Reaction Mechanism of Human DNA Repair Excision Nuclease

Mu, David; Hsu, David S.; Sancar, Aziz

doi:10.1074/jbc.271.14.8285

Cited by 352 publications

(348 citation statements)

References 60 publications

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“…How does this hypothesis compare with the behavior of NER components that bind before ERCC1-XPF? The formation of a stretch of unwound DNA is required for ERCC1-XPF to produce the 5Ј incision in the injured DNA strand (12,13). Unwinding is performed through the ATP-dependent helicase activity of TFIIH (14)(15)(16).…”

Section: Resultsmentioning

confidence: 99%

In vivo dynamics of chromatin-associated complex formation in mammalian nucleotide excision repair

Moné

Bernaś

Dinant

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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Chromatin is the substrate for many processes in the cell nucleus, including transcription, replication, and various DNA repair systems, all of which require the formation of multiprotein machineries on the chromatin fiber. We have analyzed the kinetics of in vivo assembly of the protein complex that is responsible for nucleotide excision repair (NER) in mammalian cells. Assembly is initiated by UV irradiation of a small area of the cell nucleus, after which the accumulation of GFP-tagged NER proteins in the DNAdamaged area is measured, reflecting the establishment of the dual-incision complex. The dynamic behavior of two NER proteins, ERCC1-XPF and TFIIH, was studied in detail. Results show that the repair complex is assembled with a rate of Ϸ30 complexes per second and is not diffusion limited. Furthermore, we provide in vivo evidence that not only binding of TFIIH, but also its helicase activity, is required for the recruitment of ERCC1-XPF. These studies give quantitative insight into the de novo assembly of a chromatinassociated protein complex in living cells.DNA repair ͉ ERCC1-XPF ͉ in vivo kinetics ͉ TFIIH N ucleotide excision repair (NER) is continuously safeguarding genomic integrity by means of its ability to repair several types of helix-distorting DNA damage. For the removal of, for instance, UV-induced photoproducts, placental mammals fully rely on NER (1). This multiprotein system, involving Ͼ30 gene products, recognizes damaged DNA and excises the injury as an oligonucleotide from the affected strand, after which DNA polymerase action resynthesizes the gap and the remaining nick is ligated (2-4). We have detailed knowledge of the in vitro NER mechanism, mainly from biochemical studies. However, how it operates in the context of the living cell nucleus has remained largely unsolved.In general, studies of the dynamics of nuclear processes have been performed by using photobleaching procedures, providing information about protein mobility and exchange rates of components of protein complexes (5, 6). In vivo studies of chromatinassociated processes are hampered by a lack of methods that allow analysis of de novo assembly kinetics in the nuclei of living cells, and, as a result, the dynamic behavior of the involved complexes could be investigated only under steady-state conditions. Taking advantage of the UV-inducible nature of NER, we have analyzed in detail the dynamics of the formation of the double-incision complex by visualizing protein accumulation at sites of DNA damage immediately after UV irradiation. Results obtained for the NER complex may serve as a paradigm for the assembly of other chromatin-associated multiprotein systems, such as transcription and replication complexes. MethodsCell Culture. We used 43-3B Chinese hamster ovary (CHO) cells stably expressing ERCC1-GFP as described in ref. 7. We stably transfected 27.1 cells with enhanced GFP (EGFP) (Clontech) cloned in-frame to the C terminus of the XPB subunit of TFIIH, as described in ref. 8. Cells were grown at 37°C under 5% CO 2 in a ...

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Section: Resultsmentioning

confidence: 99%

In vivo dynamics of chromatin-associated complex formation in mammalian nucleotide excision repair

Moné

Bernaś

Dinant

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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“…Upon recognition of lesion sites, the XPC complex acts as a landing platform for the recruitment of TFIIH, which among its 10 subunits comprises the two DNA helicases XPB and XPD responsible for strand separation. Further GGR players that are sequentially recruited to target sites include XPA, RPA, XPG and, finally, XPF-ERCC1 [14,15]. The DNA unwinding activity of TFIIH generates a central nucleoprotein intermediate, in which the duplex undergoes partial melting by about 25 nucleotides [16][17][18].…”

Section: Overview Of the Ggr Pathwaymentioning

confidence: 99%

Dynamic two-stage mechanism of versatile DNA damage recognition by xeroderma pigmentosum group C protein

