Interstrand cross-links arising from strand breaks at true abasic sites in duplex DNA.

Interstrand cross-links are exceptionally bioactive DNA lesions. Endogenous generation of interstrand cross-links in genomic DNA may contribute to aging, neurodegeneration, and cancer. Abasic (Ap) sites are common lesions in genomic DNA that readily undergo spontaneous and amine-catalyzed strand cleavage reactions that generate a 2,3-didehydro-2,3-dideoxyribose sugar remnant (3'ddR5p) at the 3'-terminus of the strand break. Interestingly, this strand scission process leaves an electrophilic α,ß-unsaturated aldehyde residue embedded within the resulting nicked duplex. Here we present evidence that 3'ddR5p derivatives generated by spermine-catalyzed strand cleavage at Ap sites in duplex DNA can react with adenine residues on the opposing strand to generate a complex lesion consisting of an interstrand cross-link adjacent to a strand break. The cross-link blocks DNA replication by ϕ29 DNA polymerase, a highly processive polymerase enzyme that couples synthesis with strand displacement. This suggests that 3'ddR5p-derived cross-links have the potential to block critical cellular DNA transactions that require strand separation. LC-MS/MS methods developed herein provide powerful tools for studying the occurrence and properties of these cross-links in biochemical and biological systems.

Probing Enhanced Double-Strand Break Formation at Abasic Sites within Clustered Lesions in Nucleosome Core Particles.

DNA is rapidly cleaved under mild alkaline conditions at apyrimidinic/apurinic sites, but the half-life is several weeks in phosphate buffer (pH 7.5). However, abasic sites are ∼100-fold more reactive within nucleosome core particles (NCPs). Histone proteins catalyze the strand scission, and at superhelical location 1.5, the histone H4 tail is largely responsible for the accelerated cleavage. The rate constant for strand scission at an abasic site is enhanced further in a nucleosome core particle when it is part of a bistranded lesion containing a proximal strand break. Cleavage of this form results in a highly deleterious double-strand break. This acceleration is dependent upon the position of the abasic lesion in the NCP and its structure. The enhancement in cleavage rate at an apurinic/apyrimidinic site rapidly drops off as the distance between the strand break and abasic site increases and is negligible once the two forms of damage are separated by 7 bp. However, the enhancement of the rate of double-strand break formation increases when the size of the gap is increased from one to two nucleotides. In contrast, the cleavage rate enhancement at 2-deoxyribonolactone within bistranded lesions is more modest, and it is similar in free DNA and nucleosome core particles. We postulate that the enhanced rate of double-strand break formation at bistranded lesions containing apurinic/apyrimidinic sites within nucleosome core particles is a general phenomenon and is due to increased DNA flexibility.

Pre-steady state kinetics of DNA binding and abasic site hydrolysis by tyrosyl-DNA phosphodiesterase 1.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) processes DNA 3'-end-blocking modifications, possesses DNA and RNA 3'-nucleosidase activity and is also able to hydrolyze an internal apurinic/apyrimidinic (AP) site and its synthetic analogs. The mechanism of Tdp1 interaction with DNA was analyzed using pre-steady state stopped-flow kinetics with tryptophan, 2-aminopurine and Förster resonance energy transfer fluorescence detection. Phosphorothioate or tetramethyl phosphoryl guanidine groups at the 3'-end of DNA have been used to prevent 3'-nucleosidase digestion by Tdp1. DNA binding and catalytic properties of Tdp1 and its mutants H493R (Tdp1 mutant SCAN1) and H263A have been compared. The data indicate that the initial step of Tdp1 interaction with DNA includes binding of Tdp1 to the DNA ends followed by the 3'-nucleosidase reaction. In the case of DNA containing AP site, three steps of fluorescence variation were detected that characterize (i) initial binding the enzyme to the termini of DNA, (ii) the conformational transitions of Tdp1 and (iii) search for and recognition of the AP-site in DNA, which leads to the formation of the catalytically active complex and to the AP-site cleavage reaction. Analysis of Tdp1 interaction with single- and double-stranded DNA substrates shows that the rates of the 3'-nucleosidase and AP-site cleavage reactions have similar values in the case of single-stranded DNA, whereas in double-stranded DNA, the cleavage of the AP-site proceeds two times faster than 3'-nucleosidase digestion. Therefore, the data show that the AP-site cleavage reaction is an essential function of Tdp1 which may comprise an independent of AP endonuclease 1 AP-site repair pathway.

