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1.
J Biol Chem ; 289(8): 5074-82, 2014 Feb 21.
Article in English | MEDLINE | ID: mdl-24403078

ABSTRACT

DNA repair and DNA damage checkpoints work in concert to help maintain genomic integrity. In vivo data suggest that these two global responses to DNA damage are coupled. It has been proposed that the canonical 30 nucleotide single-stranded DNA gap generated by nucleotide excision repair is the signal that activates the ATR-mediated DNA damage checkpoint response and that the signal is enhanced by gap enlargement by EXO1 (exonuclease 1) 5' to 3' exonuclease activity. Here we have used purified core nucleotide excision repair factors (RPA, XPA, XPC, TFIIH, XPG, and XPF-ERCC1), core DNA damage checkpoint proteins (ATR-ATRIP, TopBP1, RPA), and DNA damaged by a UV-mimetic agent to analyze the basic steps of DNA damage checkpoint response in a biochemically defined system. We find that checkpoint signaling as measured by phosphorylation of target proteins by the ATR kinase requires enlargement of the excision gap generated by the excision repair system by the 5' to 3' exonuclease activity of EXO1. We conclude that, in addition to damaged DNA, RPA, XPA, XPC, TFIIH, XPG, XPF-ERCC1, ATR-ATRIP, TopBP1, and EXO1 constitute the minimum essential set of factors for ATR-mediated DNA damage checkpoint response.


Subject(s)
DNA Damage , DNA Repair , Animals , Ataxia Telangiectasia Mutated Proteins/metabolism , DNA/metabolism , Exodeoxyribonucleases/metabolism , Humans , Kinetics , Mice , Models, Biological , Phosphorylation , Replication Protein A/metabolism , Signal Transduction , Tumor Suppressor Protein p53/metabolism
2.
J Biol Chem ; 288(29): 20918-20926, 2013 Jul 19.
Article in English | MEDLINE | ID: mdl-23749995

ABSTRACT

Nucleotide excision repair is the sole mechanism for removing the major UV photoproducts from genomic DNA in human cells. In vitro with human cell-free extract or purified excision repair factors, the damage is removed from naked DNA or nucleosomes in the form of 24- to 32-nucleotide-long oligomers (nominal 30-mer) by dual incisions. Whether the DNA damage is removed from chromatin in vivo in a similar manner and what the fate of the excised oligomer was has not been known previously. Here, we demonstrate that dual incisions occur in vivo identical to the in vitro reaction. Further, we show that transcription-coupled repair, which operates in the absence of the XPC protein, also generates the nominal 30-mer in UV-irradiated XP-C mutant cells. Finally, we report that the excised 30-mer is released from the chromatin in complex with the repair factors TFIIH and XPG. Taken together, our results show the congruence of in vivo and in vitro data on nucleotide excision repair in humans.


Subject(s)
DNA Damage , DNA Repair , Oligonucleotides/metabolism , Animals , Cell Line , DNA Repair/radiation effects , DNA-Binding Proteins/metabolism , Humans , Models, Biological , Mutation/genetics , Pyrimidine Dimers/metabolism , Transcription Factor TFIIH/metabolism , Transcription, Genetic/radiation effects , Ultraviolet Rays
3.
J Biol Chem ; 287(43): 36123-31, 2012 Oct 19.
Article in English | MEDLINE | ID: mdl-22948311

ABSTRACT

Replication protein A (RPA) plays essential roles in DNA metabolism, including replication, checkpoint, and repair. Recently, we described an in vitro system in which the phosphorylation of human Chk1 kinase by ATR (ataxia telangiectasia mutated and Rad3-related) is dependent on RPA bound to single-stranded DNA. Here, we report that phosphorylation of other ATR targets, p53 and Rad17, has the same requirements and that RPA is also phosphorylated in this system. At high p53 or Rad17 concentrations, RPA phosphorylation is inhibited and, in this system, RPA with phosphomimetic mutations cannot support ATR kinase function, whereas a non-phosphorylatable RPA mutant exhibits full activity. Phosphorylation of these ATR substrates depends on the recruitment of ATR and the substrates by RPA to the RPA-ssDNA complex. Finally, mutant RPAs lacking checkpoint function exhibit essentially normal activity in nucleotide excision repair, revealing RPA separation of function for checkpoint and excision repair.


