scholarly journals Identification and characterization of XPC-binding domain of hHR23B.

1997 ◽  
Vol 17 (12) ◽  
pp. 6915-6923 ◽  
Author(s):  
C Masutani ◽  
M Araki ◽  
K Sugasawa ◽  
P J van der Spek ◽  
A Yamada ◽  
...  
Keyword(s):  
Hydrophobic Interactions ◽  
Excision Repair ◽  
Binding Domain ◽  
Cell Extracts ◽  
Nucleotide Excision ◽  
Stimulation Of

hHR23B was originally isolated as a component of a protein complex that specifically complements nucleotide excision repair (NER) defects of xeroderma pigmentosum group C cell extracts in vitro and was identified as one of two human homologs of the Saccharomyces cerevisiae NER gene product Rad23. Recombinant hHR23B has previously been shown to significantly stimulate the NER activity of recombinant human XPC protein (rhXPC). In this study we identify and functionally characterize the XPC-binding domain of hHR23B protein. We prepared various internal as well as terminal deletion products of hHR23B protein in a His-tagged form and examined their binding with rhXPC by using nickel-chelating Sepharose. We demonstrate that a domain covering 56 amino acids of hHR23B is required for binding to rhXPC as well as for stimulation of in vitro NER reactions. Interestingly, a small polypeptide corresponding to the XPC-binding domain is sufficient to exert stimulation of XPC NER activity. Comparison with known crystal structures and analysis with secondary structure programs provided strong indications that the binding domain has a predominantly amphipathic alpha-helical character, consistent with evidence that the affinity with XPC is based on hydrophobic interactions. Our work shows that binding to XPC alone is required and sufficient for the role of hHR23B in in vitro NER but does not rule out the possibility that the protein has additional functions in vivo.

scholarly journals UV radiation-induced SUMOylation of DDB2 regulates nucleotide excision repair

2017 ◽  
Vol 38 (10) ◽  
pp. 976-985 ◽  
Author(s):  
Chunhua Han ◽  
Ran Zhao ◽  
John Kroger ◽  
Jinshan He ◽  
Gulzar Wani ◽  
...  
Keyword(s):  
Dna Damage ◽  
Nucleotide Excision Repair ◽  
Uv Radiation ◽  
Uv Irradiation ◽  
Excision Repair ◽  
26S Proteasome ◽  
Cell Extracts ◽  
Nucleotide Excision

Abstract Subunit 2 of DNA damage-binding protein complex (DDB2) is an early sensor of nucleotide excision repair (NER) pathway for eliminating DNA damage induced by UV radiation (UVR) and cisplatin treatments of mammalian cells. DDB2 is modified by ubiquitin and poly(ADP-ribose) (PAR) in response to UVR, and these modifications play a crucial role in regulating NER. Here, using immuno-analysis of irradiated cell extracts, we have identified multiple post-irradiation modifications of DDB2 protein. Interestingly, although the DNA lesions induced by both UVR and cisplatin are corrected by NER, only the UV irradiation, but not the cisplatin treatment, induces any discernable DDB2 modifications. We, for the first time, show that the appearance of UVR-induced DDB2 modifications depend on the binding of DDB2 to the damaged chromatin and the participation of functionally active 26S proteasome. The in vitro and in vivo analysis revealed that SUMO-1 conjugations comprise a significant portion of these UVR-induced DDB2 modifications. Mapping of SUMO-modified sites demonstrated that UVR-induced SUMOylation occurs on Lys-309 residue of DDB2 protein. Mutation of Lys-309 to Arg-309 diminished the DDB2 SUMOylation observable both in vitro and in vivo. Moreover, K309R mutated DDB2 lost its function of recruiting XPC to the DNA damage sites, as well as the ability to repair cyclobutane pyrimidine dimers following cellular UV irradiation. Taken together, our results indicate that DDB2 is modified by SUMOylation upon UV irradiation, and this post-translational modification plays an important role in the initial recognition and processing of UVR-induced DNA damage occurring within the context of chromatin.

