Regulation of DNA repair by ubiquitylation.

Cellular DNA repair is a frontline system that is responsible for maintaining genome integrity and thus preventing premature aging and cancer by repairing DNA lesions and strand breaks caused by endogenous and exogenous mutagens. However, it is also the principal cellular system in cancer cells that counteracts the killing effect of the major cancer treatments, e.g. chemotherapy and ionizing radiation. Although it is clear that an individual's DNA repair capacity varies, the mechanisms involved in the regulation of repair systems that are responsible for such variations are only just emerging. This knowledge gap is impeding the finding of new cancer therapy targets and the development of novel treatment strategies. In recent years the vital role of post-translational modifications of DNA repair proteins, including ubiquitylation and phosphorylation, has been uncovered. This review will cover recent progress in our understanding of the role of ubiquitylation in the regulation of DNA repair.

Monitoring base excision repair proteins on damaged DNA using human cell extracts.

BER (base excision repair) is a major pathway for the removal of simple lesions in DNA including base damage and base loss (abasic site). We have developed an assay, using formaldehyde cross-linking during repair in human cell extracts, to observe BER proteins involved in the repair of damaged DNA. This approach allows visualization of repair proteins on damaged DNA during BER in human cell extracts and provides a detailed view of the molecular events leading to repair.

DNA polymerase beta is the major dRP lyase involved in repair of oxidative base lesions in DNA by mammalian cell extracts.

The repair of oxidative base lesions in DNA is a coordinated chain of reactions that includes removal of the damaged base, incision of the phosphodiester backbone at the abasic sugar residue, incorporation of an undamaged nucleotide and sealing of the DNA strand break. Although removal of a damaged base in mammalian cells is initiated primarily by a damage-specific DNA glycosylase, several lyases and DNA polymerases may contribute to the later stages of repair. DNA polymerase beta (Pol beta) was implicated recently as the major polymerase involved in repair of oxidative base lesions; however, the identity of the lyase participating in the repair of oxidative lesions is unclear. We studied the mechanism by which mammalian cell extracts process DNA substrates containing a single 8-oxoguanine or 5,6-dihydrouracil at a defined position. We find that, when repair synthesis proceeds through a Pol beta-dependent single nucleotide replacement mechanism, the 5'-deoxyribosephosphate lyase activity of Pol beta is essential for repair of both lesions.

Werner syndrome protein interacts with human flap endonuclease 1 and stimulates its cleavage activity.

Werner syndrome (WS) is a human premature aging disorder characterized by chromosomal instability. The cellular defects of WS presumably reflect compromised or aberrant function of a DNA metabolic pathway that under normal circumstances confers stability to the genome. We report a novel interaction of the WRN gene product with the human 5' flap endonuclease/5'-3' exonuclease (FEN-1), a DNA structure-specific nuclease implicated in DNA replication, recombination and repair. WS protein (WRN) dramatically stimulates the rate of FEN-1 cleavage of a 5' flap DNA substrate. The WRN-FEN-1 functional interaction is independent of WRN catalytic function and mediated by a 144 amino acid domain of WRN that shares homology with RecQ DNA helicases. A physical interaction between WRN and FEN-1 is demonstrated by their co-immunoprecipitation from HeLa cell lysate and affinity pull-down experiments using a recombinant C-terminal fragment of WRN. The underlying defect of WS is discussed in light of the evidence for the interaction between WRN and FEN-1.

Interaction of human AP endonuclease 1 with flap endonuclease 1 and proliferating cell nuclear antigen involved in long-patch base excision repair.

To understand the mechanism involved in the coordination of the sequential repair reactions that lead to long-patch BER, we have investigated interactions between proteins involved in this pathway. We find that human AP endonuclease 1 (APE1) physically interacts with flap endonuclease 1 (FEN1) and with proliferating cell nuclear antigen. An oligonucleotide substrate containing a reduced abasic site, which was pre-incised with APE1, was employed to reconstitute the excision step of long-patch BER with purified human DNA polymerase beta and FEN1. We demonstrate that addition of APE1 to the excision reaction mixture slightly (1.5-2-fold) stimulates the removal of the displaced flap by FEN1. These results suggest the possibility that long-patch BER is coordinated and directed by protein-protein interactions.

Base excision repair in nuclear and mitochondrial DNA.

