A new structural insight into XPA–DNA interactions

XPA (xeroderma pigmentosum group A) protein is an essential factor for NER (nucleotide excision repair) which is believed to be involved in DNA damage recognition/verification, NER factor recruiting and stabilization of repair intermediates. Past studies on the structure of XPA have focused primarily on XPA interaction with damaged DNA. However, how XPA interacts with other DNA structures remains unknown though recent evidence suggest that these structures could be important for its roles in both NER and non-NER activities. Previously, we reported that XPA recognizes undamaged DNA ds/ssDNA (double-strand/single-strandDNA) junctions with a binding affinity much higher than its ability to bind bulky DNA damage. To understand how this interaction occurs biochemically we implemented a structural determination of the interaction using a MS-based protein footprinting method and limited proteolysis. By monitoring surface accessibility of XPA lysines to NHS-biotin modification in the free protein and the DNA junction-bound complex we show that XPA physically interacts with the DNA junctions via two lysines, K168 and K179, located in the previously known XPA(98–219) DBD (DNA-binding domain). Importantly, we also uncovered new lysine residues, outside of the known DBD, involved in the binding. We found that residues K221, K222, K224 and K236 in the C-terminal domain are involved in DNA binding. Limited proteolysis analysis of XPA–DNA interactions further confirmed this observation. Structural modelling with these data suggests a clamp-like DBD for the XPA binding to ds/ssDNA junctions. Our results provide a novel structure-function view of XPA–DNA junction interactions.     

A nonsense mutation in the Xeroderma pigmentosum complementation group F (XPF) gene is associated with gastric carcinogenesis

XPF/ERCC1 endonuclease is required for DNA lesion repair. To assess effects of a C2169A nonsense mutation in XPF at position 2169 in gastric cancer tissues and cell lines, genomic DNA was extracted from blood samples of 488 cancer patients and 64 gastric tumors. The mutation was mapped using a TaqMan MGB probe. In addition, gastric cancer cell lines were transfected with mutated XPF to explore XPF/ERCC1 interaction, XPF degradation, and DNA repair by a comet assay. The C2169A mutation was not detected in 488 samples of blood genomic DNA, yet was found in 32 of 64 gastric cancer tissue samples (50.0%), resulting in a 194C-terminal amino acid loss in XPF protein and lower expression. Laser micro-dissection confirmed that this point mutation was not present in surrounding normal tissues from the same patients. The truncated form of XPF (tXPF) impaired interaction with ERCC1, was rapidly degraded via ubiquitination, and resulted in reduced DNA repair. In gastric cancers, the mutation was monoallelic, indicating that XPF is a haplo-insufficient DNA repair gene. As the C2169A mutation is closely associated with gastric carcinogenesis in the Chinese population, our findings shine light on it as a therapeutic target for early diagnosis and treatment of gastric cancer.     

In vitro eradication of abasic site-mediated DNA–peptide/protein cross-links by Escherichia coli long-patch base excision repair

Apurinic/apyrimidinic (AP or abasic) sites are among the most abundant DNA lesions. Numerous proteins within different organisms ranging from bacteria to human have been demonstrated to react with AP sites to form covalent Schiff base DNA–protein cross-links (DPCs). These DPCs are unstable due to their spontaneous hydrolysis, but the half-lives of these cross-links can be as long as several hours. Such long-lived DPCs are extremely toxic due to their large sizes, which physically block DNA replication. Therefore, these adducts must be promptly eradicated to maintain genome integrity. Herein, we used in vitro reconstitution experiments with chemically synthesized, stable, and site-specific Schiff base AP-peptide/protein cross-link analogs to demonstrate for the first time that this type of DPC can be repaired by Escherichia coli (E. coli) long-patch base excision repair. We demonstrated that the repair process requires a minimum of three enzymes and five consecutive steps, including: (1) 5′-DNA strand incision of the DPC by endonuclease IV; (2 to 4) strand-displacement DNA synthesis, removal of the 5′-deoxyribose phosphate-peptide/protein adduct-containing flap, and gap-filling DNA synthesis by DNA polymerase I; and (5) strand ligation by a ligase. We further demonstrated that endonuclease IV plays a major role in incising an AP-peptide cross-link within E. coli cell extracts. We also report that eradicating model AP-protein (11.2–36.1 kDa) DPCs is less efficient than that of an AP-peptide_10mer cross-link, supporting the emerging model that proteolysis is likely required for efficient DPC repair.     