Subject(s)
Cell Cycle Checkpoints , Cell Cycle Proteins/chemistry , Protein Serine-Threonine Kinases/chemistry , Replication Protein A/chemistry , Signal Transduction , Ataxia Telangiectasia Mutated Proteins , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line , Cell-Free System/chemistry , Cell-Free System/metabolism , Checkpoint Kinase 1 , DNA Repair , DNA, Single-Stranded/chemistry , DNA, Single-Stranded/genetics , DNA, Single-Stranded/metabolism , Humans , Mutation , Phosphorylation/physiology , Protein Kinases/chemistry , Protein Kinases/genetics , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Replication Protein A/genetics , Replication Protein A/metabolism , Tumor Suppressor Protein p53/chemistry , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism
4.
Cell Cycle ; 11(18): 3481-91, 2012 Sep 15.
Article in English | MEDLINE | ID: mdl-22918252

ABSTRACT

The circadian clock is a global regulatory mechanism that confers daily rhythmicity on many biochemical and physiological functions, including DNA excision repair in mammalian organisms. Here, we investigated the effect of the circadian clock on the major DNA damage response pathways by using mouse cell lines mutated in genes encoding proteins in the positive (Bmal1, CLOCK) or negative (Cry 1/2, Per 1/2) arms of the transcription-translation feedback loop that generates the circadian clock. We find that cells mutated in these genes are indistinguishable from wild-type in their response to UV, ionizing radiation and mitomycin C. We conclude that either the majority of DNA damage response reactions are not controlled by the circadian clock or that, even if such a control exists at the organism level, it is supplanted by homeostatic control mechanisms at the cellular level in tissue culture. We suggest that caution must be exercised in extrapolating from experiments in tissue culture to whole animals with respect to the effect of the circadian clock on cellular response to DNA damaging agents.


Subject(s)
CLOCK Proteins/genetics , Circadian Clocks/genetics , DNA Damage/genetics , Mammals/genetics , Mammals/physiology , Mutation/genetics , Animals , Apoptosis/drug effects , Apoptosis/genetics , Apoptosis/radiation effects , Ataxia Telangiectasia Mutated Proteins , CLOCK Proteins/metabolism , Cell Cycle Checkpoints/drug effects , Cell Cycle Checkpoints/genetics , Cell Cycle Proteins/metabolism , Cell Line , Cell Survival/drug effects , Cell Survival/genetics , Cell Survival/radiation effects , Checkpoint Kinase 2 , Circadian Clocks/drug effects , Circadian Clocks/radiation effects , DNA Repair/drug effects , DNA Repair/genetics , DNA Repair/radiation effects , DNA-Binding Proteins/metabolism , Down-Regulation/drug effects , Down-Regulation/genetics , Down-Regulation/radiation effects , Enzyme Activation/drug effects , Enzyme Activation/radiation effects , Feedback, Physiological/drug effects , Feedback, Physiological/radiation effects , Humans , Mice , Mitomycin/pharmacology , Protein Serine-Threonine Kinases/metabolism , Radiation, Ionizing , Tumor Suppressor Proteins/metabolism , Ultraviolet Rays
5.
J Biol Chem ; 287(27): 22889-99, 2012 Jun 29.
Article in English | MEDLINE | ID: mdl-22573372

ABSTRACT

A wide range of environmental and carcinogenic agents form bulky lesions on DNA that are removed from the human genome in the form of short, ∼30-nucleotide oligonucleotides by the process of nucleotide excision repair. Although significant insights have been made regarding the mechanisms of damage recognition, dual incisions, and repair resynthesis during nucleotide excision repair, the fate of the dual incision/excision product is unknown. Using excision assays with both mammalian cell-free extract and purified proteins, we unexpectedly discovered that lesion-containing oligonucleotides are released from duplex DNA in complex with the general transcription and repair factor, Transcription Factor IIH (TFIIH). Release of excision products from TFIIH requires ATP but not ATP hydrolysis, and release occurs slowly, with a t(1/2) of 3.3 h. Excised oligonucleotides released from TFIIH then become bound by the single-stranded binding protein Replication Protein A or are targeted by cellular nucleases. These results provide a mechanism for release and an understanding of the initial fate of excised oligonucleotides during nucleotide excision repair.