Base excision repair mechanisms have been analyzed in nuclear and mitochondrial DNA. We measured the size and position of the newly incorporated DNA repair patch in various DNA substrates containing single oxidative lesions. Repair of 8-oxoguanine and of thymine glycol is almost exclusively via the base excision repair (BER) pathway with little or no involvement of nucleotide excision repair (NER). The repair mode is generally via the single-nucleotide replacement pathway with little incorporation into longer patches. Extension of these studies suggests that DNA polymerase beta plays a critical role not only in the short-patch repair process but also in the long-patch, PCNA-dependent pathway. Mitochondria are targets for a heavy load of oxidative DNA damage. They have efficient BER repair capacity, but cannot repair most bulky lesions normally repaired by NER. In vitro experiments performed using rat and human mitochondrial extracts suggest that the repair incorporation during the removal of uracil in DNA occurs via the short-patch repair BER pathway. Oxidative DNA damage accumulates with age in mitochondrial DNA, but this cannot be explained by an attenuation of DNA repair. In contrast, we observe that mitochondrial incision of 8-oxoG increases with age in rodents.

Securing genome stability by orchestrating DNA repair: removal of radiation-induced clustered lesions in DNA.

In addition to double- and single-strand DNA breaks and isolated base modifications, ionizing radiation induces clustered DNA damage, which contains two or more lesions closely spaced within about two helical turns on opposite DNA strands. Post-irradiation repair of single-base lesions is routinely performed by base excision repair and a DNA strand break is involved as an intermediate. Simultaneous processing of lesions on opposite DNA strands may generate double-strand DNA breaks and enhance nonhomologous end joining, which frequently results in the formation of deletions. Recent studies support the possibility that the mechanism of base excision repair contributes to genome stability by diminishing the formation of double-strand DNA breaks during processing of clustered lesions.

Human DNA polymerase beta initiates DNA synthesis during long-patch repair of reduced AP sites in DNA.

Simple base damages are repaired through a short-patch base excision pathway where a single damaged nucleotide is removed and replaced. DNA polymerase beta (Pol beta) is responsible for the repair synthesis in this pathway and also removes a 5'-sugar phosphate residue by catalyzing a beta-elimination reaction. How ever, some DNA lesions that render deoxyribose resistant to beta-elimination are removed through a long-patch repair pathway that involves strand displacement synthesis and removal of the generated flap by specific endonuclease. Three human DNA polymerases (Pol beta, Pol delta and Pol epsilon) have been proposed to play a role in this pathway, however the identity of the polymerase involved and the polymerase selection mechanism are not clear. In repair reactions catalyzed by cell extracts we have used a substrate containing a reduced apurinic/apyrimidinic (AP) site resistant to beta-elimination and inhibitors that selectively affect different DNA polymerases. Using this approach we find that in human cell extracts Pol beta is the major DNA polymerase incorporating the first nucleotide during repair of reduced AP sites, thus initiating long-patch base excision repair synthesis.

DNA synthesis and dRPase activities of polymerase beta are both essential for single-nucleotide patch base excision repair in mammalian cell extracts.

In mammalian cells the majority of altered bases in DNA are processed through a single-nucleotide patch base excision repair mechanism. Base excision repair is initiated by a DNA glycosylase that removes a damaged base and generates an abasic site (AP site). This AP site is further processed by an AP endonuclease activity that incises the phosphodiester bond adjacent to the AP site and generates a strand break containing 3'-OH and 5'-sugar phosphate ends. In mammalian cells, the 5'-sugar phosphate is removed by the AP lyase activity of DNA polymerase beta (Pol beta). The same enzyme also fills the gap, and the DNA ends are finally rejoined by DNA ligase. We measured repair of oligonucleotide substrates containing a single AP site in cell extracts prepared from normal and Pol beta-null mouse cells and show that the reduced repair in Pol beta-null extracts can be complemented by addition of purified Pol beta. Using this complementation assay, we demonstrate that mutated Pol beta without dRPase activity is able to stimulate long patch BER. Mutant Pol beta deficient in DNA synthesis, but with normal dRPase activity, does not stimulate repair in Pol beta-null cells. However, under conditions where we measure base excision repair accomplished exclusively through a single-nucleotide patch BER, neither dRPase nor DNA synthesis mutants of Pol beta alone, or the two together, were able to complement the repair defect. These data suggest that the dRPase and DNA synthesis activities of Pol beta are coupled and that both of these Pol beta functions are essential during short patch BER and cannot be efficiently substituted by other cellular enzymes.