Laryngeal cancer risk and common single nucleotide polymorphisms in nucleotide excision repair pathway genes ERCC1, ERCC2, ERCC3, ERCC4, ERCC5 and XPA

Because the molecular mechanisms underlying the development of laryngeal cancer are not well understood, we conducted a case–control study to determine the association between eight common SNPs in NER pathway genes and risk of laryngeal cancer, and the association between genetic polymorphisms and environmental factors. A 1:1 matched case–control study of 176 cases and 176 controls was conducted. Laryngeal cancer cases were more likely to smoke and drink (all P values < 0.05). Subjects with the ERCC1 rs11615 CC genotype and C allele had an increased risk of laryngeal cancer. Similarly, individuals with the ERCC5 rs17655 GG genotype and G allele had an increased risk of laryngeal cancer. Gene–gene interaction analysis showed that subjects carrying ERCC1 rs11615 C allele and XPG/ERCC5 rs17655 G allele had a greatly increased risk of breast cancer. Stratified analysis revealed that the interaction between polymorphisms of ERCC1 rs11615 and ERCC5 rs17655 and smoking on cancer risk was statistically significant, and ERCC1 rs11615 polymorphisms also had a significant interaction with drinking habit. In conclusion, our study suggests that ERCC1 rs11615 and ERCC5 rs17655 polymorphisms are associated with increased risk of laryngeal cancer, and that they confer more risk among smokers and drinkers.     

siRNA‐induced CD44 knockdown suppresses the proliferation and invasion of colorectal cancer stem cells through inhibiting epithelial–mesenchymal transition

CD44 has shown prognostic values and promising therapeutic potential in multiple human cancers; however, the effects of CD44 silencing on biological behaviors of cancer stem cells (CSCs) have not been fully understood in colorectal cancer. To examine the contribution of siRNA‐induced knockdown of CD44 to the biological features of colorectal CSCs, colorectal CSCs HCT116‐CSCs were generated, and CD44 was knocked down in HCT116‐CSCs using siRNA. The proliferation, migration and invasion of HCT116‐CSCs were measured, and apoptosis and cell‐cycle analyses were performed. The sensitivity of HCT116‐CSCs to oxaliplatin was tested, and xenograft tumor growth assay was performed to examine the role of CD44 in HCT116‐CSCs tumorigenesis in vivo. In addition, the expression of epithelial–mesenchymal transition (EMT) markers E‐cadherin, N‐cadherin and vimentin was quantified. siRNA‐induced knockdown of CD44 was found to inhibit the proliferation, migration and invasion, induce apoptosis, promote cell‐cycle arrest at the G1/G0 phase and increase the sensitivity of HCT116‐CSCs to oxaliplatin in HCT116‐CSCs, and knockdown of CD44 suppressed in vivo tumorigenesis and intrapulmonary metastasis of HCT116‐CSCs. Moreover, silencing CD44 resulted in EMT inhibition. Our findings demonstrate that siRNA‐induced CD44 knockdown suppresses the proliferation, invasion and in vivo tumorigenesis and metastasis of colorectal CSCs by inhibiting EMT.     

Repair of DNA damage induced by anthanthrene, a polycyclic aromatic hydrocarbon (PAH) without bay or fjord regions

Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants, formed during incomplete burning of coal, oil and gas. Several PAHs have carcinogenic and mutagenic potencies, but these compounds must be activated in order to exert their mutagenic effects. One of the principal pathways proposed for metabolic activation of PAHs involves the cytochrome P450 enzymes. The DNA damaging potential of cytochrome P450-activated PAHs is generally associated with their bay and fjord regions, and the DNA repair response of PAHs containing such regions has been thoroughly studied. However, little is known about the repair of DNA damage resulting from metabolites from PAHs without bay and fjord regions. We have investigated the six-ringed PAH anthanthrene (dibenzo[def,mno]chrysene), which does not posses bay or fjord motifs. We analyzed the repair profile of human cell extracts and of cell cultures in response to DNA damage induced by cytochrome P450-activated anthanthrene. In cell extracts, functional nucleotide excision repair (NER) and mismatch repair (MMR) activities were necessary to trigger a response to anthanthrene metabolite-induced DNA damage. In cell cultures, NER was responsible for the repair of anthanthrene metabolite-induced DNA damage. However, when the NER pathway was inactivated, a residual repair pathway performed the DNA repair.     

