scholarly journals Local action of the chromatin assembly factor CAF-1 at sites of nucleotide excision repair in vivo

2003 ◽  
Vol 22 (19) ◽  
pp. 5163-5174 ◽  
Author(s):  
C. M. Green
Keyword(s):  
Nucleotide Excision Repair ◽  
Excision Repair ◽  
Chromatin Assembly ◽  
Local Action ◽  
Chromatin Assembly Factor ◽  
Nucleotide Excision ◽  
Assembly Factor

scholarly journals Essential Role of Chromatin Assembly Factor-1–mediated Rapid Nucleosome Assembly for DNA Replication and Cell Division in Vertebrate Cells

2007 ◽  
Vol 18 (1) ◽  
pp. 129-141 ◽  
Author(s):  
Yasunari Takami ◽  
Tatsuya Ono ◽  
Tatsuo Fukagawa ◽  
Kei-ichi Shibahara ◽  
Tatsuo Nakayama
Keyword(s):  
Dna Replication ◽  
Essential Role ◽  
Chromatin Assembly ◽  
Nuclear Antigen ◽  
Nucleosome Assembly ◽  
Chromatin Assembly Factor ◽  
Assembly Factor

Chromatin assembly factor-1 (CAF-1), a complex consisting of p150, p60, and p48 subunits, is highly conserved from yeast to humans and facilitates nucleosome assembly of newly replicated DNA in vitro. To investigate roles of CAF-1 in vertebrates, we generated two conditional DT40 mutants, respectively, devoid of CAF-1p150 and p60. Depletion of each of these CAF-1 subunits led to delayed S-phase progression concomitant with slow DNA synthesis, followed by accumulation in late S/G2 phase and aberrant mitosis associated with extra centrosomes, and then the final consequence was cell death. We demonstrated that CAF-1 is necessary for rapid nucleosome formation during DNA replication in vivo as well as in vitro. Loss of CAF-1 was not associated with the apparent induction of phosphorylations of S-checkpoint kinases Chk1 and Chk2. To elucidate the precise role of domain(s) in CAF-1p150, functional dissection analyses including rescue assays were preformed. Results showed that the binding abilities of CAF-1p150 with CAF-1p60 and DNA polymerase sliding clamp proliferating cell nuclear antigen (PCNA) but not with heterochromatin protein HP1-γ are required for cell viability. These observations highlighted the essential role of CAF-1–dependent nucleosome assembly in DNA replication and cell proliferation through its interaction with PCNA.

scholarly journals Nucleotide Excision Repair in Human Cells

2013 ◽  
Vol 288 (29) ◽  
pp. 20918-20926 ◽  
Author(s):  
Jinchuan Hu ◽  
Jun-Hyuk Choi ◽  
Shobhan Gaddameedhi ◽  
Michael G. Kemp ◽  
Joyce T. Reardon ◽  
...  
Keyword(s):  
Nucleotide Excision Repair ◽  
Genomic Dna ◽  
Excision Repair ◽  
Human Cells ◽  
Naked Dna ◽  
Nucleotide Excision ◽  
Uv Photoproducts ◽  
Mutant Cells

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.

scholarly journals Sequential Assembly of the Nucleotide Excision Repair Factors In Vivo

2001 ◽  
Vol 8 (1) ◽  
pp. 213-224 ◽  
Author(s):  
Marcel Volker ◽  
Martijn J Moné ◽  
Parimal Karmakar ◽  
Anneke van Hoffen ◽  
Wouter Schul ◽  
...  
Keyword(s):  
Nucleotide Excision Repair ◽  
Excision Repair ◽  
Nucleotide Excision ◽  
Sequential Assembly

scholarly journals Growth Retardation, Early Death, and DNA Repair Defects in Mice Deficient for the Nucleotide Excision Repair Enzyme XPF

2004 ◽  
Vol 24 (3) ◽  
pp. 1200-1205 ◽  
Author(s):  
Ming Tian ◽  
Reiko Shinkura ◽  
Nobuhiko Shinkura ◽  
Frederick W. Alt
Keyword(s):  
Dna Repair ◽  
Nucleotide Excision Repair ◽  
Uv Irradiation ◽  
Excision Repair ◽  
Genetic Defect ◽  
Growth Defect ◽  
Human Genetic Disease ◽  
Nucleotide Excision ◽  
Deficient Mice

