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DNA-protein relationships have been studied by numerous methods, but a particular gap in methodology lies in the study of DNA adduct-specific interactions with proteins in vivo, which particularly affects the field of DNA repair. Using the repair of a well-characterized and ubiquitous adduct, the abasic (AP) site, as a model, we have developed a comprehensive method of monitoring DNA lesion-specific recruitment of proteins in vivo over time. We utilized a surrogate system in which a Cy3-labeled plasmid containing a single AP-site was transfected into cells, and the interaction of the labeled DNA with BER enzymes, including APE1, Polβ, LIG1, and FEN1, was monitored by immunofluorescent staining of the enzymes by Alexafluor-488-conjugated secondary antibody. The recruitment of enzymes was characterized by quantification of Cy3-Alexafluor-488 co-localization. To validate the microscopy-based method, repair of the transfected AP-site DNA was also quantified at various time points post-transfection using a real time PCR-based method. Notably, the recruitment time kinetics for each enzyme were consistent with AP-site repair time kinetics. This microscopy-based methodology is reliable in detecting the recruitment of proteins to specific DNA substrates and can be extended to study other in vivo DNA-protein relationships in any DNA sequence and in the context of any DNA structure in transfectable proliferating or quiescent cells. The method may be applied to a variety of disciplines of nucleic acid transaction pathways, including repair, replication, transcription, and recombination., (Copyright © 2015 Elsevier B.V. All rights reserved.)

ω-3 and ω-6 polyunsaturated fatty acids (PUFAs) play a role in the pathogenesis of colon cancer. Upon oxidation, PUFAs generate α,β-unsaturated aldehydes or enals, such as acrolein (Acr) and (E)-4-hydroxy-2-nonenal (HNE), which can form cyclic adducts of deoxyguanosine (Acr-dG and HNE-dG, respectively) in DNA. Both Acr-dG and HNE-dG adducts have been detected in human and animal tissues and are potentially mutagenic and carcinogenic. In vivo levels of Acr-dG in DNA are at least two orders of magnitude higher than those of HNE-dG. In addition to the facile reaction with Acr, the higher levels of Acr-dG than HNE-dG in vivo may be due to a lower rate of repair. Previous studies have shown that HNE-dG adducts are repaired by the NER pathway (Choudhury et al. [42]). We hypothesize that Acr-dG adducts are repaired at a slower rate than HNE-dG and that HNE-dG in DNA may influence the repair of Acr-dG. In this study, using a DNA repair synthesis assay and a LC-MS/MS method, we showed that Acr-dG in a plasmid DNA is repaired by NER proteins, but it is repaired at a much slower rate than HNE-dG in human colon cell extracts, and the slow repair of Acr-dG is likely due to poor recognition/excision of the lesions in DNA. Furthermore, using a plasmid DNA containing both adducts we found the repair of Acr-dG is significantly inhibited by HNE-dG, however, the repair of HNE-dG is not much affected by Acr-dG. This study demonstrates that the NER repair efficiencies of the two major structurally-related in vivo cyclic DNA adducts from lipid oxidation vary greatly. More importantly, the repair of Acr-dG can be significantly retarded by the presence of HNE-dG in DNA. Therefore, this study provides a mechanistic explanation for the higher levels of Acr-dG than HNE-dG observed in tissue DNA., (Copyright © 2013. Published by Elsevier B.V.)

trans-4-Hydroxynonenal (HNE) is a major peroxidation product of omega-6 polyunsaturated fatty acids. The reaction of HNE with DNA produces four diastereomeric 1,N(2)-gamma-hydroxypropano adducts of deoxyguanosine (HNE-dG); background levels of these adducts have been detected in tissues of animals and humans. There is evidence to suggest that these adducts are mutagenic and involved in liver carcinogenesis in patients with Wilson's disease and in other human cancers. Here, we present biochemical evidence that in human cell nuclear extracts the HNE-dG adducts are repaired by the nucleotide excision repair (NER) pathway. To investigate the recognition and repair of HNE-dG adducts in human cell extracts, we prepared plasmid DNA substrates modified by HNE. [(32)P]-Postlabeling/HPLC determined that the HNE-dG adduct levels were approximately 1200/10(6) dG of plasmid DNA substrate. We used this substrate in an in vitro repair-synthesis assay to study the complete repair of HNE-induced DNA adducts in cell-free extracts. We observed that nuclear extracts from HeLa cells incorporated a significant amount of alpha[(32)P]dCTP in DNA that contained HNE-dG adducts by comparison with UV-irradiated DNA as the positive control. Such repair synthesis for UV damage or HNE-dG adducts did not occur in XPA cell nuclear extracts that lack the capacity for NER. However, XPA cells complemented with XPA protein restored repair synthesis for both of these adducts. To verify that HNE-dG adducts in DNA were indeed repaired, we measured HNE-dG adducts in the post-repaired DNA substrates by the [(32)P]-postlabeling/HPLC method, showing that 50-60% of HNE-dG adducts were removed from the HeLa cell nuclear extracts after 3 h at 30 degrees C. The repair kinetics indicated that the excision rate is faster than the rate of gap-filling/DNA synthesis. Furthermore, the HNE-dG adduct isomers 2 and 4 appeared to be repaired more efficiently at early time points than isomers 1 and 3.

The cyclic 1,N(2)-propanodeoxyguanosine adducts, derived from alpha,beta-unsaturated aldehydes or enals, including acrolein (Acr), crotonaldehyde (Cro), and trans-4-hydroxy-2-nonenal (HNE), have been detected as endogenous DNA lesions in rodent and human tissues. Collective evidence has indicated that the oxidative metabolism of polyunsaturated fatty acids (PUFAs) is an important pathway for endogenous formation of these adducts. In a recent study, we examined the specific role of different types of fatty acids, omega-3 and omega-6 PUFAs, in the formation of cyclic adducts of Acr, Cro, and HNE. Our studies showed that the incubation of deoxyguanosine 5'-monophosphate with omega-3 or omega-6 fatty acids under oxidative conditions in the presence of ferrous sulfate yielded different amounts of Acr, Cro, and HNE adducts, depending on the types of fatty acids. We observed that Acr- and Cro-dG adducts are primarily formed from omega-3, and the adducts derived from longer chain enals, such as HNE, were detected exclusively from omega-6 fatty acids. Acr adducts are also formed from omega-6 fatty acids, but to a lesser extent; the yields of Acr adducts are proportional to the number of double bonds present in the PUFAs. Two previously unknown cyclic adducts, one from pentenal and the other from heptenal, were detected as products from omega-3 and omega-6 fatty acids, respectively. Because omega-6 PUFAs are known to be involved in the promotion of tumorigenesis, we investigated the role of HNE adducts in p53 gene mutation by mapping the HNE binding to the human p53 gene with UvrABC nuclease and determined the formation of HNE-dG adducts in the gene. The results showed that HNE-dG adducts are preferentially formed in a sequence-specific manner at the third base of codon 249 in the p53 gene, a mutational hotspot in human cancers. The DNA repair study using plasmid DNA containing HNE-dG adducts as a substrate in HeLa cell extracts showed that HNE adducts are readily repaired, and that nucleotide excision repair appears to be a major pathway involved. Together, results of these studies provide a better understanding of the involvement of different PUFAs in DNA damage and their possible roles in tumorigenesis.