Replication Past the γ-Radiation-Induced Guanine-Thymine Cross-Link G[8,5-Me]T by Human and Yeast DNA Polymerase η

γ-Radiation-induced intrastrand guanine-thymine cross-link, G[8,5-Me]T, hinders replication in vitro and is mutagenic in mammalian cells. Herein we report in vitro translesion synthesis of G[8,5-Me]T by human and yeast DNA polymerase η (hPol η and yPol η). dAMP misincorporation opposite the cross-linked G by yPol η was preferred over correct incorporation of dCMP, but further extension was 100-fold less efficient for G∗:A compared to G∗:C. For hPol η, both incorporation and extension were more efficient with the correct nucleotides. To evaluate translesion synthesis in the presence of all four dNTPs, we have developed a plasmid-based DNA sequencing assay, which showed that yPol η was more error-prone. Mutational frequencies of yPol η and hPol η were 36% and 14%, respectively. Targeted G → T was the dominant mutation by both DNA polymerases. But yPol η induced targeted G → T in 23% frequency relative to 4% by hPol η. For yPol η, targeted G → T and G → C constituted 83% of the mutations. By contrast, with hPol η, semi-targeted mutations (7.2%), that is, mutations at bases near the lesion, occurred at equal frequency as the targeted mutations (6.9%). The kind of mutations detected with hPol η showed significant similarities with the mutational spectrum of G[8,5-Me]T in human embryonic kidney cells

Inhibition of DNA replication fork progression and mutagenic potential of 1, N6-ethenoadenine and 8-oxoguanine in human cell extracts

Comparative mutagenesis of 1,N6-ethenoadenine (εA) and 8-oxoguanine (8-oxoG), two endogenous DNA lesions that are also formed by exogenous DNA damaging agents, have been evaluated in HeLa and xeroderma pigmentosum variant (XPV) cell extracts. Two-dimensional gel electrophoresis of the duplex M13mp2SV vector containing these lesions established that there was significant inhibition of replication fork movement past εA, whereas 8-oxoG caused only minor stalling of fork progression. In extracts of HeLa cells, εA was weakly mutagenic inducing all three base substitutions in approximately equal frequency, whereas 8-oxoG was 10-fold more mutagenic inducing primarily G→T transversions. These data suggest that 8-oxoG is a miscoding lesion that presents a minimal, if any, block to DNA replication in human cells. We hypothesized that bypass of εA proceeded principally by an error-free mechanism in which the undamaged strand was used as a template, since this lesion strongly blocked fork progression. To examine this, we determined the sequence of replication products derived from templates in which a G was placed across from the εA. Consistent with our hypothesis, 93% of the progeny were derived from replication of the undamaged strand. When translesion synthesis occurred, εA→T mutations increased 3-fold in products derived from the mismatched εA: G construct compared with those derived from the εA: T construct. More efficient repair of εA in the εA: T construct may have been responsible for lower mutation frequency. Primer extension studies with purified pol η have shown that this polymerase is highly error-prone when bypassing εA. To examine if pol η is the primary mutagenic translesion polymerase in human cells, we determined the lesion bypass characteristics of extracts derived from XPV cells, which lack this polymerase. The εA: T construct induced εA→G and εA→C mutant frequencies that were approximately the same as those observed using the HeLa extracts. However, εA→T events were increased 5-fold relative to HeLa extracts. These data support a model in which pol η-mediated translesion synthesis past this adduct is error-free in the context of semiconservative replication in the presence of fidelity factors such as PCNA

Genetic effects of oxidative DNA damages: comparative mutagenesis of the imidazole ring-opened formamidopyrimidines (Fapy lesions) and 8-oxo-purines in simian kidney cells