[The role of Ku antigen in the repair of apurinic/apyrimidinic sites in DNA].

Apurinic/apyrimidinic (AP) sites are some of the most frequent lesions in genomic DNA. It is widely accepted that, irrespective of their origin, AP sites are further processed by the base excision repair (BER) machinery, being the central intermediate of this process. Under special conditions, proteins, which recognize AP sites, are able to form covalent adducts with DNA. By combination of the cross-linking technique with mass-spectrometry analysis, Ku antigen (Ku)--the central player in nonhomologous end joining (NHEJ), the pathway of double-strand break (DSB) repair--was identified as a protein reactive to AP sites. Moreover, Ku was shown to be a 5'-dRP/AP lyase that acts near DSBs in NHEJ. The recent studies have demonstrated involvement of Ku in the different stages of BER. Here, Ku roles in NHEJ and BER pathways of DNA repair are overviewed.

Structural and kinetic analysis of nucleoside triphosphate incorporation opposite an abasic site by human translesion DNA polymerase η.

The most common lesion in DNA is an abasic site resulting from glycolytic cleavage of a base. In a number of cellular studies, abasic sites preferentially code for dATP insertion (the "A rule"). In some cases frameshifts are also common. X-ray structures with abasic sites in oligonucleotides have been reported for several microbial and human DNA polymerases (pols), e.g. Dpo4, RB69, KlenTaq, yeast pol ι, human (h) pol ι, and human pol ß. We reported previously that hpol η is a major pol involved in abasic site bypass (Choi, J.-Y., Lim, S., Kim, E. J., Jo, A., and Guengerich, F. P. (2010 J. Mol. Biol. 404, 34-44). hpol η inserted all four dNTPs in steady-state and pre-steady-state assays, preferentially inserting A and G. In LC-MS analysis of primer-template pairs, A and G were inserted but little C or T was inserted. Frameshifts were observed when an appropriate pyrimidine was positioned 5' to the abasic site in the template. In x-ray structures of hpol η with a non-hydrolyzable analog of dATP or dGTP opposite an abasic site, H-bonding was observed between the phosphate 5' to the abasic site and water H-bonded to N1 and N6 of A and N1 and O6 of G nucleoside triphosphate analogs, offering an explanation for what appears to be a "purine rule." A structure was also obtained for an A inserted and bonded in the primer opposite the abasic site, but it did not pair with a 5' T in the template. We conclude that hpol η, a major copying enzyme with abasic sites, follows a purine rule, which can also lead to frameshifts. The phenomenon can be explained with H-bonds.

[Tyrosyl-DNA Phosphodiesterase 1 Is a New Player in Repair of Apurinic/Apyrimidinic Sites].