scholarly journals The C-terminal Domain of p21 Inhibits Nucleotide Excision Repair In Vitro and In Vivo

1999 ◽  
Vol 10 (7) ◽  
pp. 2119-2129 ◽  
Author(s):  
Marcus P. Cooper ◽  
Adayabalam S. Balajee ◽  
Vilhelm A. Bohr
Keyword(s):  
Dna Repair ◽  
Nucleotide Excision Repair ◽  
Excision Repair ◽  
Binding Domain ◽  
In Vivo Studies ◽  
C Terminus ◽  
Nucleotide Excision ◽  
N Terminus

The protein p21Cip1, Waf1, Sdi1 is a potent inhibitor of cyclin-dependent kinases (CDKs). p21 can also block DNA replication through its interaction with the proliferating cell nuclear antigen (PCNA), which is an auxiliary factor for polymerase δ. PCNA is also implicated in the repair resynthesis step of nucleotide excision repair (NER). Previous studies have yielded contradictory results on whether p21 regulates NER through its interaction with PCNA. Resolution of this controversy is of interest because it would help understand how DNA repair and replication are regulated. Hence, we have investigated the effect of p21 on NER both in vitro and in vivo using purified fragments of p21 containing either the CDK-binding domain (N terminus) or the PCNA binding domain (C terminus) of the protein. In the in vitro studies, DNA repair synthesis was measured in extracts from normal human fibroblasts using plasmids damaged by UV irradiation. In the in vivo studies, we used intact and permeabilized cells. The results show that the C terminus of the p21 protein inhibits NER both in vitro and in vivo. These are the first in vivo studies in which this question has been examined, and we demonstrate that inhibition of NER by p21 is not merely an artificial in vitro effect. A 50% inhibition of in vitro NER occurred at a 50:1 molar ratio of p21 C-terminus fragment to PCNA monomer. p21 differentially regulates DNA repair and replication, with repair being much less sensitive to inhibition than replication. Our in vivo results suggest that the inhibition occurs at the resynthesis step of the repair process. It also appears that preassembly of PCNA at repair sites mitigates the inhibitory effect of p21. We further demonstrate that the inhibition of DNA repair is mediated via binding of p21 to PCNA. The N terminus of p21 had no effect on DNA repair, and the inhibition of DNA repair by the C terminus of p21 was relieved by the addition of purified PCNA protein.

scholarly journals Mutations in XPA that prevent association with ERCC1 are defective in nucleotide excision repair.

1995 ◽  
Vol 15 (4) ◽  
pp. 1993-1998 ◽  
Author(s):  
L Li ◽  
C A Peterson ◽  
X Lu ◽  
R J Legerski
Keyword(s):  
Nucleotide Excision Repair ◽  
Excision Repair ◽  
Repair Synthesis ◽  
Damage Site ◽  
Cell Extracts ◽  
Nucleotide Excision ◽  
Delta G ◽  
Dna Repair Synthesis

The human repair proteins XPA and ERCC1 have been shown to be absolutely required for the incision step of nucleotide excision repair, and recently we identified an interaction between these two proteins both in vivo and in vitro (L. Li, S. J. Elledge, C. A. Peterson, E. S. Bales, and R. J. Legerski, Proc. Natl. Acad. Sci. USA 91:5012-5016, 1994). In this report, we demonstrate the functional relevance of this interaction. The ERCC1-binding domain on XPA was previously mapped to a region containing two highly conserved XPA sequences, Gly-72 to Phe-75 and Glu-78 to Glu-84, which are termed the G and E motifs, respectively. Site-specific mutagenesis was used to independently delete these motifs and create two XPA mutants referred to as delta G and delta E. In vitro, the binding of ERCC1 to delta E was reduced by approximately 70%, and binding to delta G was undetectable; furthermore, both mutants failed to complement XPA cell extracts in an in vitro DNA repair synthesis assay. In vivo, the delta E mutant exhibited an intermediate level of complementation of XPA cells and the delta G mutant exhibited little or no complementation. In addition, the delta G mutant inhibited repair synthesis in wild-type cell extracts, indicating that it is a dominant negative mutant. The delta E and delta G mutations, however, did not affect preferential binding of XPA to damaged DNA. These results suggest that the association between XPA and ERCC1 is a required step in the nucleotide excision repair pathway and that the probable role of the interaction is to recruit the ERCC1 incision complex to the damage site. Finally, the affinity of the XPA-ERCC1 complex was found to increase as a function of salt concentration, indicating a hydrophobic interaction; the half-life of the complex was determined to be approximately 90 min.