Genetic polymorphisms in translesion synthesis genes are associated with colorectal cancer risk and metastasis in Han Chinese

Translesion synthesis (TLS) polymerases have low processivity and fidelity compared with replicative polymerases. Defective function of TLS polymerases result in chromosome instability. The aim of this study was to evaluate the effects of TLS genes on susceptibility and metastasis in colorectal cancer (CRC). Four single nucleotide polymorphisms (SNPs) (rs462779, rs11153292, rs373572 and rs2233004) of TLS genes were genotyped in the pilot cohort consisted of 516 patients with CRC and 503 controls, and then replicated in the replication cohort of 421 cases and 446 controls. The genotype frequencies of rs462779 and rs373572 were significantly different between CRC patients and controls in both two cohorts, even after it was adjusted by age, gender and smoking status. Stratified analysis showed that rs462779 and rs373572 were significantly associated with both colon and rectum cancer. In patients with metastatic CRC, the frequency of AA genotype of rs373572 was significantly increased as compared with those without metastasis CRC (P=0.001). Furthermore, rs462779 and rs373572 exhibited remarkably cumulative effect on the risk of CRC (trend P value=0.001). No significant difference was observed between other SNPs and CRC. These results suggest that polymorphisms in TLS genes are associated with susceptibility to CRC in Chinese and might be a novel biomarker for the predication of metastasis risk of CRC.     

Effects of chemical modification on the potency,serum stability,and immunostimulatory properties of short shRNAs

Small hairpin RNAs (shRNAs) with 19-base-pair, or shorter, stems (short shRNAs [sshRNAs]) have been found to constitute a class whose mechanism of action appears to be distinct from that of small interfering RNAs (siRNAs) or longer shRNAs. These sshRNAs can be as active as canonical siRNAs or longer shRNAs. Their activity is affected by whether the antisense strand is positioned 5′ or 3′ to the loop (L or R sshRNAs, respectively). Dicer seems not to be involved in the processing of sshRNAs, although the mechanism of target gene suppression by these hairpins is through Ago2-mediated mRNA cleavage. In this study, the effects of chemical modifications on the potency, serum stability, and innate immune response of sshRNAs were investigated. Deoxynucleotide substitution and 2′-O-methyl (2′-OMe) modification in the sense strand and loop did not affect silencing activity, but, unlike with siRNAs, when placed in the antisense strand these modifications were detrimental. Conjugation with bulky groups at the 5′-end of L sshRNAs or 3′-end of R sshRNAs had a negative impact on the potency. Unmodified sshRNAs in dimer form or with blunt ends were immunostimulatory. Some modifications such as 3′-end conjugation and phosphorothioate linkages on the backbone of the sshRNAs could also induce inflammatory cytokine production. However, 2′-OMe substitution of sshRNAs abrogated the innate immune response and improved the serum stability of the hairpins.     

An EMT spectrum defines an anoikis-resistant and spheroidogenic intermediate mesenchymal state that is sensitive to e-cadherin restoration by a src-kinase inhibitor,saracatinib (AZD0530)

The phenotypic transformation of well-differentiated epithelial carcinoma into a mesenchymal-like state provides cancer cells with the ability to disseminate locally and to metastasise. Different degrees of epithelial–mesenchymal transition (EMT) have been found to occur in carcinomas from breast, colon and ovarian carcinoma (OC), among others. Numerous studies have focused on bona fide epithelial and mesenchymal states but rarely on intermediate states. In this study, we describe a model system for appraising the spectrum of EMT using 43 well-characterised OC cell lines. Phenotypic EMT characterisation reveals four subgroups: Epithelial, Intermediate E, Intermediate M and Mesenchymal, which represent different epithelial–mesenchymal compositions along the EMT spectrum. In cell-based EMT-related functional studies, OC cells harbouring an Intermediate M phenotype are characterised by high N-cadherin and ZEB1 expression and low E-cadherin and ERBB3/HER3 expression and are more anoikis-resistant and spheroidogenic. A specific Src-kinase inhibitor, Saracatinib (AZD0530), restores E-cadherin expression in Intermediate M cells in in vitro and in vivo models and abrogates spheroidogenesis. We show how a 33-gene EMT Signature can sub-classify an OC cohort into four EMT States correlating with progression-free survival (PFS). We conclude that the characterisation of intermediate EMT states provides a new approach to better define EMT. The concept of the EMT Spectrum allows the utilisation of EMT genes as predictive markers and the design and application of therapeutic targets for reversing EMT in a selective subgroup of patients.     

