Reconstitution of the DNA base excision—repair pathway

doi:10.1016/S0960-9822(00)00245-1

Current Biology

Volume 4, Issue 12, December 1994, Pages 1069-1076

https://doi.org/10.1016/S0960-9822(00)00245-1 Get rights and content

Abstract

Background: The base excision–repair pathway is the major cellular defence mechanism against spontaneous DNA damage. The enzymes involved have been highly conserved during evolution. Base excision–repair has been reproduced previously with crude cell-free extracts of bacterial or human origin. To further our understanding of base excision–repair, we have attempted to reconstitute the pathway in vitro using purified enzymes.

Results We report here the successful reconstitution of the base excision–repair pathway with five purified enzymes from Escherichia coli: uracil-DNA glycosylase, a representative of the DNA glycosylases that remove various lesions from DNA; the AP endonuclease IV that specifically cleaves at abasic sites; RecJ protein which excises a 5′ terminal deoxyribose-phosphate residue; DNA polymerase I; and DNA ligase. The reaction proceeds with high efficiency in the absence of additional factors in the reconstituted system. Four of the enzymes are absolutely required for completion of the repair reaction. An unusual feature we have discovered is that the pathway branches after enzymatic incision at an abasic DNA site. RecJ protein is required for the major reaction, which involves replacement of only a single nucleotide at the damaged site; in its absence, an alternative pathway is observed, with generation of longer repair patches by the 5′ nuclease function of DNA polymerase I.

Conclusion Repair of uracil in DNA is achieved by a very short-patch excision–repair process involving five different enzymes. No additional protein factors seem to be required. There is a minor, back-up pathway that uses replication factors to generate longer repair patches.

Section snippets

Background:

Three different excision–repair pathways are universally present in living organisms, and constitute the main cellular strategies for removing lesions and pre-mutagenic errors from DNA. Strand-specific mismatch repair provides protection against occasional rare errors that occur during DNA replication and have escaped proof-reading mechanisms. Nucleotide excision–repair (NER) is responsible for correction of damage that causes major helix distortion, in particular the dipyrimidine adducts

Requirement for a DNA glycosylase in the in vitro reaction

Uracil residues in DNA can be generated by cytosine deamination and are repaired efficiently in vivo by a BER process initiated by uracil-DNA glycosylase [10]. Double-stranded oligonucleotides containing a centrally placed G.U base pair, surrounded by appropriate restriction enzyme sites, have been employed as substrates for in vitro reactions [4]. In previous work with E. coli extracts, the dUMP residue was replaced efficiently by a dCMP residue. No incorporation of newly synthesized material

Discussion

A general model for the BER process (Figure 6) may be proposed on the basis of the results described above and recent results obtained with cell extracts and partially purified components [4], [8]. This model is similar to previous schemes [18], [19], but the existence of a branched pathway and the events associated with removal of the deoxyribose-phosphate residue at an incised abasic site are new features. The main route, which has been reproduced with purified enzymes in the present study,

Reagent enzymes

The following E. coli enzymes were purified from enzyme-overproducing strains as described: uracil-DNA glycosylase [33], endonuclease IV [34], RecJ protein [13], [14] and Fpg protein [15]. E. coli DNA polymerase I, DNA ligase, exonuclease III and T4 polynucleotide kinase were obtained from Boehringer-Mannheim. Restriction endonuclease HpaII was purchased from New England Biolabs, Inc.

Cell extracts and oligonucleotide substrates

E. coli NH5033 (recB, sbcB, endA) was obtained from S.C. West, and E. coli BD10 (ung, thyA, deoC) and its ung⁺

Grigory Dianov and Tomas Lindahl (corresponding author), Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire, EN6 3LD, UK.

Present address for Grigory Dianov: Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Texas 75235,USA.