ABSTRACT Xeroderma pigmentosum (XP) is a human genetic disease which is caused by defects in nucleotide excision repair. Since this repair pathway is responsible for removing UV irradiation-induced damage to DNA, XP patients are hypersensitive to sunlight and are prone to develop skin cancer. Based on the underlying genetic defect, the disease can be divided into the seven complementation groups XPA through XPG. XPF, in association with ERCC1, constitutes a structure-specific endonuclease that makes an incision 5′ to the photodamage. XPF-ERCC1 has also been implicated in both removal of interstrand DNA cross-links and homology-mediated recombination and in immunoglobulin class switch recombination (CSR). To study the function of XPF in vivo, we inactivated the XPF gene in mice. XPF-deficient mice showed a severe postnatal growth defect and died approximately 3 weeks after birth. Histological examination revealed that the liver of mutant animals contained abnormal cells with enlarged nuclei. Furthermore, embryonic fibroblasts defective in XPF are hypersensitive to UV irradiation and mitomycin C treatment. No defect in CSR was detected, suggesting that the nuclease is dispensable for this recombination process. These phenotypes are identical to those exhibited by the ERCC1-deficient mice, consistent with the functional association of the two proteins. The complex phenotype suggests that XPF-ERCC1 is involved in multiple DNA repair processes.

scholarly journals Recruitment of the Nucleotide Excision Repair Endonuclease XPG to Sites of UV-Induced DNA Damage Depends on Functional TFIIH

2006 ◽  
Vol 26 (23) ◽  
pp. 8868-8879 ◽  
Author(s):  
Angelika Zotter ◽  
Martijn S. Luijsterburg ◽  
Daniël O. Warmerdam ◽  
Shehu Ibrahim ◽  
Alex Nigg ◽  
...  
Keyword(s):  
Dna Damage ◽  
Nucleotide Excision Repair ◽  
Fluorescent Protein ◽  
Core Protein ◽  
Excision Repair ◽  
Mammalian Cell Lines ◽  
Nucleotide Excision ◽  
Green Fluorescent ◽  
Damaged Dna

ABSTRACT The structure-specific endonuclease XPG is an indispensable core protein of the nucleotide excision repair (NER) machinery. XPG cleaves the DNA strand at the 3′ side of the DNA damage. XPG binding stabilizes the NER preincision complex and is essential for the 5′ incision by the ERCC1/XPF endonuclease. We have studied the dynamic role of XPG in its different cellular functions in living cells. We have created mammalian cell lines that lack functional endogenous XPG and stably express enhanced green fluorescent protein (eGFP)-tagged XPG. Life cell imaging shows that in undamaged cells XPG-eGFP is uniformly distributed throughout the cell nucleus, diffuses freely, and is not stably associated with other nuclear proteins. XPG is recruited to UV-damaged DNA with a half-life of 200 s and is bound for 4 min in NER complexes. Recruitment requires functional TFIIH, although some TFIIH mutants allow slow XPG recruitment. Remarkably, binding of XPG to damaged DNA does not require the DDB2 protein, which is thought to enhance damage recognition by NER factor XPC. Together, our data present a comprehensive view of the in vivo behavior of a protein that is involved in a complex chromatin-associated process.

scholarly journals Centrin 2 Stimulates Nucleotide Excision Repair by Interacting with Xeroderma Pigmentosum Group C Protein

2005 ◽  
Vol 25 (13) ◽  
pp. 5664-5674 ◽  
Author(s):  
Ryotaro Nishi ◽  
Yuki Okuda ◽  
Eriko Watanabe ◽  
Toshio Mori ◽  
Shigenori Iwai ◽  
...  
Keyword(s):  
Nucleotide Excision Repair ◽  
Xeroderma Pigmentosum ◽  
Excision Repair ◽  
Physical Interaction ◽  
C Protein ◽  
Damage Recognition ◽  
C Terminus ◽  
Nucleotide Excision ◽  
Dna Damage Recognition

ABSTRACT Xeroderma pigmentosum group C (XPC) protein plays a key role in DNA damage recognition in global genome nucleotide excision repair (NER). The protein forms in vivo a heterotrimeric complex involving one of the two human homologs of Saccharomyces cerevisiae Rad23p and centrin 2, a centrosomal protein. Because centrin 2 is dispensable for the cell-free NER reaction, its role in NER has been unclear. Binding experiments with a series of truncated XPC proteins allowed the centrin 2 binding domain to be mapped to a presumed α-helical region near the C terminus, and three amino acid substitutions in this domain abrogated interaction with centrin 2. Human cell lines stably expressing the mutant XPC protein exhibited a significant reduction in global genome NER activity. Furthermore, centrin 2 enhanced the cell-free NER dual incision and damaged DNA binding activities of XPC, which likely require physical interaction between XPC and centrin 2. These results reveal a novel vital function for centrin 2 in NER, the potentiation of damage recognition by XPC.

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