Genetic polymorphisms of xeroderma pigmentosum group D gene Asp312Asn and Lys751Gln and susceptibility to prostate cancer: A systematic review and meta-analysis

Many studies have reported the role of xeroderma pigmentosum group D (XPD) with prostate cancer risk, but the results remained controversial. To derive a more precise estimation of the relationship, a meta-analysis was performed. Odds ratios (ORs) with 95% confidence intervals (CIs) were estimated to assess the association between XPD Asp312Asn and Lys751Gln polymorphisms and prostate cancer risk. A total of 8 studies including 2620 cases and 3225 controls described Asp312Asn genotypes, among which 10 articles involving 3230 cases and 3582 controls described Lys751Gln genotypes and were also involved in this meta-analysis. When all the eligible studies were pooled into this meta-analysis, a significant association between prostate cancer risk and XPD Asp312Asn polymorphism was found. For Asp312Asn polymorphism, in the stratified analysis by ethnicity and source of controls, prostate cancer risk was observed in co-dominant, dominant and recessive models, while no evidence of any associations of XPD Lys751Gln polymorphism with prostate cancer was found in the overall or subgroup analyses. Our meta-analysis supports that the XPD Asp312Asn polymorphism contributed to the risk of prostate cancer from currently available evidence. However, a study with a larger sample size is needed to further evaluate gene–environment interaction on XPD Asp312Asn and Lys751Gln polymorphisms and prostate cancer risk.     

Design,synthesis, and in vitro evaluation of BP-1-102 analogs with modified hydrophobic fragments for STAT3 inhibition

Twelve novel analogs of STAT3 inhibitor BP-1-102 were designed and synthesised with the aim to modify hydrophobic fragments of the molecules that are important for interaction with the STAT3 SH2 domain. The cytotoxic activity of the reference and novel compounds was evaluated using several human and two mouse cancer cell lines. BP-1-102 and its two analogs emerged as effective cytotoxic agents and were further tested in additional six human and two murine cancer cell lines, in all of which they manifested the cytotoxic effect in a micromolar range. Reference compound S3I-201.1066 was found ineffective in all tested cell lines, in contrast to formerly published data. The ability of selected BP-1-102 analogs to induce apoptosis and inhibition of STAT3 receptor-mediated phosphorylation was confirmed. The structure–activity relationship confirmed a demand for two hydrophobic substituents, i.e. the pentafluorophenyl moiety and another spatially bulky moiety, for effective cytotoxic activity and STAT3 inhibition.     

No evidence of association between the synonymous polymorphisms in XRCC1 and ERCC2 and breast cancer susceptibility among nonsmoking Chinese

DNA repair proficiency has also been proposed as a potential susceptibility factor for breast cancer. Synonymous polymorphism roles of the DNA repair genes in relation to breast cancer remain largely unknown. Nonsmokers are a good model in which to investigate genetic susceptibility to cancer because they are at low-dose carcinogen exposure. To validate genetic biomarkers of the disease, we explored the effects of the two synonymous polymorphisms [Pro206Pro (rs915927) and Arg156Arg (rs238406)] in the DNA repair genes XRCC1 and ERCC2 at chromosome 19q13.2-3 on breast cancer susceptibility among nonsmoking Chinese. The study recruited 243 patients with breast cancer and 234 cancer-free controls matched to the cases by age (±3years), gender, nonsmoking status and ethnicity. Genotypes were determined using polymerase chain reaction-restriction fragment length polymorphism. No associations were observed between both individual single nucleotide polymorphisms or haplotypes and breast cancer susceptibility. After stratification, no effects were detected for age-dependent effects or menopause status in relation to breast cancer occurrence. No evidence of gene-gene interaction in breast cancer susceptibility was revealed. The two loci were at weak linkage disequilibrium (D' value=0.244, P=0.07). The present data suggest that XRCC1 Pro206Pro and ERCC2 Arg156Arg do not substantially influence breast cancer susceptibility among nonsmoking Chinese.     

Viruses,cancer and non-self recognition

Virus–host interactions form an essential part of every aspect of life, and this review is aimed at looking at the balance between the host and persistent viruses with a focus on the immune system. The virus–host interaction is like a cat-and-mouse game and viruses have developed ingenious mechanisms to manipulate cellular pathways, most notably the major histocompatibility (MHC) class I pathway, to reside within infected cell while evading detection and destruction by the immune system. However, some of the signals sensing and responding to viral infection are derived from viruses and the fact that certain viruses can prevent the infection of others, highlights a more complex coexistence between the host and the viral microbiota. Viral immune evasion strategies also illustrate that processes whereby cells detect and present non-self genetic material to the immune system are interlinked with other cellular pathways. Immune evasion is a target also for cancer cells and a more detailed look at the interfaces between viral factors and components of the MHC class I peptide-loading complex indicates that these interfaces are also targets for cancer mutations. In terms of the immune checkpoint, however, viral and cancer strategies appear different.     