scholarly journals HHR23B, a human Rad23 homolog, stimulates XPC protein in nucleotide excision repair in vitro.

1996 ◽  
Vol 16 (9) ◽  
pp. 4852-4861 ◽  
Author(s):  
K Sugasawa ◽  
C Masutani ◽  
A Uchida ◽  
T Maekawa ◽  
P J van der Spek ◽  
...  
Keyword(s):  
Gene Product ◽  
Nucleotide Excision Repair ◽  
Excision Repair ◽  
General Role ◽  
Cell Extracts ◽  
C Cell ◽  
Nucleotide Excision ◽  
Repair Defect

A protein complex which specifically complements defects of XP-C cell extracts in vitro was previously purified to near homogeneity from HeLa cells. The complex consists of two tightly associated proteins: the XPC gene product and HHR23B, one of two human homologs of the Saccharomyces cerevisiae repair gene product Rad23 (Masutani et al., EMBO J. 13:1831-1843, 1994). To elucidate the roles of these proteins in "genome-overall" repair, we expressed the XPC protein in a baculovirus system and purified it to near homogeneity. The recombinant human XPC (rhXPC) protein exhibited a high level of affinity for single-stranded DNA and corrected the repair defect in XP-C whole-cell extracts without extra addition of recombinant HHR23B (rHHR23B) protein. However, Western blot (immunoblot) experiments revealed that XP-C cell extracts contained excess endogenous HHR23B protein, which might be able to form a complex upon addition of the rhXPC protein. To investigate the role of HHR23B, we fractionated the XP-C cell extracts and constructed a reconstituted system in which neither endogenous XPC nor HHR23B proteins were present. In this assay system, rhXPC alone weakly corrected the repair defect, while significant enhancement of the correcting activity was observed upon coaddition of rHHR23B protein. Stimulation of XPC by HHR23B was found with simian virus 40 minichromosomes as well as with naked plasmid DNA and with UV- as well as N-acetoxy-2- acetylfluorene-induced DNA lesions, indicating a general role of HHR23B in XPC functioning in the genome-overall nucleotide excision repair subpathway.

scholarly journals Role of the Escherichia coli Nucleotide Excision Repair Proteins in DNA Replication

2000 ◽  
Vol 182 (20) ◽  
pp. 5706-5714 ◽  
Author(s):  
Geri F. Moolenaar ◽  
Celine Moorman ◽  
Nora Goosen
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Nucleotide Excision Repair ◽  
Excision Repair ◽  
Inhibitory Effect ◽  
Binding Domain ◽  
Nucleotide Excision ◽  
Polymerase I

ABSTRACT DNA polymerase I (PolI) functions both in nucleotide excision repair (NER) and in the processing of Okazaki fragments that are generated on the lagging strand during DNA replication.Escherichia coli cells completely lacking the PolI enzyme are viable as long as they are grown on minimal medium. Here we show that viability is fully dependent on the presence of functional UvrA, UvrB, and UvrD (helicase II) proteins but does not require UvrC. In contrast, ΔpolA cells grow even better when theuvrC gene has been deleted. Apparently UvrA, UvrB, and UvrD are needed in a replication backup system that replaces the PolI function, and UvrC interferes with this alternative replication pathway. With specific mutants of UvrC we could show that the inhibitory effect of this protein is related to its catalytic activity that on damaged DNA is responsible for the 3′ incision reaction. Specific mutants of UvrA and UvrB were also studied for their capacity to support the PolI-independent replication. Deletion of the UvrC-binding domain of UvrB resulted in a phenotype similar to that caused by deletion of the uvrC gene, showing that the inhibitory incision activity of UvrC is mediated via binding to UvrB. A mutation in the N-terminal zinc finger domain of UvrA does not affect NER in vivo or in vitro. The same mutation, however, does give inviability in combination with the ΔpolA mutation. Apparently the N-terminal zinc-binding domain of UvrA has specifically evolved for a function outside DNA repair. A model for the function of the UvrA, UvrB, and UvrD proteins in the alternative replication pathway is discussed.

