency. However, little is known about the effects of XPC-DNA damage recognition kinetics on hNER. Although association of XPC is important, our current work shows that the XPC-DNA dissociation rate also plays a pivotal role in achieving NER efficiency. We characterized for the first time the binding of XPC to mono- versus di-AAF-modified sequences by using the real time monitoring surface plasmon resonance technique. Strikingly, the half-life (t1/2 or the retention time of XPC in association with damaged DNA) shares an inverse relationship with NER efficiency. This is particularly true when XPC remained bound to clustered adducts for a much longer period of time as compared to mono-adducts. Our results suggest that XPC dissociation from the damage site could become a rate-limiting step in NER of certain types of DNA adducts, leading to repression of NER

Evaluation of the complexation behaviour among functionalized diphenyl viologens and cucurbit[7] and [8]urils

Abstract The complexation behaviour of Diphenyl viologens (DPVs) with Cucurbit[n]urils (CB[n]) was evaluated in detail and the results were reported. In this work, we present the synthesis of various DPVs functionalised with electron withdrawing and electron donating groups (EWGs & EDGs) and investigate their complexation behaviour with CB[7] and CB [8]. Carboxylic acid functionalized DPV’s (DPV-COOH) complexation with CB[8] gives additional insights, i.e., indicates hydrogen bonding plays an effective role in the complexation. The formation of a 2:2 quaternary complex of DPV-COOH/CB[8] under neutral pH conditions was supported by various analytical techniques. The complexation of DPVs with CB[7] specifies that irrespective of the functional group attached, they all form a 1:2 ternary complex, but the findings elaborate that the pattern followed in the complexation depends on the EW or EDG attached to the DPVs. The competition experiments conducted between functionalized DPVs and CB[7], CB[8] shows that they have more affinity towards CB[8] than CB[7] because of the better macrocyclic confinement effect of CB[8], as confirmed using UV–Vis spectroscopy. The binding affinity among EWG and EDG functionalised DPVs with CB[8] concludes EDG functionalised DPVs show better affinity towards CB[8], because they can form a charge transfer complex inside the CB[8] cavity. Exploring these host–guest interactions in more complex biological or environmental settings and studying their impact on the functionality of DPVs could be an exciting avenue for future research

Conformational insights into the lesion and sequence effects for arylamine-induced translesion DNA synthesis: \u3csup\u3e19\u3c/sup\u3eF NMR, surface plasmon resonance, and primer kinetic studies

Adduct-induced DNA damage can affect transcription efficiency and DNA replication and repair. We previously investigated the effects of the 3′-next flanking base (GCT vs GCA; G, FABP, N-(2′-deoxyguanosin-8- yl)-4′-fluoro-4-aminobiphenyl; FAF, N-(2′-deoxyguanosin-8-yl)-7- fluoro-2-aminofluorene) on the conformation of arylamine-DNA lesions in relation to E. coli nucleotide excision repair (Jain, V., Hilton, B., Lin, B., Patnaik, S., Liang, F., Darian, E., Zou, Y., Mackerell, A. D., Jr., and Cho, B. P. (2013) Nucleic Acids Res., 41, 869-880). Here, we report the differential effects of the same pair of sequences on DNA replication in vitro by the polymerases exofree Klenow fragment (Kf-exo-) and Dpo4. We obtained dynamic 19F NMR spectra for two 19-mer modified templates during primer elongation: GCA [d(5′-CTTACCATCGCAACCATTC-3′)] and GCT [d(5′-CTTACCATCGCTACCATTC-3′)]. We found that lesion stacking is favored in the GCT sequence compared to the GCA counterpart. Surface plasmon resonance binding results showed consistently weaker affinities for the modified DNA with the binding strength in the order of FABP \u3e FAF and GCA \u3e GCT. Primer extension was stalled at (n) and near (n - 1 and n + 1) the lesion site, and the extent of blockage and the extension rates across the lesion were influenced by not only the DNA sequences but also the nature of the adduct\u27s chemical structure (FAF vs FABP) and the polymerase employed (Kf-exo- vs Dpo4). Steady-state kinetics analysis with Kf-exo- revealed the most dramatic sequence and lesion effects at the lesion (n) and postinsertion (n + 1) sites, respectively. Taken together, these results provide insights into the important role of lesion-induced conformational heterogeneity in modulating translesion DNA synthesis. © 2014 American Chemical Society