Single nucleotide patch base excision repair is the major pathway for removal of thymine glycol from DNA in human cell extracts.

The repair pathways involved in the removal of thymine glycol (TG) from DNA by human cell extracts have been examined. Closed circular DNA constructs containing a single TG at a defined site were used as substrates to determine the patch size generated after in vitro repair by cell extracts. Restriction analysis of the repair incorporation in the vicinity of the lesion indicated that the majority of TG was repaired through the base excision repair (BER) pathways. Repair incorporation 5' to the lesion, characteristic for the nucleotide excision repair pathway, was not found. More than 80% of the TG repair was accomplished by the single-nucleotide repair mechanism, and the remaining TGs were removed by the long patch BER pathway. We also analyzed the role of the xeroderma pigmentosum, complementation group G (XPG) protein in the excision step of BER. Cell extracts deficient in XPG protein had an average 25% reduction in TG incision. These data show that BER is the primary pathway for repair of TG in DNA and that XPG protein may be involved in repair of TG as an accessory factor.

FEN1 stimulation of DNA polymerase beta mediates an excision step in mammalian long patch base excision repair.

In mammalian cells, single-base lesions, such as uracil and abasic sites, appear to be repaired by at least two base excision repair (BER) subpathways: "single-nucleotide BER" requiring DNA synthesis of just one nucleotide and "long patch BER" requiring multi-nucleotide DNA synthesis. In single-nucleotide BER, DNA polymerase beta (beta-pol) accounts for both gap filling DNA synthesis and removal of the 5'-deoxyribose phosphate (dRP) of the abasic site, whereas the involvement of various DNA polymerases in long patch BER is less well understood. Recently, we found that beta-pol plays a role in mammalian cell extract-mediated long patch BER, in that formation of a key excision product, 5'-dRP-trinucleotide (5'-dRP-N(3)), is dependent upon beta-pol (Dianov, G. L., Prasad, R., Wilson, S. H., and Bohr, V.A. (1999) J. Biol. Chem. 274, 13741-13743). The structure-specific endonuclease flap endonuclease 1 (FEN1) has also been suggested to be involved in long patch BER excision. Here, we demonstrate by immunodepletion experiments that 5'-dRP-N(3) excision in long patch BER of uracil-DNA in a human lymphoid cell extract is, indeed, dependent upon FEN1. Next, we reconstituted the excision step of long patch BER using purified human proteins and an oligonucleotide substrate with 5'-dRP at the margin of a one-nucleotide gap. Formation of the excision product 5'-dRP-N(3) was dependent upon both strand displacement DNA synthesis by beta-pol and FEN1 excision. FEN1 stimulated strand displacement DNA synthesis of beta-pol. FEN1 acting either alone, or without DNA synthesis by beta-pol, produced a two-nucleotide excision product, 5'-dRP-N(1), but not 5'-dRP-N(3). These results demonstrate that human FEN1 and beta-pol can cooperate in long patch BER excision and specify the predominant excision product seen with a cell extract.

DNA base excision repair in human malaria parasites is predominantly by a long-patch pathway.

Mammalian cells repair apurinic/apyrimidinic (AP) sites in DNA by two distinct pathways: a polymerase beta (pol beta)-dependent, short- (one nucleotide) patch base excision repair (BER) pathway, which is the major route, and a PCNA-dependent, long- (several nucleotide) patch BER pathway. The ability of a cell-free lysate prepared from asexual Plasmodium falciparum malaria parasites to remove uracil and repair AP sites in a variety of DNA substrates was investigated. We found that the lysate contained uracil DNA glycosylase, AP endonuclease, DNA polymerase, flap endonuclease, and DNA ligase activities. This cell-free lysate effectively repaired a regular or synthetic AP site on a covalently closed circular (ccc) duplex plasmid molecule or a long (382 bp), linear duplex DNA fragment, or a regular or reduced AP site in short (28 bp), duplex oligonucleotides. Repair of the AP sites in the various DNA substrates involved a long-patch BER pathway. This biology is different from mammalian cells, yeast, Xenopus, and Escherichia coli, which predominantly repair AP sites by a one-nucleotide patch BER pathway. The apparent absence of a short-patch BER pathway in P. falciparum may provide opportunities to develop antimalarial chemotherapeutic strategies for selectively damaging the parasites in vivo and will allow the characterization of the long-patch BER pathway without having to knock-out or inactivate a short-patch BER pathway, which is necessary in mammalian cells.