Subject(s)
DNA Damage/genetics , DNA Repair/genetics , DNA-Binding Proteins/metabolism , Oligonucleotides/genetics , Cell-Free System , DNA/genetics , Deoxyribonucleases/metabolism , HeLa Cells , Humans , Phosphorus Radioisotopes , Replication Protein A/metabolism , Transcription Factor TFIIH/metabolism
6.
Nucleic Acids Res ; 39(8): 3176-87, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21193487

ABSTRACT

The XPA (Xeroderma pigmentosum A) protein is one of the six core factors of the human nucleotide excision repair system. In this study we show that XPA is a rate-limiting factor in all human cell lines tested, including a normal human fibroblast cell line. The level of XPA is controlled at the transcriptional level by the molecular circadian clock and at the post-translational level by a HECT domain family E3 ubiquitin ligase called HERC2. Stabilization of XPA by downregulation of HERC2 moderately enhances excision repair activity. Conversely, downregulation of XPA by siRNA reduces excision repair activity in proportion to the level of XPA. Ubiquitination and proteolysis of XPA are inhibited by DNA damage that promotes tight association of the protein with chromatin and its dissociation from the HERC2 E3 ligase. Finally, in agreement with a recent report, we find that XPA is post-translationally modified by acetylation. However, contrary to the previous claim, we find that in mouse liver only a small fraction of XPA is acetylated and that downregulation of SIRT1 deacetylase in two human cell lines does not affect the overall repair rate. Collectively, the data reveal that XPA is a limiting factor in excision repair and that its level is coordinately regulated by the circadian clock, the ubiquitin-proteasome system and DNA damage.


Subject(s)
DNA Repair , Xeroderma Pigmentosum Group A Protein/metabolism , Acetylation , Animals , Cell Line , Circadian Clocks/genetics , DNA Damage , Fibroblasts/metabolism , Humans , Mice , Transcription, Genetic , Ubiquitin-Protein Ligases/metabolism , Ubiquitination , Xeroderma Pigmentosum Group A Protein/genetics
7.
Cancer Res ; 70(12): 4922-30, 2010 Jun 15.
Article in English | MEDLINE | ID: mdl-20501836

ABSTRACT

Sunlight UV exposure produces DNA photoproducts in skin that are repaired solely by nucleotide excision repair in humans. A significant fraction of melanomas are thought to result from UV-induced DNA damage that escapes repair; however, little evidence is available about the functional capacity of normal human melanocytes, malignant melanoma cells, and metastatic melanoma cells to repair UV-induced photoproducts in DNA. In this study, we measured nucleotide excision repair in both normal melanocytes and a panel of melanoma cell lines. Our results show that in 11 of 12 melanoma cell lines tested, UV photoproduct repair occurred as efficiently as in primary melanocytes. Importantly, repair capacity was not affected by mutation in the N-RAS or B-RAF oncogenes, nor was a difference observed between a highly metastatic melanoma cell line (A375SM) or its parental line (A375P). Lastly, we found that although p53 status contributed to photoproduct removal efficiency, its role did not seem to be mediated by enhanced expression or activity of DNA binding protein DDB2. We concluded that melanoma cells retain capacity for nucleotide excision repair, the loss of which probably does not commonly contribute to melanoma progression.


