Multiple nuclear localization signals in XPG nuclease

Knauf, Jeffrey A.; Pendergrass, Stephanie H.; Marrone, Babetta L.; Strniste, G.F.; MacInnes, Mark A.; Park, Min

doi:10.1016/0921-8777(95)00062-3

Cited by 26 publications

(21 citation statements)

References 31 publications

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“…5). For comparison, XPG immunofluorescence redistribution data given previously in Table 1 (31). An analogous focal localization of the DNA synthesis protein-proliferating cell nuclear antigen-specifically during S phase of the cell cycle, and its dispersion after UV exposure have been reported (41).…”

Section: Resultsmentioning

confidence: 71%

Ultraviolet-induced movement of the human DNA repair protein, Xeroderma pigmentosum type G, in the nucleus.

Park

Knauf

Pendergrass

et al. 1996

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

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Xeroderma pigmentosum type G (XPG) is a human genetic disease exhibiting extreme sensitivity to sunlight. XPG patients are defective XPG endonuclease, which is an enzyme essential for DNA repair of the major kinds of solar ultraviolet (UV)-induced DNA damages. Here we describe a novel dynamics of this protein within the cell nucleus after UV irradiation of human cells. Using confocal microscopy, we have localized the immunofluorescent, antigenic signal of XPG protein to foci throughout the cell nucleus. Our biochemical studies also established that XPG protein forms a tight association with nuclear structure(s). In human skin fibroblast cells, the number of XPG foci decreased within 2 h after UV irradiation, whereas total nuclear XPG fluorescence intensity remained constant, suggesting redistribution of XPG from a limited number of nuclear foci to the nucleus overall. Within 8 h after UV, most XPG antigenic signal was found as foci. Using j8-galactosidase-XPG fusion constructs (,B-gal-XPG) In recent decades, there has been substantial progress in understanding the basic structure of the cell nucleus and its implications in functional compartmentation (12). A proteinaceous framework known as the nuclear matrix or scaffold has been implicated in a variety of nuclear processes including DNA replication (13), excision repair (14, 15), and RNA transcription and processing (16-18). There is good evidence that regulatory proteins are situated near to the attachment sites of DNA loops to the nuclear matrix (19,20). This type of geometrical organization is thought to facilitate nuclear processes by efficiently reducing the search volume of transcriptional regulators for their cognate DNA elements (19). This principle may also apply to transcription-coupled NER by a mechanism that pre-positions DNA repair enzymes in transcriptionally active chromatin regions via association with nuclear matrix.The

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Section: Resultsmentioning

confidence: 71%

Ultraviolet-induced movement of the human DNA repair protein, Xeroderma pigmentosum type G, in the nucleus.

Park

Knauf

Pendergrass

et al. 1996

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

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“…Noncovalently bound proteins were eluted with Laemmli sample buffer, resolved on 4 -12% polyacrylamide gradient gels, and transferred to Immobilon-P (Millipore, Bedford, MA) polyvinylidene fluoride membrane for Western blot analysis. The membrane was sequentially incubated with 10% nonfat dry milk, 1:2000 rabbit polyclonal XPG1322 (IgG fraction) raised against an XPG amino acid 1147-1163 peptide (20,21), and 1:2000 peroxidase-conjugated goat anti-rabbit IgG (Life Technologies, Inc.), and then XPG bands were detected by enhanced chemiluminescence (Amersham Corp.).…”

Section: Methodsmentioning

confidence: 99%

“…Absence of repair activity is shown by cells transfected with truncated XPG lacking residues 948 -1186. The deletion of 3Ј-untranslated sequence in this mutant probably destabilizes the mRNA, and the coding region truncation removes the nuclear localization signal to encode a mutant protein unable to enter the nucleus (20,21). Lying between these benchmark indicators of maximum and minimum repair activity were those cells dependent upon R992A and R992E XPG mutants for NER function.…”

Section: Identification Of a Pcna-binding Domain In Xpg-mentioning

confidence: 99%

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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

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225

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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“…The C-terminus also contains two putative nuclear localization signals at residues 1051-1084 and 1169-1186. 31,32 The role of XPG in nucleotide excision repair NER operates by two distinct pathways, global genome repair (GG-NER) and transcriptioncoupled repair (TC-NER) and XPG plays a key role in both of them. GG-NER is the betterunderstood pathway of the two and involves the removal of lesions from all sites of the genome, while TC-NER refers to the preferential repair of lesion from the transcribed strands of active genes.…”

Section: Discovery and Cloning Of Xpgmentioning

confidence: 99%

XPG: Its Products and Biological Roles

Schärer

Molecular Mechanisms of Xeroderma Pigmentosum

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Xeroderma pigmetosum patients of the complementation group G are rare. One group of XP-G patients displays a rather mild and typical XP phenotype. Mutations in these patients interfere with the function of XPG in the nucleotide excision repair, where it has a structural role in the assembly of the preincision complex and a catalytic role in making the incision 3' to the damaged site in DNA. Another set of XP-G patient is much more severely affected, displaying combined symptoms of xeroderma pigmentosum and Cockayne syndrome, referred to as XP/CS complex. Although the molecular basis leading to the XP/CS complex has not yet been fully established, current evidence suggests that these patients suffer from a mild defect in transcription in addition to a repair defect. Here, the history of how the XPG gene was discovered, the biochemical properties of the XPG protein, and the molecular defects found in XP-G patients and mouse models are reviewed.

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Multiple nuclear localization signals in XPG nuclease

Cited by 26 publications

References 31 publications

Ultraviolet-induced movement of the human DNA repair protein, Xeroderma pigmentosum type G, in the nucleus.

Ultraviolet-induced movement of the human DNA repair protein, Xeroderma pigmentosum type G, in the nucleus.

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

XPG: Its Products and Biological Roles

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