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.

Replication protein A stimulates proliferating cell nuclear antigen-dependent repair of abasic sites in DNA by human cell extracts.

Base excision repair (BER) pathway is the major cellular process for removal of endogenous base lesions and apurinic/apyrimidinic (AP) sites in DNA. There are two base excision repair subpathways in mammalian cells, characterized by the number of nucleotides synthesized into the excision patch. They are the "single-nucleotide" (one nucleotide incorporated) and the "long-patch" (several nucleotides incorporated) BER pathways. Proliferating cell nuclear antigen (PCNA) is known to be an essential factor in long-patch base excision repair. We have studied the role of replication protein A (RPA) in PCNA-dependent, long-patch BER of AP sites in human cell extracts. PCNA and RPA were separated from the other BER proteins by fractionation of human whole-cell extract on a phosphocellulose column. The protein fraction PC-FII (phosphocellulose fraction II), which does not contain RPA and PCNA but otherwise contains all core BER proteins required for PCNA-dependent BER (AP endonuclease, DNA polymerases delta, beta and DNA ligase, and FEN1 endonuclease), had reduced ability to repair plasmid DNA containing AP sites. Purified PCNA or RPA, when added separately, could only partially restore the PC-FII repair activity of AP sites. However, additions of both proteins together greatly stimulated AP site repair by PC-FII. These results demonstrate a role for RPA in PCNA-dependent BER of AP sites.

Repair of oxidative DNA base lesions induced by fluorescent light is defective in xeroderma pigmentosum group A cells.

Fluorescent light (FL) has been shown to generate free radicals within cells, however, the specific chemical nature of DNA damage induced by FL has not previously been determined. Using gas chromatography/isotope dilution mass spectrometry, we have detected induction of the oxidative DNA lesions 5-hydroxycytosine (5-OH-Cyt), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 4, 6-diamino-5-formamidopyrimidine (FapyAde) in cultured cells irradiated with FL. We followed the repair of these lesions in normal and xeroderma pigmentosum group A (XP-A) cells. 5-OH-Cyt and FapyGua were repaired efficiently in normal cells within 6 h following FL exposure. XP-A cells were unable to repair these oxidative DNA base lesions. Additionally, to compare the repair of oxidative lesions induced by various sources, in vitro repair studies were performed using plasmid DNA damaged by FL, gamma-irradiation or OsO(4)treatment. Whole cell extracts from normal cells repaired damaged substrates efficiently, whereas there was little repair in XP-A extracts. Our data demon-strate defective repair of oxidative DNA base lesions in XP-A cells in vivo and in vitro.

Role of DNA polymerase beta in the excision step of long patch mammalian base excision repair.

The two base excision repair (BER) subpathways in mammalian cells are characterized by the number of nucleotides synthesized into the excision patch. They are the "single-nucleotide" BER pathway and the "long patch" (several nucleotides incorporated) BER pathway. Both of these subpathways involve excision of a damaged base and/or nearby nucleotides and DNA synthesis to fill the excision gap. Whereas DNA polymerase beta (pol beta) is known to participate in the single-nucleotide BER pathway, the identity of polymerases involved in long patch BER has remained unclear. By analyzing products of long patch excision generated during BER of a uracil-containing DNA substrate in mammalian cell extracts we find that long patch excision depends on pol beta. We show that the excision of the characteristic 5'-deoxyribose phosphate containing oligonucleotide (dRP-oligo) is deficient in extracts from pol beta null cells and is rescued by addition of purified pol beta. Also, pol beta-neutralizing antibody inhibits release of the dRP-oligo in wild-type cell extracts, and the addition of pol beta after inhibition with antibody completely restores the excision reaction. The results indicate that pol beta plays an essential role in long patch BER by conducting strand displacement synthesis and controlling the size of the excised flap.

Oxidative DNA damage processing in nuclear and mitochondrial DNA.