Subject(s)
DNA Damage/radiation effects , DNA Repair , Genes, ras/physiology , Melanocytes/physiology , Melanoma/genetics , Proto-Oncogene Proteins B-raf/metabolism , Skin Neoplasms/genetics , Cells, Cultured , Collagen/metabolism , DNA, Neoplasm/radiation effects , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Drug Combinations , Humans , Laminin/metabolism , Melanoma/metabolism , Mutation/genetics , Proteoglycans/metabolism , Proto-Oncogene Proteins B-raf/genetics , Skin Neoplasms/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Ultraviolet Rays
8.
Proc Natl Acad Sci U S A ; 107(11): 4890-5, 2010 Mar 16.
Article in English | MEDLINE | ID: mdl-20304803

ABSTRACT

Cisplatin is one of the most commonly used anticancer drugs. It kills cancer cells by damaging their DNA, and hence cellular DNA repair capacity is an important determinant of its efficacy. Here, we investigated the repair of cisplatin-induced DNA damage in mouse liver and testis tissue extracts prepared at regular intervals over the course of a day. We find that the XPA protein, which plays an essential role in repair of cisplatin damage by nucleotide excision repair, exhibits circadian oscillation in the liver but not in testis. Consequently, removal of cisplatin adducts in liver extracts, but not in testis extracts, exhibits a circadian pattern with zenith at approximately 5 pm and nadir at approximately 5 am. Furthermore, we find that the circadian oscillation of XPA is achieved both by regulation of transcription by the core circadian clock proteins including cryptochrome and by regulation at the posttranslational level by the HERC2 ubiquitin ligase. These findings may be used as a guide for timing of cisplatin chemotherapy.


Subject(s)
Cisplatin/pharmacology , Cryptochromes/metabolism , DNA Damage , DNA Repair/drug effects , Guanine Nucleotide Exchange Factors/metabolism , Xeroderma Pigmentosum Group A Protein/metabolism , Animals , Biological Clocks/drug effects , Cell Line , Circadian Rhythm/drug effects , DNA Adducts/metabolism , Down-Regulation/drug effects , Humans , Liver/drug effects , Liver/metabolism , Male , Mice , Protein Binding/drug effects , Protein Processing, Post-Translational/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Testis/drug effects , Testis/metabolism , Time Factors , Tissue Extracts , Ubiquitin-Protein Ligases , Xeroderma Pigmentosum Group A Protein/genetics
9.
FEBS Lett ; 584(12): 2618-25, 2010 Jun 18.
Article in English | MEDLINE | ID: mdl-20227409

ABSTRACT

Mammalian cells possess a cell-autonomous molecular clock which controls the timing of many biochemical reactions and hence the cellular response to environmental stimuli including genotoxic stress. The clock consists of an autoregulatory transcription-translation feedback loop made up of four genes/proteins, BMal1, Clock, Cryptochrome, and Period. The circadian clock has an intrinsic period of about 24 h, and it dictates the rates of many biochemical reactions as a function of the time of the day. Recently, it has become apparent that the circadian clock plays an important role in determining the strengths of cellular responses to DNA damage including repair, checkpoints, and apoptosis. These new insights are expected to guide development of novel mechanism-based chemotherapeutic regimens.


Subject(s)
Circadian Rhythm/physiology , DNA Damage , Models, Biological , Animals , Apoptosis/genetics , Apoptosis/physiology , Cell Cycle/genetics , Cell Cycle/physiology , Circadian Rhythm/genetics , DNA Repair/genetics , DNA Repair/physiology , DNA Repair Enzymes/genetics , DNA Repair Enzymes/metabolism , Drug Chronotherapy , Humans , Mutation , Neoplasms/drug therapy , Neoplasms/etiology
10.
J Biol Chem ; 285(7): 4788-97, 2010 Feb 12.
Article in English | MEDLINE | ID: mdl-19996105