Fapy·dG and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) are formed in DNA by hydroxyl radical damage. In order to study replication past these lesions in cells, we constructed a single-stranded shuttle vector containing the lesion in 5′-TGT and 5′-TGA sequence contexts. Replication of the modified vector in simian kidney (COS-7) cells showed that Fapy·dG is mutagenic inducing primarily targeted Fapy·G→T transversions. In the 5′-TGT sequence mutational frequency of Fapy·dG was ∼30%, whereas in the 5′-TGA sequence it was ∼8%. In parallel studies 8-oxo-dG was found to be slightly less mutagenic than Fapy·dG, though it also exhibited a similar context effect: 4-fold G→T transversions (24% versus 6%) occurred in the 5′-TGT sequence relative to 5′-TGA. To investigate a possible structural basis for the higher G→T mutations induced by both lesions when their 3′ neighbor was T, we carried out a molecular modeling investigation in the active site of DNA polymerase β, which is known to incorporate both dCTP (no mutation) and dATP (G→T substitution) opposite 8-oxo-G. In pol β, the syn-8-oxo-G:dATP pair showed greater stacking with the 3′-T:A base pair in the 5′-TGT sequence compared with the 3′-A:T in the 5′-TGA sequence, whereas stacking for the anti-8-oxo-G:dCTP pair was similar in both 5′-TGT and 5′-TGA sequences. Similarly, syn-Fapy·G:dATP pairing showed greater stacking in the 5′-TGT sequence compared with the 5′-TGA sequence, while stacking for anti-Fapy·G:dCTP pairs was similar in the two sequences. Thus, for both lesions less efficient base stacking between the lesion:dATP pair and the 3′-A:T base pair in the 5′-TGA sequence might cause lower G→T mutational frequencies in the 5′-TGA sequence compared to 5′-TGT. The corresponding lesions derived from 2′-deoxyadenosine, Fapy·dA and 8-oxo-dA, were not detectably mutagenic in the 5′-TAT sequence, and were only weakly mutagenic (<1%) in the 5′-TAA sequence context, where both lesions induced targeted A→C transversions. To our knowledge this is the first investigation using extrachromosomal probes containing a Fapy·dG or Fapy·dA site-specifically incorporated, which showed unequivocally that in simian kidney cells Fapy·G→T substitutions occur at a higher frequency than 8-oxo-G→T and that Fapy·dA is very weakly mutagenic, as is 8-oxo-dA

Genetic effects of oxidative DNA damages: comparative mutagenesis of the imidazole ring-opened formamidopyrimidines (Fapy lesions) and 8-oxo-purines in simian kidney cells

Fapy·dG and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) are formed in DNA by hydroxyl radical damage. In order to study replication past these lesions in cells, we constructed a single-stranded shuttle vector containing the lesion in 5′-TGT and 5′-TGA sequence contexts. Replication of the modified vector in simian kidney (COS-7) cells showed that Fapy·dG is mutagenic inducing primarily targeted Fapy·G→T transversions. In the 5′-TGT sequence mutational frequency of Fapy·dG was ∼30%, whereas in the 5′-TGA sequence it was ∼8%. In parallel studies 8-oxo-dG was found to be slightly less mutagenic than Fapy·dG, though it also exhibited a similar context effect: 4-fold G→T transversions (24% versus 6%) occurred in the 5′-TGT sequence relative to 5′-TGA. To investigate a possible structural basis for the higher G→T mutations induced by both lesions when their 3′ neighbor was T, we carried out a molecular modeling investigation in the active site of DNA polymerase β, which is known to incorporate both dCTP (no mutation) and dATP (G→T substitution) opposite 8-oxo-G. In pol β, the syn-8-oxo-G:dATP pair showed greater stacking with the 3′-T:A base pair in the 5′-TGT sequence compared with the 3′-A:T in the 5′-TGA sequence, whereas stacking for the anti-8-oxo-G:dCTP pair was similar in both 5′-TGT and 5′-TGA sequences. Similarly, syn-Fapy·G:dATP pairing showed greater stacking in the 5′-TGT sequence compared with the 5′-TGA sequence, while stacking for anti-Fapy·G:dCTP pairs was similar in the two sequences. Thus, for both lesions less efficient base stacking between the lesion:dATP pair and the 3′-A:T base pair in the 5′-TGA sequence might cause lower G→T mutational frequencies in the 5′-TGA sequence compared to 5′-TGT. The corresponding lesions derived from 2′-deoxyadenosine, Fapy·dA and 8-oxo-dA, were not detectably mutagenic in the 5′-TAT sequence, and were only weakly mutagenic (<1%) in the 5′-TAA sequence context, where both lesions induced targeted A→C transversions. To our knowledge this is the first investigation using extrachromosomal probes containing a Fapy·dG or Fapy·dA site-specifically incorporated, which showed unequivocally that in simian kidney cells Fapy·G→T substitutions occur at a higher frequency than 8-oxo-G→T and that Fapy·dA is very weakly mutagenic, as is 8-oxo-dA

