DNA base excision repair of uracil residues in reconstituted nucleosome core particles

The human base excision repair machinery must locate and repair DNA base damage present in chromatin, of which the nucleosome core particle is the basic repeating unit. Here, we have utilized fragments of the Lytechinus variegatus 5S rRNA gene containing site-specific U:A base pairs to investigate the base excision repair pathway in reconstituted nucleosome core particles in vitro. The human uracil-DNA glycosylases, UNG2 and SMUG1, were able to remove uracil from nucleosomes. Efficiency of uracil excision from nucleosomes was reduced 3- to 9-fold when compared with naked DNA, and was essentially uniform along the length of the DNA substrate irrespective of rotational position on the core particle. Furthermore, we demonstrate that the excision repair pathway of an abasic site can be reconstituted on core particles using the known repair enzymes, AP-endonuclease 1, DNA polymerase beta and DNA ligase III. Thus, base excision repair can proceed in nucleosome core particles in vitro, but the repair efficiency is limited by the reduced activity of the uracil-DNA glycosylases and DNA polymerase beta on nucleosome cores.     

Mitochondrial base excision repair of uracil and AP sites takes place by single-nucleotide insertion and long-patch DNA synthesis

Base excision repair (BER) corrects a variety of small base lesions in DNA. The UNG gene encodes both the nuclear (UNG2) and the mitochondrial (UNG1) forms of the human uracil-DNA glycosylase (UDG). We prepared mitochondrial extracts free of nuclear BER proteins from human cell lines. Using these extracts we show that UNG is the only detectable UDG in mitochondria, and mitochondrial BER (mtBER) of uracil and AP sites occur by both single-nucleotide insertion and long-patch repair DNA synthesis. Importantly, extracts of mitochondria carry out repair of modified AP sites which in nuclei occurs through long-patch BER. Such lesions may be rather prevalent in mitochondrial DNA because of its proximity to the electron transport chain, the primary site of production of reactive oxygen species. Furthermore, mitochondrial extracts remove 5' protruding flaps from DNA which can be formed during long-patch BER, by a "flap endonuclease like" activity, although flap endonuclease (FEN1) is not present in mitochondria. In conclusion, combined short- and long-patch BER activities enable mitochondria to repair a broader range of lesions in mtDNA than previously known.     

Characterization of nascent DNA fragments produced by excision of uracil residues in DNA.

Nascent short DNA chains could result from repair of incorporated uracil residues or be intermediates in discontinuous replication. We have characterized short DNA chains having apyrimidinic/apurinic-sites at 5' ends, the expected intermediates of repair, to distinguish them from RNA-linked replication intermediates. We have synthesized model substrates for the repair products; d(pRib[32P]poly(T)) and d(Rib[32P]poly(T)). Alkaline hydrolysis of both substrates has produced [5'-32P]poly(dT). Nascent short DNA was prepared from an Escherichia coli sof (dut) mutant, in this strain fragments from excision repair of uracil residues accumulate. The products of alkaline treatment are hardly digested by spleen exonuclease which selectively degrades 5'-hydroxyl-terminated DNA. These two results show that alkaline hydrolysis of the uracil repair fragments produces 5'-phosphoryl-terminated DNA, whereas it is known that 5'-hydroxyl-terminated DNA is generated from RNA-linked DNA molecules. The two types of nascent fragments thus can be distinguished by the 5'-terminal structure produced by an alkaline hydrolysis.     

Multiple conformational states of repair patches in chromatin during DNA excision repair