Synthesis, spectral characterization, protein binding and cytotoxic evaluation of new cobalt(II) and cobalt(III) complexes containing benzimidazolylterpyridine as ligand

Two new six-coordinate cobalt(II) [Co(bitpy)2]Cl2 (1) and cobalt(III) [Co(bitpy)2](ClO4)3 (2) complexes containing benzimidazolylterpyridine (bitpy) ligand have been synthesized and characterized using spectroscopic and electrochemical techniques. The g‖ and g⊥ values in the EPR spectrum for 1 were 2.215 and 2.012, respectively, and clearly authenticated the formation of stable octahedral low spin Co(II) meridional isomer complex. Similarly, 1H NMR spectrum of 2 revealed low spin and its diamagnetic nature. The interaction of the complexes with bovine serum albumin (BSA) has been investigated and revealed that the cobalt complexes quenched the intrinsic fluorescence by following the static quenching mechanism. MTT assay was used to check the anticancer potential of 2 against human breast cancer cells (MCF-7) and human cervical cancer cells (HeLa). The IC50 values of 2 were lower than 7 µM in both the cancer cells and higher than 100 µM for non-cancerous cells (human embryonic kidney cells), indicating the specificity of the complex toward cancer cells. Furthermore, the morphological changes induced by 2 on the tested cell lines were monitored by AO-EB (acridine orange-ethidium bromide) and DAPI (4′,6-diamidino-2-phenylindole) staining methods.</p

Oxidative Cleavage of DNA by Ruthenium(II) Complexes Containing a Ferrocene/Non-Ferrocene Conjugated Imidazole Phenol Ligand

Three mixed-ligand ruthenium­(II) complexes with general formula [Ru­(bpy)₂L]­(PF₆) (<b>1</b>–<b>3</b>), where L = 2-{4,5-bis­[(<i>E</i>)-2-ferrocenylvinyl]-1<i>H</i>-imidazol-2-yl}­phenol (<b>1</b>), 2-{4,5-bis­[(<i>E</i>)-2-ferrocenylvinyl]-1<i>H</i>-imidazol-2-yl)-4,6-dichlorophenol (<b>2</b>), 2-{4,5-bis­[(<i>E</i>)-2-(4-chlorophenyl)­ethenyl]-1<i>H</i>-imidazol-2-yl}­phenol (<b>3</b>), have been synthesized and characterized. All the three complexes bring about DNA cleavage in the presence of H₂O₂. Due to the presence of three redox-active metal centers in complexes <b>1</b> and <b>2</b> these two complexes show enhanced DNA cleaving activity in comparison to that exhibited by complex <b>3</b>, which contains only one redox-active metal center

Oxidative Cleavage of DNA by Ruthenium(II) Complexes Containing a Ferrocene/Non-Ferrocene Conjugated Imidazole Phenol Ligand

Three mixed-ligand ruthenium­(II) complexes with general formula [Ru­(bpy)₂L]­(PF₆) (<b>1</b>–<b>3</b>), where L = 2-{4,5-bis­[(<i>E</i>)-2-ferrocenylvinyl]-1<i>H</i>-imidazol-2-yl}­phenol (<b>1</b>), 2-{4,5-bis­[(<i>E</i>)-2-ferrocenylvinyl]-1<i>H</i>-imidazol-2-yl)-4,6-dichlorophenol (<b>2</b>), 2-{4,5-bis­[(<i>E</i>)-2-(4-chlorophenyl)­ethenyl]-1<i>H</i>-imidazol-2-yl}­phenol (<b>3</b>), have been synthesized and characterized. All the three complexes bring about DNA cleavage in the presence of H₂O₂. Due to the presence of three redox-active metal centers in complexes <b>1</b> and <b>2</b> these two complexes show enhanced DNA cleaving activity in comparison to that exhibited by complex <b>3</b>, which contains only one redox-active metal center

Dissociation Dynamics of XPC-RAD23B from Damaged DNA Is a Determining Factor of NER Efficiency.