CD151 promotes Colorectal Cancer progression by a crosstalk involving CEACAM6, LGR5 and Wnt signaling via TGFβ1

CD151 impacts various signaling pathways in different cancers, and promotes colorectal cancer (CRC) cell malignancy by yet undefined mechanisms. This study aimed to comprehensively assess CD151''s function in CRC. CD151 levels were significantly higher in CRC tissues and cells compared with controls in the tissue microarray. Cell viability, migration and invasion were suppressed by CD151 downregulation in CRC cells. Consistently, mouse xenografts were inhibited by CD151 silencing. RNA-seq revealed that multiple genes were significantly altered by CD151 knockdown in cultured CRC cells and xenografts. Particularly, transforming growth factor β1 (TGFβ1), carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) and leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) alongside CD151 were downregulated both in vitro and in vivo. Co-immunoprecipitation and mass spectrometry results were validated by qRT-PCR and immunoblot. Moreover, pull-down assay and immunofluorescence confirmed the associations of TGFβ1, CEACAM6 and LGR5 with CD151. This study demonstrated CEACAM6, LGR5 and Wnt pathway suppression by CD151 silencing might occur through TGFβ1 regulation, offering a comprehensive view of CD151''s roles in colorectal carcinogenesis. Our findings provide an insight into the CD151-involved signaling network in CRC oncogenesis, which could be utilized to design novel targeted therapies against CD151-based signaling in treatment for CRC.     

Diverse Ras-related GTPase DIRAS2, downregulated by PSMD2 in a proteasome-mediated way,inhibits colorectal cancer proliferation by blocking NF-κB signaling

Colorectal cancer (CRC) is the most common gastrointestinal cancer, with a high mortality rate but limited therapeutic targets. DIRAS family GTPase 2 (DIRAS2) is a member of the Ras-related small G-protein family whose biological functions and underlying mechanism in CRC remain poorly understood. In this study, we identified the crucial roles of DIRAS2 in CRC. DIRAS2 expression was downregulated in CRC and closely correlated with poor prognosis. Functionally, DIRAS2 inhibited CRC cell proliferation and affected cell-cycle protein expression. Mechanistically, DIRAS2 blocked nuclear factor kappa light-chain enhancer of activated B-cell signaling pathways, inducing G0/G1 arrest. Moreover, DIRAS2 interacted with 26S proteasome non-ATPase regulatory subunit 2, which facilitates the degradation of DIRAS2 in a proteasome-mediated way. Together, these results demonstrate potential functions of DIRAS2 as a tumor-suppressor gene in CRC and reveal a distinct mechanism of DIRAS2 in CRC tumorigenesis, indicating its role as a potential biomarker and target for CRC therapy.     

Promoter polymorphisms in DNA repair gene ERCC5 and susceptibility to gastric cancer in Chinese

Genetic variations in excision repair cross-complementing group 5 (ERCC5) might influence individual vulnerability to gastric cancer (GC). We investigated effects of two putatively functional polymorphisms in ERCC5 promoter region, rs751402 (+ 25A > G) and rs2296147 (+ 202C > T), and their potential interaction with environment factors on the risk of developing GC. We performed a sex- and age-matched case–control design with 400 GC cases and 400 healthy controls for rs751402 and 403 GC cases and 403 healthy controls for rs2296147. Our results showed that rs751402 were associated with increased GC risk (AA vs. GG: OR = 1.99, 95%CI: 1.20–3.31, P = 0.008; AG + AA vs. GG: OR = 1.41, 95%CI: 1.07–1.86, P = 0.016), and rs2296147 was also associated with increased cancer risk (CC vs. TT: OR = 2.17, 95%CI: 1.04–4.54, P = 0.039; CC vs. CT + TT: OR = 2.26, 95%CI: 1.09–4.69, P = 0.028). In a stratified analysis, rs751402 (AG + AA vs. GG: OR = 1.44, 95%CI: 1.02–2.02, P = 0.037) and rs2296147 (CC vs. CT + TT: OR = 2.33, 95%CI: 1.00–5.44, P = 0.050) were also found to be associated with diffuse-type GC risk. The most common GT haplotype (rs751402–rs2296147) showed protective effect for GC development (OR = 0.73, 95%CI: 0.58–0.91, P = 0.005), and especially for diffuse-type GC (OR = 0.68, 95%CI: 0.52–0.90, P = 0.006). Genetic effects on increased GC risk seemed to be enhanced by Helicobacter pylori infection, smoking and alcohol drinking, with corresponding adjusted ORs of 4.57, 2.42 and 2.50 for the rs751402 AG/AA variants, and of 5.32, 3.20 and 6.87 for the rs2296147 CC variant, but their interaction effects on GC risk didn't reach statistically significance. ERCC5 rs751402 and rs2296147 polymorphisms might alter the risk of developing GC and especially the diffuse subtype. Further validation of our results in larger populations and additional studies evaluating their function impact are required.     