In mammalian cells, newly synthesized DNA repair patches are highly sensitive to digestion by staphylococcal nuclease (SN), but with time, they acquire approximately the same nuclease resistance as the DNA in bulk chromatin. We refer to the process which restores native SN sensitivity to repaired DNA as chromatin rearrangement. We find that during repair of ultraviolet damage in human fibroblasts, repair patch synthesis and ligation occur at approximately the same rate, with ligation delayed by about 4 min, but that chromatin rearrangement is only 75% as rapid. Thus, repair-incorporated nucleotides can exist in at least three distinct states: unligated/unrearranged, ligated/unrearranged, and ligated/rearranged. Inhibition of repair patch synthesis by aphidicolin or hydroxyurea results in inhibition of both patch ligation and chromatin rearrangement, confirming that repair patch completion and/or ligation are prerequisites for rearrangement. We also analyze the kinetics of SN digestion of repair-incorporated nucleotides at various extents of rearrangement and find the data to be consistent with the existence of two or more forms of unrearranged repair patch which have different sensitivities to digestion by SN. These data indicate that the chromatin rearrangement which restores native SN sensitivity to repaired DNA is a multistep process. The multiple forms of unrearranged chromatin with different SN sensitivities may include the unligated/unrearranged and ligated/unrearranged states. If so, the differences in SN sensitivity must arise from differences in chromatin structure, because SN does not differentiate between ligated and unligated repair patches in naked DNA.     

Inefficient excision of uracil from loop regions of DNA oligomers by E. coli uracil DNA glycosylase.

Kinetic parameters for uracil DNA glycosylase (E. coli)-catalysed excision of uracil from DNA oligomers containing dUMP in different structural contexts were determined. Our results show that single-stranded oligonucleotides (unstructured) are used as somewhat better substrates than the double-stranded oligonucleotides. This is mainly because of the favourable Vmax value of the enzyme for single-stranded substrates. More interestingly, however, we found that uracil release from loop regions of DNA hairpins is extremely inefficient. The poor efficiency with which uracil is excised from loop regions is a result of both increased Km and lowered Vmax values. This observation may have significant implications in uracil DNA glycosylase-directed repair of DNA segments that can be extruded as hairpins. In addition, these studies are useful in designing oligonucleotides for various applications in DNA research where the use of uracil DNA glycosylase is sought.     

Single-nucleotide patch base excision repair of uracil in DNA by mitochondrial protein extracts.

Mammalian mitochondria contain several 16.5 kb circular DNAs (mtDNA) encoding electron transport chain proteins. Reactive oxygen species formed as byproducts from oxidative phosphorylation in these organelles can cause oxidative deamination of cytosine and lead to uracil in mtDNA. Upon mtDNA replication, these lesions, if unrepaired, can lead to mutations. Until recently, it was thought that there was no DNA repair in mitochondria, but lately there is evidence that some lesions are efficiently repaired in these organelles. In the study of nuclear DNA repair, the in vitro repair measurements in cell extracts have provided major insights into the mechanisms. The use of whole-cell extract based DNA repair methods has revealed that mammalian nuclear base excision repair (BER) diverges into two pathways: the single-nucleotide replacement and long patch repair mechanisms. Similar in vitro methods have not been available for the study of mitochondrial BER. We have established an in vitro DNA repair system supported by rat liver mitochondrial protein extract and DNA substrates containing a single uracil opposite to a guanine. Using this approach, we examined the repair pathways and the identity of the DNA polymerase involved in mitochondrial BER (mtBER). Employing restriction analysis of in vitro repaired DNA to map the repair patch size, we demonstrate that only one nucleotide is incorporated during the repair process. Thus, in contrast to BER in the nucleus, mtBER of uracil in DNA is solely accomplished by single-nucleotide replacement.     

Specificities and kinetics of uracil excision from uracil-containing DNA oligomers by Escherichia coli uracil DNA glycosylase

Uracil DNA glycosylase excises uracil residues from DNA that can arise as a result of deamination of cytosine or incorporation of dUMP residues by DNA polymerase. We have carried out a detailed study to define the specificities and the kinetic parameters for its substrates by using a number of synthetic oligodeoxyribonucleotides of varying lengths and containing uracil residue(s) in various locations. The results show that the Escherichia coli enzyme can remove a 5'-terminal U from an oligomer only if the 5'-end is phosphorylated. The enzyme does not remove U residues from a 3'-terminal position, but U residues can be excised from oligonucleotides with either pd(UN)p or pd(UNN) 3'-termini. The oligomer d(UUUUT) can have the second or third U residues from the 5'-end excised even when the neighboring site is an abasic site (3' or 5', respectively). On the basis of these findings, pd(UN)p was anticipated to be the smallest size substrate. Results show detectable amounts of U release from the substrate pd(UT)p; however, significantly higher amounts of U release were observed from pd(UT-sugar) or pd(UTT). Determinations of the Km and Vmax values show that the different rates of U excision from oligomers of different sizes (trimeric to pentameric) but containing U in the same position are largely due to the differences in the Km values, whereas the different rates of U excision from the substrates of the same size but containing U in different positions are largely due to different Vmax values.     