Genomic DNA is constantly damaged by the action of exogenous factors and endogenous reactive metabolites. Apurinic/apyrimidinic sites (AP sites), which occur as a result of DNA glycosylase induced or spontaneous hydrolysis of the N-glycosidic bonds, are the most common damages of DNA. The chemical reactivity of AP sites is the cause of DNA breaks, and DNA-protein and DNA-DNA crosslinks. Repair of AP sites is one of the most important mechanisms for maintaining genome stability. Despite the fact that the main participants of the AP site repair are very well studied, the new proteins that could be involved potentially in this process as "back up" players or perform certain specialized functions are being found. This review is dedicated to one of these proteins, tyrosyl-DNA phosphodiesterase 1 (Tdp1), for which we have recently shown that in addition to its main activity of specific cleavage of the tyrosyl-DNA bond formed via a covalent attachment of topoisomerase 1 (Top1) to DNA, Tdp1 is able to initiate the cleavage of the internal AP sites in DNA and their following repair. Tdp1 was discovered in Saccharomyces cerevisiae yeast as an enzyme hydrolyzing the covalent bond between tyrosyl residue of topoisomerase 1 and 3'-phosphate group in DNA. Tdp1 is the major enzyme which carries out the repair of the irreversible complexes of DNA and topoisomerase 1, which appear. in the presence of Top 1 inhibitors, such as camptothecin, therefore Tdp1 is a very important target for the development of inhibitors--anticancer drugs. Besides, Tdp1 hydrolyzes a wide range of 3'-terminal DNA modifications and the 3'-end nucleosides and its derivatives to form a 3'-phosphate. Tdp1 ability to cleave AP sites suggests its involvement in the base excision repair as an alternative enzyme to cleave AP sites instead of AP endonuclease 1--the major enzyme hydrolyzing AP sites in DNA repair process.

Interaction of apurinic/apyrimidinic endonuclease 2 (Apn2) with Myh1 DNA glycosylase in fission yeast.

Oxidative DNA damage is repaired primarily by the base excision repair (BER) pathway in a process initiated by removal of base lesions or mismatched bases by DNA glycosylases. MutY homolog (MYH, MUTYH, or Myh1) is a DNA glycosylase which excises adenine paired with the oxidative lesion 8-oxo-7,8-dihydroguanine (8-oxoG, or G°), thus reducing G:C to T:A mutations. The resulting apurinic/apyrimidinic (AP) site is processed by an AP-endonuclease or a bifunctional glycosylase/lyase. We show here that the major Schizosaccharomyces pombe AP endonuclease, Apn2, binds to the inter-domain connector located between the N- and C-terminal domains of Myh1. This Myh1 inter-domain connector also interacts with the Hus1 subunit of the Rad9-Rad1-Hus1 checkpoint clamp. Mutagenesis studies indicate that Apn2 and Hus1 bind overlapping but different sequence motifs on Myh1. Mutation on I(261) of Myh1 reduces its interaction with Hus1, but only slightly attenuates its interaction with Apn2. However, E(262) of Myh1 is a key determinant for both Apn2 and Hus1 interactions. Like human APE1, Apn2 has 3'-phosphodiesterase activity. However, unlike hAPE1, Apn2 has a weak AP endonuclease activity which cleaves the AP sites generated by Myh1 glycosylase. Functionally, Apn2 stimulates Myh1 glycosylase activity and Apn2 phosphodiesterase activity is stimulated by Myh1. The cross stimulation of Myh1 and Apn2 enzymatic activities is dependent on their physical interaction. Thus, Myh1 and Apn2 constitute an initial BER complex.

PrimPol, an archaic primase/polymerase operating in human cells.

We describe a second primase in human cells, PrimPol, which has the ability to start DNA chains with deoxynucleotides unlike regular primases, which use exclusively ribonucleotides. Moreover, PrimPol is also a DNA polymerase tailored to bypass the most common oxidative lesions in DNA, such as abasic sites and 8-oxoguanine. Subcellular fractionation and immunodetection studies indicated that PrimPol is present in both nuclear and mitochondrial DNA compartments. PrimPol activity is detectable in mitochondrial lysates from human and mouse cells but is absent from mitochondria derived from PRIMPOL knockout mice. PRIMPOL gene silencing or ablation in human and mouse cells impaired mitochondrial DNA replication. On the basis of the synergy observed with replicative DNA polymerases Polγ and Polε, PrimPol is proposed to facilitate replication fork progression by acting as a translesion DNA polymerase or as a specific DNA primase reinitiating downstream of lesions that block synthesis during both mitochondrial and nuclear DNA replication.