ABSTRACT

Replication protein A (RPA) is a heterotrimeric protein complex required for a large number of DNA metabolic processes, including DNA replication and repair. An alternative form of RPA (aRPA) has been described in which the RPA2 subunit (the 32-kDa subunit of RPA and product of the RPA2 gene) of canonical RPA is replaced by a homologous subunit, RPA4. The normal function of aRPA is not known; however, previous studies have shown that it does not support DNA replication in vitro or S-phase progression in vivo. In this work, we show that the RPA4 gene is expressed in normal human tissues and that its expression is decreased in cancerous tissues. To determine whether aRPA plays a role in cellular physiology, we investigated its role in DNA repair. aRPA interacted with both Rad52 and Rad51 and stimulated Rad51 strand exchange. We also showed that, by using a reconstituted reaction, aRPA can support the dual incision/excision reaction of nucleotide excision repair. aRPA is less efficient in nucleotide excision repair than canonical RPA, showing reduced interactions with the repair factor XPA and no stimulation of XPF-ERCC1 endonuclease activity. In contrast, aRPA exhibits higher affinity for damaged DNA than canonical RPA, which may explain its ability to substitute for RPA in the excision step of nucleotide excision repair. Our findings provide the first direct evidence for the function of aRPA in human DNA metabolism and support a model for aRPA functioning in chromosome maintenance functions in nonproliferating cells.


Subject(s)
DNA Repair/physiology , Replication Protein A/metabolism , DNA Repair/genetics , Enzyme-Linked Immunosorbent Assay , Humans , Immunoblotting , In Vitro Techniques , Polymerase Chain Reaction , Protein Binding/genetics , Protein Binding/physiology , Rad51 Recombinase/metabolism , Rad52 DNA Repair and Recombination Protein/metabolism , Replication Protein A/genetics
11.
Chem Res Toxicol ; 22(8): 1464-72, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19601657

ABSTRACT

Tobacco-specific nitrosamines, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N'-nitrosonornicotine, are considered to be human carcinogens. Both compounds are metabolized to pyridyloxobutylating intermediates that react with DNA to form adducts such as 7-[4-(3-pyridyl)-4-oxobut-1-yl]guanine, O(2)-[4-(3-pyridyl)-4-oxobut-1-yl]cytosine, O(2)-[4-(3-pyridyl)-4-oxobut-1-yl]-2'-deoxythymidine (O(2)-pobdT), O(6)-[4-(3-pyridyl)-4-oxobut-1-yl]-2'-deoxyguanosine (O(6)-pobdG), and 4-hydroxy-1-(3-pyridyl)-1-butanone-releasing adducts. The role of specific DNA adducts in the overall genotoxic activity of the pyridyloxobutylation pathway is not known. One adduct, O(6)-pobdG, is mutagenic. To characterize the mutagenic and cytotoxic properties of pyridyloxobutyl DNA adducts, the impact of DNA repair pathways on the cytotoxic and mutagenic properties of the model pyridyloxobutylating agent, 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc), was investigated in Chinese hamster ovary cell lines proficient or deficient in O(6)-alkylguanine DNA alkyltransferase (AGT), deficient in both AGT and base excision repair (BER), or deficient in both AGT and nucleotide excision repair (NER). The repair of the four pyridyloxobutyl DNA adducts was determined in the same cell lines via sensitive LC-MS/MS methods. NNKOAc was more cytotoxic in the cell lines lacking AGT, BER, and NER repair pathways. It also induced more mutations in the hprt gene in the BER- and NER-deficient cell lines. However, AGT expression did not influence NNKOAc's mutagenicity despite efficient repair of O(6)-pobdG. Analysis of the hprt mutational spectra indicated that NNKOAc primarily caused point mutations at AT base pairs. GC to AT transition mutations were a minor contributor to the overall mutation spectrum, providing a rationale for the observation that AGT does not protect against the overall mutagenic properties of NNKOAc in this model system. The only adduct affected by the absence of effective NER was O(2)-pobdT. Slower repair of O(2)-pobdT in NER-deficient cells was associated with increased AT to TA transversion mutations, supporting the hypothesis that these mutations are caused by O(2)-pobdT. Together, these data support a hypothesis that the pyridyloxobutylation pathway generates multiple mutagenic and toxic adducts.