Structural basis for poor uracil excision from hairpin DNA. An NMR study.

Two-dimensional NMR and molecular dynamics simulations have been used to determine the three-dimensional structures of two hairpin DNA structures: d-CTAGAG GATCCUTTTGGATCCT (abbreviated as U1-hairpin) and d-CTAGAGGATCCTTUTGGATCCT (abbreviated as U3-hairpin). The 1H resonances of both of these hairpin structures have been assigned almost completely. NMR restrained molecular dynamics and energy minimization procedures have been used to describe the three-dimensional structures of these hairpins. This study and concurrent NMR structural studies on two other d-CTAGAGGA TCCTUTTGGATCCT (abbreviated as U2-hairpin) and d-CTAGAGGATCCTTTUGGATCCT (abbreviated as U4-hairpin) have shed light upon various interactions reported between Echerichia coli uracil DNA glycosylase (UDG) and uracil-containing DNA. The backbone torsion angles, which partially influence the local conformation of U12 and U14 in U1 and U3-hairpins, respectively, are probably locked in the trans conformation as in the case of U13 in the U2-hairpin. Such a stretched-out backbone conformation in the vicinity of U12 and U14 is thought to be the reason why the Km value is poor for U1- and U3-hairpins as it is for the U2-hairpin. Furthermore, the bases U12 and U14 in both U1- and U3-hairpins adopt an anti conformation, in contrast with the base conformation of U13 in the U2-hairpin, which adopts a syn conformation. The clear discrepancy observed in the U-base orientation with respect to the sugar moieties could explain why the Vmax value is 10- to 20-fold higher for the U1- and U3-hairpins compared with the U2-hairpin. Taken together, these observations support our interpretation that the unfavourable backbone results in a poor Km value, whereas the unfavourable nucleotide conformation results in a poor Vmax value. These two parameters therefore make the U1- and U3-hairpins better substrates for UDG compared with the U2-hairpin, as reported earlier [Kumar, N. V. & Varshney, U. (1997) Nucleic Acids Res. 25, 2336-2343.].     

Enzymatic excision of uracil residues in nucleosomes depends on the local DNA structure and dynamics

The excision of uracil bases from DNA is accomplished by the enzyme uracil DNA glycosylase (UNG). Recognition of uracil bases in free DNA is facilitated by uracil base pair dynamics, but it is not known whether this same mechanistic feature is relevant for detection and excision of uracil residues embedded in nucleosomes. Here we investigate this question using nucleosome core particles (NCPs) generated from Xenopus laevis histones and the high-affinity "Widom 601" positioning sequence. The reactivity of uracil residues in NCPs under steady-state multiple-turnover conditions was generally decreased compared to that of free 601 DNA, mostly because of anticipated steric effects of histones. However, some sites in NCPs had equal or even greater reactivity than free DNA, and the observed reactivities were not readily explained by simple steric considerations or by global DNA unwrapping models for nucleosome invasion. In particular, some reactive uracils were found in occluded positions, while some unreactive uracils were found in exposed positions. One feature of many exposed reactive sites is a wide DNA minor groove, which allows penetration of a key active site loop of the enzyme. In single-turnover kinetic measurements, multiphasic reaction kinetics were observed for several uracil sites, where each kinetic transient was independent of the UNG concentration. These kinetic measurements, and supporting structural analyses, support a mechanism in which some uracils are transiently exposed to UNG by local, rate-limiting nucleosome conformational dynamics, followed by rapid trapping of the exposed state by the enzyme. We present structural models and plausible reaction mechanisms for the reaction of UNG at three distinct uracil sites in the NCP.     