Kinetic mechanism of nick sealing by T4 RNA ligase 2 and effects of 3'-OH base mispairs and damaged base lesions.

T4 RNA ligase 2 (Rnl2) repairs 3'-OH/5'-PO4 nicks in duplex nucleic acids in which the broken 3'-OH strand is RNA. Ligation entails three chemical steps: reaction of Rnl2 with ATP to form a covalent Rnl2-(lysyl-Nζ)-AMP intermediate (step 1); transfer of AMP to the 5'-PO4 of the nick to form an activated AppN- intermediate (step 2); and attack by the nick 3'-OH on the AppN- strand to form a 3'-5' phosphodiester (step 3). Here we used rapid mix-quench methods to analyze the kinetic mechanism and fidelity of single-turnover nick sealing by Rnl2-AMP. For substrates with correctly base-paired 3'-OH nick termini, kstep2 was fast (9.5 to 17.9 sec(-1)) and similar in magnitude to kstep3 (7.9 to 32 sec(-1)). Rnl2 fidelity was enforced mainly at the level of step 2 catalysis, whereby 3'-OH base mispairs and oxoguanine, oxoadenine, or abasic lesions opposite the nick 3'-OH elicited severe decrements in the rate of 5'-adenylylation and relatively modest slowing of the rate of phosphodiester synthesis. The exception was the noncanonical A:oxoG base pair, which Rnl2 accepted as a correctly paired end for rapid sealing. These results underscore (1) how Rnl2 requires proper positioning of the 3'-terminal ribonucleoside at the nick for optimal 5'-adenylylation and (2) the potential for nick-sealing ligases to embed mutations during the repair of oxidative damage.

DNA-N-glycosylases process novel O-glycosidic sites in DNA.

After the hydrolysis of the N-glycosyl bond between a damaged base and C1' of a deoxyribosyl moiety of DNA, human alkyladenine DNA glycosylase (AAG) and Escherichia coli 3-methyladenine DNA glycosylase II (AlkA) bind tightly to their abasic DNA products, potentially protecting these reactive species. Here we show that both AAG and AlkA catalyze reactions between bound abasic DNA and small, primary alcohols to form novel DNA-O-glycosides. The synthesis reactions are reversible, as the DNA-O-glycosides are converted back into abasic DNA upon being incubated with AAG or AlkA in the absence of alcohol. AAG and AlkA are therefore able to hydrolyze O-glycosidic bonds in addition to N-glycosyl bonds. The newly discovered DNA-O-glycosidase activities of both enzymes compare favorably with their known DNA-N-glycosylase activities: AAG removes both methanol and 1,N(6)-ethenoadenine (εA) from DNA with single-turnover rate constants that are 2.9 × 10(5)-fold greater than the corresponding uncatalyzed rates, whereas the rate enhancement of 3.7 × 10(7) for removal of methanol from DNA by AlkA is 300-fold greater than its rate enhancement for removal of εA from DNA. Although the biological significance of the DNA-O-glycosidase reactions is not known, the evolution of new DNA repair pathways may be aided by enzymes that practice catalytic promiscuity, such as these two unrelated DNA glycosylases.

The structural location of DNA lesions in nucleosome core particles determines accessibility by base excision repair enzymes.