Living organisms are constantly exposed to oxidative stress from environmental agents and from endogenous metabolic processes. The resulting oxidative modifications occur in proteins, lipids and DNA. Since proteins and lipids are readily degraded and resynthesized, the most significant consequence of the oxidative stress is thought to be the DNA modifications, which can become permanent via the formation of mutations and other types of genomic instability. Many different DNA base changes have been seen following some form of oxidative stress, and these lesions are widely considered as instigators for the development of cancer and are also implicated in the process of aging. Several studies have documented that oxidative DNA lesions accumulate with aging, and it appears that the major site of this accumulation is mitochondrial DNA rather than nuclear DNA. The DNA repair mechanisms involved in the removal of oxidative DNA lesions are much more complex than previously considered. They involve base excision repair (BER) pathways and nucleotide excision repair (NER) pathways, and there is currently a great deal of interest in clarification of the pathways and their interactions. We have used a number of different approaches to explore the mechanism of the repair processes, to examine the repair of different types of oxidative lesions and to measure different steps of the repair processes. Furthermore, we can measure the DNA damage processing in the nuclear DNA and separately, in the mitochondrial DNA. Contrary to widely held notions, mitochondria have efficient DNA repair of oxidative DNA damage.

An oxidative damage-specific endonuclease from rat liver mitochondria.

Reactive oxygen species have been shown to generate mutagenic lesions in DNA. One of the most abundant lesions in both nuclear and mitochondrial DNA is 7,8-dihydro-8-oxoguanine (8-oxoG). We report here the partial purification and characterization of a mitochondrial oxidative damage endonuclease (mtODE) from rat liver that recognizes and incises at 8-oxoG and abasic sites in duplex DNA. Rat liver mitochondria were purified by differential and Percoll gradient centrifugation, and mtODE was extracted from Triton X-100-solubilized mitochondria. Incision activity was measured using a radiolabeled double-stranded DNA oligonucleotide containing a unique 8-oxoG, and reaction products were separated by polyacrylamide gel electrophoresis. Gel filtration chromatography predicts mtODE's molecular mass to be between 25 and 30 kDa. mtODE has a monovalent cation optimum between 50 and 100 mM KCl and a pH optimum between 7.5 and 8. mtODE does not require any co-factors and is active in the presence of 5 mM EDTA. It is specific for 8-oxoG and preferentially incises at 8-oxoG:C base pairs. mtODE is a putative 8-oxoG glycosylase/lyase enzyme, because it can be covalently linked to the 8-oxoG oligonucleotide by sodium borohydride reduction. Comparison of mtODE's activity with other known 8-oxoG glycosylases/lyases and mitochondrial enzymes reveals that this may be a novel protein.

Reduced RNA polymerase II transcription in extracts of cockayne syndrome and xeroderma pigmentosum/Cockayne syndrome cells.

The hereditary disease Cockayne syndrome (CS) is a complex clinical syndrome characterized by arrested post-natal growth as well as neurological and other defects. The CSA and CSB genes are implicated in this disease. The clinical features of CS can also accompany the excision repair-defective hereditary disorder xeroderma pigmentosum (XP) from genetic complementation groups B, D or G. The XPB and XPD proteins are subunits of RNA polymerase II (RNAP II) transcription factor IIH (TFIIH). We show here that extracts of CS-A and CS-B cells, as well as those from XP-B/CS cells, support reduced levels of RNAP II transcription in vitro and that this feature is dependent on the state or quality of the template.

Reduced RNA polymerase II transcription in intact and permeabilized Cockayne syndrome group B cells.

Cockayne syndrome (CS) is characterized by increased photosensitivity, growth retardation, and neurological and skeletal abnormalities. The recovery of RNA synthesis is abnormally delayed in CS cells after exposure to UV radiation. Gene-specific repair studies have shown a defect in the transcription-coupled repair (TCR) of active genes in CS cells from genetic complementation groups A and B (CS-A and CS-B). We have analyzed transcription in vivo in intact and permeabilized CS-B cells. Uridine pulse labeling in intact CS-B fibroblasts and lymphoblasts shows a reduction of approximately 50% compared with various normal cells and with cells from a patient with xeroderma pigmentosum (XP) group A. In permeabilized CS-B cells transcription in chromatin isolated under physiological conditions is reduced to about 50% of that in normal chromatin and there is a marked reduction in fluorescence intensity in transcription sites in interphase nuclei. Transcription in CS-B cells is sensitive to alpha-amanitin, suggesting that it is RNA polymerase II-dependent. The reduced transcription in CS-B cells is complemented in chromatin by the addition of normal cell extract, and in intact cells by transfection with the CSB gene. CS-B may be a primary transcription deficiency.