Micronuclei in humans induced by exposure to low level of ionizing radiation: influence of polymorphisms in DNA repair genes

Understanding the risks deriving from protracted exposure to low doses of ionizing radiation has remarkable societal importance in view of the large number of work settings in which sources of IR are encountered. To address this question, we studied the frequency of micronuclei (MN), which is an indicator of DNA damage, in a population exposed to low levels of ionizing radiation and in matched controls. In both exposed population and controls, the possible influence of single nucleotide polymorphisms in XRCC1, XRCC3 and XPD genes on the frequency of micronuclei was also evaluated. We also considered the effects of confounding factors, like smoking status, age and gender. The results indicated that MN frequency was significantly higher in the exposed workers than in the controls [8.62+/-2.80 versus 6.86+/-2.65; P=0.019]. Radiological workers with variant alleles for XRCC1 or XRCC3 polymorphisms or wild-type alleles for XPD exon 23 or 10 polymorphisms showed a significantly higher MN frequency than controls with the same genotypes. Smoking status did not affect micronuclei frequency either in exposed workers or controls, while age was associated with increased MN frequency in the exposed only. In the combined population, gender but not age exerted an influence on the yield of MN, being higher in females than in males. Even though there is a limitation in this study due to the small number of subjects, these results suggest that even exposures to low level of ionizing radiation could have genotoxic effects and that XRCC3, XRCC1 and XPD polymorphisms might contribute to the increased genetic damage in susceptible individuals occupationally exposed to chronic low levels of ionizing radiation. For a clear conclusion on the induction of DNA damage caused by protracted exposure to low doses of ionizing radiation and the possible influence of genetic polymorphism in DNA repair genes larger studies are needed.     

Dynamics of a benzo[a]pyrene-derived guanine DNA lesion in TGT and CGC sequence contexts: enhanced mobility in TGT explains conformational heterogeneity, flexible bending, and greater susceptibility to nucleotide excision repair

The nucleotide excision repair (NER) machinery excises a variety of bulky DNA lesions, but with varying efficiencies. The structural features of the DNA lesions that govern these differences are not well understood. An intriguing model system for studying structure-function relationships in NER is the major adduct derived from the reaction of the highly tumorigenic metabolite of benzo[a]pyrene, (+)-anti-benzo[a]pyrene diol epoxide, with the exocyclic amino group of guanine ((+)-trans-anti-[BP]-N²-dG, or G*). The rates of incision of the stereochemically identical lesions catalyzed by the prokaryotic UvrABC system was shown to be greater by a factor of 2.3 ± 0.3 in the TG*T than in the CG*C sequence context [Biochemistry 46 (2007) 7006-7015]. Here we employ molecular dynamics simulations to elucidate the origin of the greater excision efficiency in the TG*T case and, more broadly, to delineate structural parameters that enhance NER. Our results show that the BP aromatic ring system is 5′-directed along the modified strand in the B-DNA minor groove in both sequence contexts. However, the TG*T modified duplex is much more dynamically flexible, featuring more perturbed and mobile Watson-Crick hydrogen bonding adjacent to the lesion, a greater impairment in stacking interactions, more dynamic local roll/bending, and more minor groove flexibility. These characteristics explain a number of experimental observations concerning the (+)-trans-anti-[BP]-N²-dG adduct in double-stranded DNA with the TG*T sequence context: its conformational heterogeneity in NMR solution studies, its highly flexible bend, and its lower thermal stability. By contrast, the CG*C modified duplex is characterized by a single BP conformation and a rigid bend. While current recognition models of bulky lesions by NER factors have stressed the importance of impaired Watson-Crick pairing/stacking and bending, our results highlight the likelihood of an important role for the local dynamics in the vicinity of the lesion.