BACKGROUND: Base excision repair is hindered by nucleosomes. RESULTS: Outwardly oriented uracils near the nucleosome center are efficiently cleaved; however, polymerase ß is strongly inhibited at these sites. CONCLUSION: The histone octamer presents different levels of constraints on BER, dependent on the structural requirements for enzyme activity. SIGNIFICANCE: Chromatin remodeling is necessary to prevent accumulation of aborted intermediates in nucleosomes. Packaging of DNA into chromatin affects accessibility of DNA regulatory factors involved in transcription, replication, and repair. Evidence suggests that even in the nucleosome core particle (NCP), accessibility to damaged DNA is hindered by the presence of the histone octamer. Base excision repair is the major pathway in mammalian cells responsible for correcting a large number of chemically modified bases. We have measured the repair of site-specific uracil and single nucleotide gaps along the surface of the NCP. Our results indicate that removal of DNA lesions is greatly dependent on their rotational and translational positioning in NCPs. Significantly, the rate of uracil removal with outwardly oriented DNA backbones is 2-10-fold higher than those with inwardly oriented backbones. In general, uracils with inwardly oriented backbones farther away from the dyad center of the NCP are more accessible than those near the dyad. The translational positioning of outwardly oriented gaps is the key factor driving gap filling activity. An outwardly oriented gap near the DNA ends exhibits a 3-fold increase in gap filling activity as compared with one near the dyad with the same rotational orientation. Near the dyad, uracil DNA glycosylase/APE1 removes an outwardly oriented uracil efficiently; however, polymerase ß activity is significantly inhibited at this site. These data suggest that the hindrance presented by the location of a DNA lesion is dependent on the structural requirements for enzyme catalysis. Therefore, remodeling at DNA damage sites in NCPs is critical for preventing accumulation of aborted intermediates and ensuring completion of base excision repair.

Endonuclease IV discriminates mismatches next to the apurinic/apyrimidinic site in DNA strands: constructing DNA sensing platforms with extremely high selectivity.

A unique capability of Endonuclease IV in discrimination of mismatches neighboring a natural abasic site in DNA strands has been demonstrated, which enables genotyping of SNPs with high discrimination factors and differentiation of as low as 0.1-0.01% of target DNA strands from a large background of single-base different interfering strands.

Non-productive DNA damage binding by DNA glycosylase-like protein Mag2 from Schizosaccharomyces pombe.

Schizosaccharomyces pombe contains two paralogous proteins, Mag1 and Mag2, related to the helix-hairpin-helix (HhH) superfamily of alkylpurine DNA glycosylases from yeast and bacteria. Phylogenetic analysis of related proteins from four Schizosaccharomyces and other fungal species shows that the Mag1/Mag2 duplication is unique to the genus Schizosaccharomyces and most likely occurred in its ancestor. Mag1 excises N3- and N7-alkylguanines and 1,N(6)-ethenoadenine from DNA, whereas Mag2 has been reported to have no detectible alkylpurine base excision activity despite high sequence and active site similarity to Mag1. To understand this discrepancy we determined the crystal structure of Mag2 bound to abasic DNA and compared it to our previously determined Mag1-DNA structure. In contrast to Mag1, Mag2 does not flip the abasic moiety into the active site or stabilize the DNA strand 5' to the lesion, suggesting that it is incapable of forming a catalytically competent protein-DNA complex. Subtle differences in Mag1 and Mag2 interactions with the DNA duplex illustrate how Mag2 can stall at damage sites without fully engaging the lesion. We tested our structural predictions by mutational analysis of base excision and found a single amino acid responsible at least in part for Mag2's lack of activity. Substitution of Mag2 Asp56, which caps the helix at the base of the DNA intercalation loop, with the corresponding serine residue in Mag1 endows Mag2 with ɛA excision activity comparable to Mag1. This work provides novel insight into the chemical and physical determinants by which the HhH glycosylases engage DNA in a catalytically productive manner.

Sculpting of DNA at abasic sites by DNA glycosylase homolog mag2.

Modifications and loss of bases are frequent types of DNA lesions, often handled by the base excision repair (BER) pathway. BER is initiated by DNA glycosylases, generating abasic (AP) sites that are subsequently cleaved by AP endonucleases, which further pass on nicked DNA to downstream DNA polymerases and ligases. The coordinated handover of cytotoxic intermediates between different BER enzymes is most likely facilitated by the DNA conformation. Here, we present the atomic structure of Schizosaccharomyces pombe Mag2 in complex with DNA to reveal an unexpected structural basis for nonenzymatic AP site recognition with an unflipped AP site. Two surface-exposed loops intercalate and widen the DNA minor groove to generate a DNA conformation previously only found in the mismatch repair MutS-DNA complex. Consequently, the molecular role of Mag2 appears to be AP site recognition and protection, while possibly facilitating damage signaling by structurally sculpting the DNA substrate.