Subject(s)
Metabolic Networks and Pathways , Nitrosamines/metabolism , Alkylating Agents , Animals , Cricetinae , Cricetulus , DNA , Female , Humans , Mutagenicity Tests , Nitrosamines/pharmacology , Thymidine/metabolism , Nicotiana
12.
Proc Natl Acad Sci U S A ; 106(8): 2864-7, 2009 Feb 24.
Article in English | MEDLINE | ID: mdl-19164551

ABSTRACT

The circadian clock regulates the daily rhythms in the physiology and behavior of many organisms, including mice and humans. These cyclical changes at molecular and macroscopic levels affect the organism's response to environmental stimuli such as light and food intake and the toxicity and efficacy of chemo- and radiotherapeutic agents. In this work, we investigated the circadian behavior of the nucleotide excision repair capacity in the mouse cerebrum to gain some insight into the optimal circadian time for favorable therapeutic response with minimal side effects in cancer treatment with chemotherapeutic drugs that produce bulky adducts in DNA. We find that nucleotide excision repair activity in the mouse cortex is highest in the afternoon/evening hours and is at its lowest in the night/early morning hours. The circadian oscillation of the repair capacity is caused at least in part by the circadian oscillation of the xeroderma pigmentosum A DNA damage recognition protein.


Subject(s)
Brain/physiology , Circadian Rhythm , DNA Repair , Animals , CLOCK Proteins , Cell Cycle Proteins/metabolism , Male , Mice , Mice, Inbred C57BL , Recombinant Proteins/metabolism , Trans-Activators/metabolism , Xeroderma Pigmentosum Group A Protein/metabolism
13.
Proc Natl Acad Sci U S A ; 105(26): 8902-7, 2008 Jul 01.
Article in English | MEDLINE | ID: mdl-18579768

ABSTRACT

We have identified unique chemical and biological properties of a cationic monofunctional platinum(II) complex, cis-diammine(pyridine)chloroplatinum(II), cis-[Pt(NH(3))(2)(py)Cl](+) or cDPCP, a coordination compound previously identified to have significant anticancer activity in a mouse tumor model. This compound is an excellent substrate for organic cation transporters 1 and 2, also designated SLC22A1 and SLC22A2, respectively. These transporters are abundantly expressed in human colorectal cancers, where they mediate uptake of oxaliplatin, cis-[Pt(DACH)(oxalate)] (DACH = trans-R,R-1,2-diaminocyclohexane), an FDA-approved first-line therapy for colorectal cancer. Unlike oxaliplatin, however, cDPCP binds DNA monofunctionally, as revealed by an x-ray crystal structure of cis-{Pt(NH(3))(2)(py)}(2+) bound to the N7 atom of a single guanosine residue in a DNA dodecamer duplex. Although the quaternary structure resembles that of B-form DNA, there is a base-pair step to the 5' side of the Pt adduct with abnormally large shift and slide values, features characteristic of cisplatin intrastrand cross-links. cDPCP effectively blocks transcription from DNA templates carrying adducts of the complex, unlike DNA lesions of other monofunctional platinum(II) compounds like {Pt(dien)}(2+). cDPCP-DNA adducts are removed by the nucleotide excision repair apparatus, albeit much less efficiently than bifunctional platinum-DNA intrastrand cross-links. These exceptional characteristics indicate that cDPCP and related complexes merit consideration as therapeutic options for treating colorectal and other cancers bearing appropriate cation transporters.


Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/metabolism , Organoplatinum Compounds/chemistry , Organoplatinum Compounds/metabolism , Animals , Antineoplastic Agents/pharmacology , Binding Sites , Cell Line , Cell Survival/drug effects , Cisplatin/chemistry , Crystallography, X-Ray , DNA/chemistry , DNA/metabolism , DNA Adducts/chemistry , DNA Repair/drug effects , Dogs , Drug Screening Assays, Antitumor , Humans , Organic Cation Transporter 1/metabolism , Organoplatinum Compounds/pharmacology , Oxaliplatin , RNA Polymerase II/chemistry , RNA Polymerase II/metabolism , Solutions , Transcription, Genetic/drug effects
14.
J Theor Biol ; 249(2): 361-75, 2007 Nov 21.
Article in English | MEDLINE | ID: mdl-17869273