Seasonal intercomparison of observational rainfall datasets over India during the southwest monsoon season

The Indian monsoon is an important component of Earth's climate system, accurate forecasting of its mean rainfall being essential for regional food and water security. Accurate measurement of the rainfall is essential for various water-related applications, the evaluation of numerical models and detection and attribution of trends, but a variety of different gridded rainfall datasets are available for these purposes. In this study, six gridded rainfall datasets are compared against the India Meteorological Department (IMD) gridded rainfall dataset, chosen as the most representative of the observed system due to its high gauge density. The datasets comprise those based solely on rain gauge observations and those merging rain gauge data with satellite-derived products. Various skill scores and subjective comparisons are carried out for the Indian region during the south-west monsoon season (June to September). Relative biases and skill metrics are documented at all-India and sub-regional scales. In the gauge-based (land-only) category, Asian Precipitation-Highly-Resolved Observational Data Integration Towards Evaluation of water resources (APHRODITE) and Global Precipitation Climatology Center (GPCC) datasets perform better relative to the others in terms of a variety of skill metrics. In the merged category, the Global Precipitation Climatology Project (GPCP) dataset is shown to perform better than the Climate Prediction Center Merged Analysis of Precipitation (CMAP) for the Indian monsoon in terms of various metrics, when compared with the IMD gridded data. Most of the datasets have difficulty in representing rainfall over orographic regions including the Western Ghats mountains, in north-east India and the Himalayan foothills. The wide range of skill scores seen among the datasets and even the change of sign of bias found in some years are causes of concern. This uncertainty between datasets is largest in north-east India. These results will help those studying the Indian monsoon region to select an appropriate dataset depending on their application and focus of research

Repair of mitomycin C mono- and interstrand cross-linked DNA adducts by UvrABC: a new model

Mitomycin C induces both MC-mono-dG and cross-linked dG-adducts in vivo. Interstrand cross-linked (ICL) dG-MC-dG-DNA adducts can prevent strand separation. In Escherichia coli cells, UvrABC repairs ICL lesions that cause DNA bending. The mechanisms and consequences of NER of ICL dG-MC-dG lesions that do not induce DNA bending remain unclear. Using DNA fragments containing a MC-mono-dG or an ICL dG-MC-dG adduct, we found (i) UvrABC incises only at the strand containing MC-mono-dG adducts; (ii) UvrABC makes three types of incisions on an ICL dG-MC-dG adduct: type 1, a single 5′ incision on 1 strand and a 3′ incision on the other; type 2, dual incisions on 1 strand and a single incision on the other; and type 3, dual incisions on both strands; and (iii) the cutting kinetics of type 3 is significantly faster than type 1 and type 2, and all of 3 types of cutting result in producing DSB. We found that UvrA, UvrA + UvrB and UvrA + UvrB + UvrC bind to MC-modified DNA specifically, and we did not detect any UvrB- and UvrB + UvrC–DNA complexes. Our findings challenge the current UvrABC incision model. We propose that DSBs resulted from NER of ICL dG-MC-dG adducts contribute to MC antitumor activity and mutations

(5′S)-8,5′-Cyclo-2′-deoxyguanosine Is a Strong Block to Replication, a Potent pol V-Dependent Mutagenic Lesion, and Is Inefficiently Repaired in Escherichia coli