RNA oxidation catalyzed by cytochrome c leads to its depurination and cross-linking, which may facilitate cytochrome c release from mitochondria.

Growing evidence indicates that RNA oxidation is correlated with a number of age-related neurodegenerative diseases, and RNA oxidation has also been shown to induce dysfunction in protein synthesis. Here we study in vitro RNA oxidation catalyzed by cytochrome c (cyt c)/H(2)O(2) or by the Fe(II)/ascorbate/H(2)O(2) system. Our results reveal that the products of RNA oxidation vary with the oxidant used. Guanosine residues are preferentially oxidized by cyt c/H(2)O(2) relative to the Fe(II)/ascorbate/H(2)O(2) system. GC/MS and LC/MS analyses demonstrated that the guanine base was not only oxidized but also depurinated to form an abasic sugar moiety. Results from gel electrophoresis and HPLC analyses show that RNA formed a cross-linked complex with cyt c in an H(2)O(2) concentration-dependent manner. Furthermore, when cyt c was associated with liposomes composed of cardiolipin/phosphatidylcholine, and incubated with RNA and H(2)O(2), it was found cross-linked with the oxidized RNA and dissociated from the liposome. Results of the quantitative analysis indicate that the release of the cyt c from the liposome is facilitated by the formation of an RNA-cyt c cross-linked complex. Thus, RNA oxidation may facilitate the release of cyt c from the mitochondrial membrane to induce apoptosis in response to oxidative stress.

Tyrosyl-DNA phosphodiesterase 1 initiates repair of apurinic/apyrimidinic sites.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) catalyzes the hydrolysis of the phosphodiester linkage between the DNA 3' phosphate and a tyrosine residue as well as a variety of other DNA 3' damaged termini. Recently we have shown that Tdp1 can liberate the 3' DNA phosphate termini from apurinic/apyrimidinic (AP) sites. Here, we found that Tdp1 is more active in the cleavage of the AP sites inside bubble-DNA structure in comparison to ssDNA containing AP site. Furthermore, Tdp1 hydrolyzes AP sites opposite to bulky fluorescein adduct faster than AP sites located in dsDNA. Whilst the Tdp1 H493R (SCAN1) and H263A mutants retain the ability to bind an AP site-containing DNA, both mutants do not reveal endonuclease activity, further suggesting the specificity of the AP cleavage activity. We suggest that this Tdp1 activity can contribute to the repair of AP sites particularly in DNA structures containing ssDNA region or AP sites in the context of clustered DNA lesions.

Effect of the multifunctional proteins RPA, YB-1, and XPC repair factor on AP site cleavage by DNA glycosylase NEIL1.

DNA glycosylases are key enzymes in the first step of base excision DNA repair, recognizing DNA damage and catalyzing the release of damaged nucleobases. Bifunctional DNA glycosylases also possess associated apurinic/apyrimidinic (AP) lyase activity that nick the damaged DNA strand at an abasic (or AP) site, formed either spontaneously or at the first step of repair. NEIL1 is a bifunctional DNA glycosylase capable of processing lesions, including AP sites, not only in double-stranded but also in single-stranded DNA. Here, we show that proteins participating in DNA damage response, YB-1 and RPA, affect AP site cleavage by NEIL1. Stimulation of the AP lyase activity of NEIL1 was observed when an AP site was located in a 60 nt-long double-stranded DNA. Both RPA and YB-1 inhibited AP site cleavage by NEIL1 when the AP site was located in single-stranded DNA. Taking into account a direct interaction of YB-1 with the AP site, located in single-stranded DNA, and the high affinity of both YB-1 and RPA for single-stranded DNA, this behavior is presumably a consequence of a competition with NEIL1 for the DNA substrate. Xeroderma pigmentosum complementation group C protein (XPC), a key protein of another DNA repair pathway, was shown to interact directly with AP sites but had no effect on AP site cleavage by NEIL1.