ABSTRACT

A mathematical model of human nucleotide excision repair was constructed and validated. The model incorporates cooperative damage recognition by RPA, XPA, and XPC followed by three kinetic proofreading steps by the TFIIH transcription/repair factor. The model yields results consistent with experimental data regarding excision rates of UV photoproducts by the reconstituted human excision nuclease system as well as the excision of oligonucleotides from undamaged DNA. The model predicts the effect that changes in the initial concentrations of repair factors have on the excision rate of damaged DNA and provides a testable hypothesis on the biochemical mechanism of cooperativity in protein assembly, suggesting experiments to determine if cooperativity in protein assembly results from an increased association rate or a decreased dissociation rate. Finally, a comparison between the random order assembly with kinetic proofreading model and a sequential assembly model is made. This investigation reveals the advantages of the random order assembly/kinetic proofreading model.


Subject(s)
DNA Damage , DNA Repair , Models, Genetic , DNA/radiation effects , DNA-Binding Proteins/genetics , Humans , Radiation Injuries/genetics , Transcription Factor TFIIH/genetics , Ultraviolet Rays
15.
Methods Enzymol ; 408: 189-213, 2006.
Article in English | MEDLINE | ID: mdl-16793370

ABSTRACT

Nucleotide excision repair is a multicomponent, multistep enzymatic system that removes a wide spectrum of DNA damage by dual incisions in the damaged strand on both sides of the lesion. The basic steps are damage recognition, dual incisions, resynthesis to replace the excised DNA, and ligation. Each step has been studied in vitro using cell extracts or highly purified repair factors and radiolabeled DNA of known sequence with DNA damage at a defined site. This chapter describes procedures for preparation of DNA substrates designed for analysis of damage recognition, either the 5' or the 3' incision event, excision (resulting from concerted dual incisions), and repair synthesis. Excision in Escherichia coli is accomplished by the three-subunit Uvr(A)BC excision nuclease and in humans by six repair factors: XPA, RPA, XPChR23B, TFIIH, XPFERCC1, and XPG. This chapter outlines methods for expression and purification of these essential repair factors and provides protocols for performing each of the in vitro repair assays with either the E. coli or the human excision nuclease.


Subject(s)
DNA Repair , Escherichia coli , Animals , Cell Line , DNA/chemistry , DNA/metabolism , DNA Damage , DNA-Binding Proteins/genetics , DNA-Binding Proteins/isolation & purification , DNA-Binding Proteins/metabolism , Endodeoxyribonucleases/genetics , Endodeoxyribonucleases/isolation & purification , Endodeoxyribonucleases/metabolism , Escherichia coli/enzymology , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Escherichia coli Proteins/isolation & purification , Escherichia coli Proteins/metabolism , Humans , Nucleic Acid Conformation , Recombinant Proteins , Replication Protein A/genetics , Replication Protein A/isolation & purification , Replication Protein A/metabolism , Transcription Factor TFIIH/genetics , Transcription Factor TFIIH/isolation & purification , Transcription Factor TFIIH/metabolism , Xeroderma Pigmentosum Group A Protein/genetics , Xeroderma Pigmentosum Group A Protein/isolation & purification , Xeroderma Pigmentosum Group A Protein/metabolism
16.
Cell Cycle ; 5(13): 1366-70, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16775425

ABSTRACT

DNA-protein cross-links are generated by both endogenous and exogenous DNA damaging agents, as intermediates during normal DNA metabolism, and during abortive base excision repair. Cross-links are relatively common lesions that are lethal when they block progression of DNA polymerases. DNA-protein cross-links may be broadly categorized into four groups by the DNA and protein chemistries near the cross-link and by the source of the cross-link: DNA-protein cross-links may be found (1) in nicked DNA at the 3' end of one strand (topo I), (2) in nicked DNA at the 5' end of one strand (pol beta), (3) at the 5' ends of both strands adjacent to nicks in close proximity (topo II; Spo 11), and (4) in one strand of duplex DNA (UV irradiation; bifunctional carcinogens and chemotherapeutic agents). Repair mechanisms are reasonably well-defined for groups 1 and 3, and suggested for groups 2 and 4. Our work is focused on the recognition and removal of DNA-protein cross-links in duplex DNA (group 4).