Incorporation of biotin-labeled deoxyuridine triphosphate into DNA during excision repair and electron microscopic visualization of repair patches

Biotin-labeled deoxyuridine triphosphate (BiodUTP) has the potential to be a useful affinity probe for studies on DNA repair, if it can be incorporated into DNA repair patches and does not inhibit subsequent steps in the excision repair pathway. We have synthesized BiodUTP by an improved procedure and have used permeable normal human fibroblasts to determine the effect of substituting BiodUTP for thymidine triphosphate on several steps in the excision repair pathway: incision, polymerization, ligation, and nucleosome rearrangement. The results demonstrate that BiodUTP is efficiently incorporated into repair patches and has little or no effect on the repair process. The presence of BiodUMP in ligated repair patches has been used to visualize the repair patches by electron microscopy following incubation with ferritin-labeled avidin. This approach has been used to estimate the maximum size of repair patches induced by ultraviolet radiation.     

Deployment of DNA polymerases beta and lambda in single-nucleotide and multinucleotide pathways of mammalian base excision DNA repair

There exist two major base excision DNA repair (BER) pathways, namely single-nucleotide or “short-patch” (SP-BER), and “long-patch” BER (LP-BER). Both pathways appear to be involved in the repair of small base lesions such as uracil, abasic sites and oxidized bases. In addition to DNA polymerase β (Polβ) as the main BER enzyme for repair synthesis, there is evidence for a minor role for DNA polymerase lambda (Polλ) in BER. In this study we explore the potential contribution of Polλ to both SP- and LP-BER in cell-free extracts. We measured BER activity in extracts of mouse embryonic fibroblasts using substrates with either a single uracil or the chemically stable abasic site analog tetrahydrofuran residue. The addition of purified Polλ complemented the pronounced BER deficiency of POLB-null cell extracts as efficiently as did Polβ itself. We have developed a new approach for determining the relative contributions of SP- and LP-BER pathways, exploiting mass-labeled nucleotides to distinguish single- and multinucleotide repair patches. Using this method, we found that uracil repair in wild-type and in Polβ-deficient cell extracts supplemented with Polλ was ∼80% SP-BER. The results show that recombinant Polλ can contribute to both SP- and LP-BER. However, endogenous Polλ, which is present at a level ˜50% that of Polβ in mouse embryonic fibroblasts, appears to make little contribution to BER in extracts. Thus Polλ in cells appears to be under some constraint, perhaps sequestered in a complex with other proteins, or post-translationally modified in a way that limits its ability to participate effectively in BER.     

Inroads into base excision repair II. The discovery of DNA glycosylases. "An N-glycosidase from Escherichia coli that releases free uracil from DNA containing deaminated cytosine residues," Proc. Nat. Acad. Sci. USA, 1974

The discovery of a DNA glycosylase that specifically removes uracil from DNA, opened the door for uncovering a large class of such enzymes that are fundamental to the process of base excision repair of DNA.     

Periodic changes of chromatin organization associated with rearrangement of repair patches accompany DNA excision repair of mammalian cells

We have used 8-methoxypsoralen to probe the chromatin structure of mammalian cells in situ while they repair pyrimidine dimers or bulky lesions in DNA. We observed that excision repair of these DNA lesions is accompanied by periodic alterations of chromatin organization. In parallel, fluctuations of the rates of repair patch synthesis accompanied these structural changes. Taking advantage of the accessibility of free DNA domains for psoralen intercalation, we have developed a technique to quantitatively isolate the micrococcal nuclease-sensitive, free DNA fraction of native bulk chromatin. We have determined the location of newly synthesized repair patches relative to free DNA domains as a function of repair time. Extensive rearrangements of repair patches from these domains into micrococcal nuclease-resistant DNA were observed. Our results indicate that periodic changes of chromatin organization associated with rearrangement of repair patches accompany the process of excision repair in mammalian cells.     