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Sensitive detection of uracil-DNA glycosylase based on AND-gate triggers
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As a DNA repair enzyme, uracil-DNA glycosylase (UDG) removes uracil bases from DNA and starts the base excision repair pathway to maintain genomic integrity. It is meaningful to detect UDG activity sensitively because abnormal UDG activity is associated with cancer, immunodeficiency and many other diseases. Fluorescent detection methods have been rapidly developed because of their simplicity and sensitivity. However, in these ways, triggers used to initiate fluorescent signals only come from a single source, either the primer or the enzyme. The accidental leakage of the trigger leads to an unwanted background. To suppress the background signal further, we proposed an improved fluorescent strategy based on AND-gate triggers. In this strategy, a uracil-containing DNA oligo could inhibit the archaeal family B DNA polymerase. After UDG cleavage, the polymerase was activated (trigger-1). Meanwhile, an apurinic/apyrimidinic site was formed, then excised by Endonuclease IV, making the uracil-containing oligo become a primer with 3′-OH end (trigger-2) for further amplification. Then, an exponential amplified fluorescent signal could be generated with nicking endonuclease and well-designed templates. As the two triggers worked as an AND-gate, the leakage of any one of them was insufficient to initiate amplification reactions, resulting in low background. This AND-gate triggers based two-step amplification strategy showed a time-dependent output, with a detection limit of 1.5 × 10⁻⁴ U/mL. In addition, this solution also demonstrated a high selectivity toward UDG. Hence, this proposed strategy can be served as a promising platform for UDG activity detection and associated biomedical studies.
In vitro eradication of abasic site-mediated DNA–peptide/protein cross-links by Escherichia coli long-patch base excision repair
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Citation Excerpt :
This is possibly due to the steric hindrance of the cross-linked 10-mer peptide. The 5′-dRP has also been reported to be excised by E. coli RecJ, a 5′ to 3′ exonuclease (10). However, such activity is controversial as it was not observed by Lloyd et al. (41).
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.
The multifaceted roles of DNA repair and replication proteins in aging and obesity
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Briefly, a lesion-specific glycosylase recognizes and removes the damaged base by cleaving the glycosidic bond between the damaged base and phosphate backbone, leaving an apurinic/apyrimidinic (AP) site. AP Endonuclease 1 (APE1) then makes an incision in the backbone generating 3’hydroxyl (3’OH) and 5’deoxyribosephosphate (5’dRP) ends [13]. In most cases, the 5’dRP moiety can be removed by polymerase β (pol β), which then uses the opposite DNA strand as a template to fill in the nucleotide gap.
Efficient mechanisms for genomic maintenance (i.e., DNA repair and DNA replication) are crucial for cell survival. Aging and obesity can lead to the dysregulation of genomic maintenance proteins/pathways and are significant risk factors for the development of cancer, metabolic disorders, and other genetic diseases. Mutations in genes that code for proteins involved in DNA repair and DNA replication can also exacerbate aging- and obesity-related disorders and lead to the development of progeroid diseases. In this review, we will discuss the roles of various DNA repair and replication proteins in aging and obesity as well as investigate the possible mechanisms by which aging and obesity can lead to the dysregulation of these proteins and pathways.
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DNA carries the genetic information that directs complex biological processes; thus, maintaining a stable genome is critical for individual growth and development and for human health. DNA repair is a fundamental and conserved mechanism responsible for mending damaged DNA and restoring genomic stability, while its deficiency is closely related to multiple human disorders. In recent years, remarkable progress has been made in the field of DNA repair and aging. Here, we will extensively discuss the relationship among DNA damage, DNA repair, aging and aging-associated diseases based on the latest research. In addition, the possible role of DNA repair in several potential rejuvenation strategies will be discussed. Finally, we will also review the emerging methods that may facilitate future research on DNA repair.
Using single-molecule FRET to probe the nucleotide-dependent conformational landscape of polymerase b-DNA complexes
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Eukaryotic DNA polymerase β (Pol β) plays an important role in cellular DNA repair, as it fills short gaps in dsDNA that result from removal of damaged bases. Since defects in DNA repair may lead to cancer and genetic instabilities, Pol β has been extensively studied, especially its mechanisms for substrate binding and a fidelity-related conformational change referred to as “fingers closing.” Here, we applied single-molecule FRET to measure distance changes associated with DNA binding and prechemistry fingers movement of human Pol β. First, using a doubly labeled DNA construct, we show that Pol β bends the gapped DNA substrate less than indicated by previously reported crystal structures. Second, using acceptor-labeled Pol β and donor-labeled DNA, we visualized dynamic fingers closing in single Pol β-DNA complexes upon addition of complementary nucleotides and derived rates of conformational changes. We further found that, while incorrect nucleotides are quickly rejected, they nonetheless stabilize the polymerase-DNA complex, suggesting that Pol β, when bound to a lesion, has a strong commitment to nucleotide incorporation and thus repair. In summary, the observation and quantification of fingers movement in human Pol β reported here provide new insights into the delicate mechanisms of prechemistry nucleotide selection.
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Reactive oxygen species drive the oxidation of guanine to 8-oxoguanine (8oxoG), which threatens genome integrity. The repair of 8oxoG is carried out by base excision repair enzymes in Bacteria and Eukarya, however, little is known about archaeal 8oxoG repair. This study identifies a member of the Ogg-subfamily archaeal GO glycosylase (AGOG) in Thermococcus kodakarensis, an anaerobic, hyperthermophilic archaeon, and delineates its mechanism, kinetics, and substrate specificity. TkoAGOG is the major 8oxoG glycosylase in T. kodakarensis, but is non-essential. In addition to TkoAGOG, the major apurinic/apyrimidinic (AP) endonuclease (TkoEndoIV) required for archaeal base excision repair and cell viability was identified and characterized. Enzymes required for the archaeal oxidative damage base excision repair pathway were identified and the complete pathway was reconstituted. This study illustrates the conservation of oxidative damage repair across all Domains of life.

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Grigory Dianov and Tomas Lindahl (corresponding author), Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire, EN6 3LD, UK.

Present address for Grigory Dianov: Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Texas 75235,USA.

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Current Biology

Research PaperReconstitution of the DNA base excision—repair pathway

Abstract

Section snippets

Background:

Requirement for a DNA glycosylase in the in vitro reaction

Discussion

Reagent enzymes

Cell extracts and oligonucleotide substrates

Cell

FEBS Lett

J Biol Chem

J Biol Chem

Cell

J Biol Chem

J Biol Chem

J Biol Chem

J Biol Chem

Instability and decay of the primary structure of DNA

Nature

Endonuclease IV (nfo) mutant of Escherichia coli.

J Bacteriol

Generation of single-nucleotide repair patches following excision of uracil residues from DNA

Mol Cell Biol

DNA repair synthesis during base excision repair in vitro is catalyzed by DNA polymerase ϵ and is influenced by DNA polymerases α and δ in Saccharomyces cerevisiae.

Mol Cell Biol

Repair of a synthetic abasic site in DNA in a Xenopus laevis oocyte extract

Mol Cell Biol

Repair of a synthetic abasic site involves concerted reactions of DNA synthesis followed by excision and ligation

Mol Cell Biol

Proliferating cell nuclear antigen-dependent abasic site repair in Xenopus laevis oocytes: an alternative pathway of base excision DNA repair

Mol Cell Biol

Enzymatic release of 5′-terminal deoxyribose phosphate residues from damaged DNA in human cells

Biochemistry

Research Paper
Reconstitution of the DNA base excision—repair pathway