Subject(s)
Cross-Linking Reagents/chemistry , DNA Repair/genetics , DNA/genetics , DNA/metabolism , Proteins/metabolism , Animals , Humans , Models, Genetic
17.
Proc Natl Acad Sci U S A ; 103(11): 4056-61, 2006 Mar 14.
Article in English | MEDLINE | ID: mdl-16537484

ABSTRACT

DNA-protein crosslinks are relatively common DNA lesions that form during the physiological processing of DNA by replication and recombination proteins, by side reactions of base excision repair enzymes, and by cellular exposure to bifunctional DNA-damaging agents such as platinum compounds. The mechanism by which pathological DNA-protein crosslinks are repaired in humans is not known. In this study, we investigated the mechanism of recognition and repair of protein-DNA and oligopeptide-DNA crosslinks by the human excision nuclease. Under our assay conditions, the human nucleotide excision repair system did not remove a 16-kDa protein crosslinked to DNA at a detectable level. However, 4- and 12-aa-long oligopeptides crosslinked to the DNA backbone were recognized by some of the damage recognition factors of the human excision nuclease with moderate selectivity and were excised from DNA at relatively efficient rates. Our data suggest that, if coupled with proteolytic degradation of the crosslinked protein, the human excision nuclease may be the major enzyme system for eliminating protein-DNA crosslinks from the genome.


Subject(s)
DNA Repair Enzymes/metabolism , DNA Repair/physiology , DNA/chemistry , DNA/metabolism , Peptides/chemistry , Peptides/metabolism , Amino Acid Sequence , Animals , Base Sequence , CHO Cells , Cricetinae , Cross-Linking Reagents , DNA/genetics , DNA Repair Enzymes/chemistry , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Escherichia coli/enzymology , HeLa Cells , Humans , In Vitro Techniques , Kinetics , Models, Biological , Protein Subunits , Substrate Specificity , Xeroderma Pigmentosum Group A Protein/chemistry , Xeroderma Pigmentosum Group A Protein/metabolism
18.
Mol Cell Biol ; 25(22): 9784-92, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16260596

ABSTRACT

Xeroderma pigmentosum is characterized by increased sensitivity of the affected individuals to sunlight and light-induced skin cancers and, in some cases, to neurological abnormalities. The disease is caused by a mutation in genes XPA through XPG and the XP variant (XPV) gene. The proteins encoded by the XPA, -B, -C, -D, -F, and -G genes are required for nucleotide excision repair, and the XPV gene encodes DNA polymerase eta, which carries out translesion DNA synthesis. In contrast, the mechanism by which the XPE gene product prevents sunlight-induced cancers is not known. The gene (XPE/DDB2) encodes the small subunit of a heterodimeric DNA binding protein with high affinity to UV-damaged DNA (UV-damaged DNA binding protein [UV-DDB]). The DDB2 protein exists in at least four forms in the cell: monomeric DDB2, DDB1-DDB2 heterodimer (UV-DDB), and as a protein associated with both the Cullin 4A (CUL4A) complex and the COP9 signalosome. To better define the role of DDB2 in the cellular response to DNA damage, we purified all four forms of DDB2 and analyzed their DNA binding properties and their effects on mammalian nucleotide excision repair. We find that DDB2 has an intrinsic damaged DNA binding activity and that under our assay conditions neither DDB2 nor complexes that contain DDB2 (UV-DDB, CUL4A, and COP9) participate in nucleotide excision repair carried out by the six-factor human excision nuclease.


Subject(s)
DNA Damage , DNA Repair , DNA-Binding Proteins/genetics , DNA-Binding Proteins/physiology , DNA/chemistry , Animals , Apoptosis , CHO Cells , COP9 Signalosome Complex , Cell Cycle , Cell Line , Chromatography, Affinity , Cricetinae , DNA-Directed DNA Polymerase/chemistry , Dimerization , Dose-Response Relationship, Drug , Humans , Insecta , Multiprotein Complexes/metabolism , Peptide Hydrolases/metabolism , Ultraviolet Rays
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