Lesion bypass by S. cerevisiae Pol ζ alone.

DNA polymerase zeta (Pol ζ) participates in translesion synthesis (TLS) of DNA adducts that stall replication fork progression. Previous studies have led to the suggestion that the primary role of Pol ζ in TLS is to extend primers created when another DNA polymerase inserts nucleotides opposite lesions. Here we test the non-exclusive possibility that Pol ζ can sometimes perform TLS in the absence of any other polymerase. To do so, we quantified the efficiency with which S. cerevisiae Pol ζ bypasses abasic sites, cis-syn cyclobutane pyrimidine dimers and (6-4) photoproducts. In reactions containing dNTP concentrations that mimic those induced by DNA damage, a Pol ζ derivative with phenylalanine substituted for leucine 979 at the polymerase active site bypasses all three lesions at efficiencies between 27 and 73%. Wild-type Pol ζ also bypasses these lesions, with efficiencies that are lower and depend on the sequence context in which the lesion resides. The results are consistent with the hypothesis that, in addition to extending aberrant termini created by other DNA polymerases, Pol ζ has the potential to be the sole DNA polymerase involved in TLS.

An assay for RNA oxidation induced abasic sites using the Aldehyde Reactive Probe.

There have been several reports describing elevation of oxidized RNA in ageing or age-related diseases, however RNA oxidation has been assessed solely based on 8-hydroxy-guanosine levels. In this study, Aldehyde Reactive Probe (ARP), which was originally developed to detect DNA abasic sites, was used to assess RNA oxidation. It was found that ARP reacted with depurinated tRNA(Phe) or chemically synthesized RNA containing abasic sites quantitatively to as little as 10 fmoles, indicating that abasic RNA is recognized by ARP. RNA oxidized by Fenton-type reactions, γ-irradiation or peroxynitrite increased ARP reactivity dose-dependently, indicating that ARP is capable of monitoring oxidized RNA mediated by reactive oxygen species or reactive nitrogen species. Furthermore, oxidative stress increased levels of ARP reactive RNA in cultured cells. These results indicate the versatility of the assay method for biologically relevant oxidation of RNA. Thus, this study developed a sensitive assay for analysis of oxidized RNA.

Rapid DNA-protein cross-linking and strand scission by an abasic site in a nucleosome core particle.

Apurinic/apyrimidinic (AP) sites are ubiquitous DNA lesions that are highly mutagenic and cytotoxic if not repaired. In addition, clusters of two or more abasic lesions within one to two turns of DNA, a hallmark of ionizing radiation, are repaired much less efficiently and thus present greater mutagenic potential. Abasic sites are chemically labile, but naked DNA containing them undergoes strand scission slowly with a half-life on the order of weeks. We find that independently generated AP sites within nucleosome core particles are highly destabilized, with strand scission occurring ∼60-fold more rapidly than in naked DNA. The majority of core particles containing single AP lesions accumulate DNA-protein cross-links, which persist following strand scission. The N-terminal region of histone protein H4 contributes significantly to DNA-protein cross-links and strand scission when AP sites are produced approximately 1.5 helical turns from the nucleosome dyad, which is a known hot spot for nucleosomal DNA damage. Reaction rates for AP sites at two positions within this region differ by ∼4-fold. However, the strand scission of the slowest reacting AP site is accelerated when it is part of a repair resistant bistranded lesion composed of two AP sites, resulting in rapid formation of double strand breaks in high yields. Multiple lysine residues within a single H4 protein catalyze double strand cleavage through a mechanism believed to involve a templating effect. These results show that AP sites within the nucleosome produce significant amounts of DNA-protein cross-links and generate double strand breaks, the most deleterious form of DNA damage.