Clement

Camenisch

Jia

et al. 2010

Mutation Research/Fundamental and Molecular Mechanisms of Mutag

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“…The results shown represent one out of a total of six independent experiments that were conducted; in all six experiments, R992A and R992E each exhibited a repair deficiency similar in magnitude to that shown. tion of dual incision (34,35). This indicates that factors in addition to those traditionally considered as constituents of the excision reaction may be needed to effect disassembly of the complex and facilitate reutilization of the constituents at a new damage site.…”

Section: Figmentioning

confidence: 99%

The DNA Repair Endonuclease XPG Binds to Proliferating Cell Nuclear Antigen (PCNA) and Shares Sequence Elements with the PCNA-binding Regions of FEN-1 and Cyclin-dependent Kinase Inhibitor p21

Gary¹,

Ludwig

Cornelius

et al. 1997

Journal of Biological Chemistry

226

165

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Proliferating cell nuclear antigen (PCNA) is a DNA polymerase accessory factor that is required for DNA replication during S phase of the cell cycle and for resynthesis during nucleotide excision repair of damaged DNA. PCNA binds to flap endonuclease 1 (FEN-1), a structure-specific endonuclease involved in DNA replication. Here we report the direct physical interaction of PCNA with xeroderma pigmentosum (XP) G, a structurespecific repair endonuclease that is homologous to FEN-1. We have identified a 28-amino acid region of human FEN-1 (residues 328 -355) and a 29-amino acid region of human XPG (residues 981-1009) that contains the PCNA binding activity. These regions share key hydrophobic residues with the PCNA-binding domain of the cyclin-dependent kinase inhibitor p21 Waf1/Cip1 , and all three competed with one another for binding to PCNA. A conserved arginine in FEN-1 (Arg 339 ) and XPG (Arg 992 ) was found to be crucial for PCNA binding activity. R992A and R992E mutant forms of XPG failed to fully reconstitute nucleotide excision repair in an in vivo complementation assay. These results raise the possibility of a mechanistic linkage between excision and repair synthesis that is mediated by PCNA.Exposure to UV light causes damage to DNA primarily in the form of cyclobutane pyrimidine dimers and (6-4) photoproducts. These types of DNA lesions, as well as bulky adducts produced by some chemical mutagens, are processed by nucleotide excision repair (NER). 1 The human genetic disorder xeroderma pigmentosum (XP) is the result of defects in this DNA damage repair pathway. Symptoms of XP include extreme sensitivity to sunlight exposure and a greatly elevated risk of skin cancer. In the past few years, much progress has been made in understanding the molecular events associated with NER (1). The DNA-binding protein XPA is involved in damage recognition. In concert with replication protein A, which binds singlestranded DNA, and helicases XPB and XPD, a ϳ27-29-base oligonucleotide segment containing the lesion is excised as the result of dual incision by structure-specific endonucleases XPF-ERCC1 and XPG. The XPF-ERCC1 complex cleaves the damaged strand at a 5Ј site about 23 nucleotides from the lesion, whereas XPG cleaves the strand approximately 5 nucleotides to the 3Ј side of the damage. The resultant gap is filled in by the action of DNA polymerase ␦ or ⑀, and then DNA ligase seals the nick to complete repair. The resynthesis step requires proliferating cell nuclear antigen (PCNA; Refs. 2 and 3), a ring-shaped homotrimeric protein that encircles DNA and acts as a "sliding clamp" that links the polymerase to the DNA template (4). PCNA performs the same essential function in replicative DNA synthesis during S phase of the cell cycle. PCNA requires replication factor C, a primer recognition protein that loads the PCNA trimer onto DNA in an ATP-dependent manner (5-7).XPG is homologous to another structure-specific endonuclease, FEN-1. FEN-1 is involved in Okazaki fragment processing during DNA replication (8), and it i...

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Reaction Mechanism of Human DNA Repair Excision Nuclease

Cited by 352 publications

References 60 publications

In vivo dynamics of chromatin-associated complex formation in mammalian nucleotide excision repair

In vivo dynamics of chromatin-associated complex formation in mammalian nucleotide excision repair

Dynamic two-stage mechanism of versatile DNA damage recognition by xeroderma pigmentosum group C protein

The DNA Repair Endonuclease XPG Binds to Proliferating Cell Nuclear Antigen (PCNA) and Shares Sequence Elements with the PCNA-binding Regions of FEN-1 and Cyclin-dependent Kinase Inhibitor p21

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