8,5′-Cyclopurines, making up an important class of ionizing radiation-induced tandem DNA damage, are repaired only by nucleotide excision repair (NER). They accumulate in NER-impaired cells, as in Cockayne syndrome group B and certain Xeroderma Pigmentosum patients. A plasmid containing (5′S)-8,5′-cyclo-2′-deoxyguanosine (S-cdG) was replicated in Escherichia coli with specific DNA polymerase knockouts. Viability was \u3c1% in the wild-type strain, which increased to 5.5% with SOS. Viability decreased further in a pol II- strain, whereas it increased considerably in a pol IV- strain. Remarkably, no progeny was recovered from a pol V- strain, indicating that pol V is absolutely required for bypassing S-cdG. Progeny analyses indicated that S-cdG is significantly mutagenic, inducing ∼34% mutation with SOS. Most mutations were S-cdG → A mutations, though S-cdG → T mutation and deletion of 5′C also occurred. Incisions of purified UvrABC nuclease on S-cdG, S-cdA, and C8-dG-AP on a duplex 51-mer showed that the incision rates are C8-dG-AP \u3e S-cdA \u3e S-cdG. In summary, S-cdG is a major block to DNA replication, highly mutagenic, and repaired slowly in E. coli

"The fruits of independence": Satyajit Ray, Indian nationhood and the spectre of empire

Challenging the longstanding consensus that Satyajit Ray's work is largely free of ideological concerns and notable only for its humanistic richness, this article shows with reference to representations of British colonialism and Indian nationhood that Ray's films and stories are marked deeply and consistently by a distinctively Bengali variety of liberalism. Drawn from an ongoing biographical project, it commences with an overview of the nationalist milieu in which Ray grew up and emphasizes the preoccupation with colonialism and nationalism that marked his earliest unfilmed scripts. It then shows with case studies of Kanchanjangha (1962), Charulata (1964), First Class Kamra (First-Class Compartment, 1981), Pratidwandi (The Adversary, 1970), Shatranj ke Khilari (The Chess Players, 1977), Agantuk (The Stranger, 1991) and Robertsoner Ruby (Robertson's Ruby, 1992) how Ray's mature work continued to combine a strongly anti-colonial viewpoint with a shifting perspective on Indian nationhood and an unequivocal commitment to cultural cosmopolitanism. Analysing how Ray articulated his ideological positions through the quintessentially liberal device of complexly staged debates that were apparently free, but in fact closed by the scenarist/director on ideologically specific notes, this article concludes that Ray's reputation as an all-forgiving, ‘everybody-has-his-reasons’ humanist is based on simplistic or even tendentious readings of his work

Repair of mitomycin C mono- and interstrand cross-linked DNA adducts by UvrABC: a new model

Mitomycin C induces both MC-mono-dG and cross-linked dG-adducts in vivo. Interstrand cross-linked (ICL) dG-MC-dG-DNA adducts can prevent strand separation. In Escherichia coli cells, UvrABC repairs ICL lesions that cause DNA bending. The mechanisms and consequences of NER of ICL dG-MC-dG lesions that do not induce DNA bending remain unclear. Using DNA fragments containing a MC-mono-dG or an ICL dG-MC-dG adduct, we found (i) UvrABC incises only at the strand containing MC-mono-dG adducts; (ii) UvrABC makes three types of incisions on an ICL dG-MC-dG adduct: type 1, a single 5′ incision on 1 strand and a 3′ incision on the other; type 2, dual incisions on 1 strand and a single incision on the other; and type 3, dual incisions on both strands; and (iii) the cutting kinetics of type 3 is significantly faster than type 1 and type 2, and all of 3 types of cutting result in producing DSB. We found that UvrA, UvrA + UvrB and UvrA + UvrB + UvrC bind to MC-modified DNA specifically, and we did not detect any UvrB- and UvrB + UvrC–DNA complexes. Our findings challenge the current UvrABC incision model. We propose that DSBs resulted from NER of ICL dG-MC-dG adducts contribute to MC antitumor activity and mutations