Removal of uracil by uracil DNA glycosylase limits pemetrexed cytotoxicity: overriding the limit with methoxyamine to inhibit base excision repair

Uracil DNA glycosylase (UDG) specifically removes uracil bases from DNA, and its repair activity determines the sensitivity of the cell to anticancer agents that are capable of introducing uracil into DNA. In the present study, the participation of UDG in the response to pemetrexed-induced incorporation of uracil into DNA was studied using isogenic human tumor cell lines with or without UDG (UDG^+/+/UDG^−/−). UDG^−/− cells were very sensitive to pemetrexed. Cell killing by pemetrexed was associated with genomic uracil accumulation, stalled DNA replication, and catastrophic DNA strand breaks. By contrast, UDG^+/+ cells were >10 times more resistant to pemetrexed due to the rapid removal of uracil from DNA by UDG and subsequent repair of the resultant AP sites (abasic sites) via the base excision repair (BER). The resistance to pemetrexed in UDG^+/+ cells could be reversed by the addition of methoxyamine (MX), which binds to AP sites and interrupts BER pathway. Furthermore, MX-bound AP sites induced cell death was related to their cytotoxic effect of dual inactivation of UDG and topoisomerase IIα, two genes that are highly expressed in lung cancer cells in comparison with normal cells. Thus, targeting BER-based therapy exhibits more selective cytotoxicity on cancer cells through a synthetic lethal mechanism.     

Repair of deaminated cytosine residues of DNA: biological significance of the absence of uracil from DNA.

Uracil-DNA glycosylase of $\text{Bacillus}\text{subtilis}$ is involved in repair of deaminated cytosine residues of DNA. Survivals of SPO2 phage after treatment with bisulfite and weak alkali are considerably higher in wild type strains than in $\text{urg}$ mutants, which are deficient in the enzyme activity, whereas survivals of bisulfite/alkali-treated PBS1 phage in the two types of cells are essentially the same. The spontaneous mutation frequency of a $\text{urg}$ mutant is three fold higher than is that of a wild type strain.     

An alternative eukaryotic DNA excision repair pathway.

DNA lesions induced by UV light, cyclobutane pyrimidine dimers, and (6-4)pyrimidine pyrimidones are known to be repaired by the process of nucleotide excision repair (NER). However, in the fission yeast Schizosaccharomyces pombe, studies have demonstrated that at least two mechanisms for excising UV photo-products exist; NER and a second, previously unidentified process. Recently we reported that S. pombe contains a DNA endonuclease, SPDE, which recognizes and cleaves at a position immediately adjacent to cyclobutane pyrimidine dimers and (6-4)pyrimidine pyrimidones. Here we report that the UV-sensitive S. pombe rad12-502 mutant lacks SPDE activity. In addition, extracts prepared from the rad12-502 mutant are deficient in DNA excision repair, as demonstrated in an in vitro excision repair assay. DNA repair activity was restored to wild-type levels in extracts prepared from rad12-502 cells by the addition of partially purified SPDE to in vitro repair reaction mixtures. When the rad12-502 mutant was crossed with the NER rad13-A mutant, the resulting double mutant was much more sensitive to UV radiation than either single mutant, demonstrating that the rad12 gene product functions in a DNA repair pathway distinct from NER. These data directly link SPDE to this alternative excision repair process. We propose that the SPDE-dependent DNA repair pathway is the second DNA excision repair process present in S. pombe.     

DNA excision repair at telomeres

DNA damage is caused by either endogenous cellular metabolic processes such as hydrolysis, oxidation, alkylation, and DNA base mismatches, or exogenous sources including ultraviolet (UV) light, ionizing radiation, and chemical agents. Damaged DNA that is not properly repaired can lead to genomic instability, driving tumorigenesis. To protect genomic stability, mammalian cells have evolved highly conserved DNA repair mechanisms to remove and repair DNA lesions. Telomeres are composed of long tandem TTAGGG repeats located at the ends of chromosomes. Maintenance of functional telomeres is critical for preventing genome instability. The telomeric sequence possesses unique features that predispose telomeres to a variety of DNA damage induced by environmental genotoxins. This review briefly describes the relevance of excision repair pathways in telomere maintenance, with the focus on base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR). By summarizing current knowledge on excision repair of telomere damage and outlining many unanswered questions, it is our hope to stimulate further interest in a better understanding of excision repair processes at telomeres and in how these processes contribute to telomere maintenance.