XPC-RAD23B (XPC) plays a critical role in human nucleotide excision repair (hNER) as this complex recognizes DNA adducts to initiate NER. To determine the mutagenic potential of structurally different bulky DNA damages, various studies have been conducted to define the correlation of XPC-DNA damage equilibrium binding affinity with NER efficiency. However, little is known about the effects of XPC-DNA damage recognition kinetics on hNER. Although association of XPC is important, our current work shows that the XPC-DNA dissociation rate also plays a pivotal role in achieving NER efficiency. We characterized for the first time the binding of XPC to mono- versus di-AAF-modified sequences by using the real time monitoring surface plasmon resonance technique. Strikingly, the half-life (t1/2 or the retention time of XPC in association with damaged DNA) shares an inverse relationship with NER efficiency. This is particularly true when XPC remained bound to clustered adducts for a much longer period of time as compared to mono-adducts. Our results suggest that XPC dissociation from the damage site could become a rate-limiting step in NER of certain types of DNA adducts, leading to repression of NER

Conformational Insights into the Lesion and Sequence Effects for Arylamine-Induced Translesion DNA Synthesis: ¹⁹F NMR, Surface Plasmon Resonance, and Primer Kinetic Studies

Adduct-induced DNA damage can affect transcription efficiency and DNA replication and repair. We previously investigated the effects of the 3′-next flanking base (G*C<i><u>T</u></i> vs G*C<i><u>A</u></i>; G*, FABP, <i>N</i>-(2′-deoxyguanosin-8-yl)-4′-fluoro-4-aminobiphenyl; FAF, <i>N</i>-(2′-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene) on the conformation of arylamine-DNA lesions in relation to <i>E. coli</i> nucleotide excision repair (Jain, V., Hilton, B., Lin, B., Patnaik, S., Liang, F., Darian, E., Zou, Y., Mackerell, A. D., Jr., and Cho, B. P. (2013) Nucleic Acids Res., 41, 869−880). Here, we report the differential effects of the same pair of sequences on DNA replication <i>in vitro</i> by the polymerases exofree Klenow fragment (Kf-exo^–) and Dpo4. We obtained dynamic ¹⁹F NMR spectra for two 19-mer modified templates during primer elongation: G*C<i>A</i> [d­(5′-CTTACCATCG*CAACCATTC-3′)] and G*C<i>T</i> [d­(5′-CTTACCATCG*CTACCATTC-3′)]. We found that lesion stacking is favored in the G*C<i>T</i> sequence compared to the G*C<i>A</i> counterpart. Surface plasmon resonance binding results showed consistently weaker affinities for the modified DNA with the binding strength in the order of FABP > FAF and G*C<i><u>A</u></i> > G*C<i><u>T</u></i>. Primer extension was stalled at (<i>n</i>) and near (n – 1 and <i>n</i> + 1) the lesion site, and the extent of blockage and the extension rates across the lesion were influenced by not only the DNA sequences but also the nature of the adduct’s chemical structure (FAF vs FABP) and the polymerase employed (Kf-exo^– vs Dpo4). Steady-state kinetics analysis with Kf-exo^– revealed the most dramatic sequence and lesion effects at the lesion (<i>n</i>) and postinsertion (<i>n</i> + 1) sites, respectively. Taken together, these results provide insights into the important role of lesion-induced conformational heterogeneity in modulating translesion DNA synthesis