scholarly journals Structural studies on M. tuberculosis O6-methylguanine methyltransferase

2014 ◽  
Vol 70 (a1) ◽  
pp. C832-C832
Author(s):  
Menico Rizzi ◽  
Riccardo Miggiano ◽  
Samarpita Lahiri ◽  
Giuseppe Perugino ◽  
Maria Ciaramella ◽  
...  
Keyword(s):  
Excision Repair ◽  
Single Step ◽  
Double Stranded Dna ◽  
Nucleotide Excision ◽  
Enzymatic Systems ◽  
Methylguanine Methyltransferase ◽  
Mutagenic Effects ◽  
Wild Type Form

Mycobacterium tuberculosis (MTB) is an extremely well adapted human pathogen capable to survive for decades inside the hostile environment represented by the host's infected macrophages despite exposure to multiple potential DNA-damaging stresses. In order to maintain a remarkable low level of genetic diversity, MTB deploys different strategies of DNA repair, including multi-enzymatic systems, such as Nucleotide Excision Repair, and single-step repair. In particular, to counteract the mutagenic effects of DNA alkylation, MTB performs the direct alkylated-base reversal by sacrificing one molecule of a DNA-protein alkyltransferase, such as O6-methylguanine methyltransferase (OGT; orf: Rv1316c). We present here the biochemical and structural characterization of recombinant mycobacterial OGT (MtOGT) in its wild-type form along with its mutated variants mimicking the ones occurring in relevant clinical strains (i.e. MtOGT-T15S and MtOGT-R37L). Our studies reveal that MtOGT-R37L is severely impaired in its activity as consequence of its ten-fold lower affinity for modified double-stranded DNA (dsDNA) (1). Further investigations on a new structure-based panel of OGT versions, designed to explore different molecular environment at position 37, allowed us a better understanding of the functional role of the MtOGT Arg37-bearing loop during catalysis. Moreover, we solved the crystal structure of MtOGT in covalent complex with modified dsDNA that reveals an unprecedented MtOGT::DNA architecture, suggesting that the MtOGT monomer performing the catalysis needs assisting unreacted subunits during cooperative DNA binding. This work is supported by European Community FP7 program SYSTEMTB (Health-F4-2010-241587)

Possible role of cyclic GMP in stimulus-secretion coupling by salt gland of the duck

1979 ◽  
Vol 237 (5) ◽  
pp. C200-C204 ◽  
Author(s):  
D. J. Stewart ◽  
J. Sax ◽  
R. Funk ◽  
A. K. Sen
Keyword(s):  
Cyclic Amp ◽  
Guanylate Cyclase ◽  
Cyclic Gmp ◽  
Salt Gland ◽  
Domestic Ducks ◽  
Stimulus Secretion Coupling ◽  
Stimulation Of

Stimulation of salt galnd secretion in domestic ducks in vivo increased the cyclic GMP concentration of the tissue, but had no effect on cyclic AMP levels. Methacholine, which is known to stimulate sodium transport by the glands both in vivo and in vitro, stimulated ouabain-sensitive respiration in salt gland slices. Cyclic GMP stimulated ouabain-sensitive respiration to the same extent as methacholine. Guanylate cyclase stimulators, hydroxylamine and sodium azide, also stimulated ouabain-sensitive respiration. The stimulation of ouabain-sensitive respiration by methacholine was blocked either by atropine or by removal of calcium from the incubation medium. The stimulation of ouabain-sensitive respiration by cyclic GMP still occurred in the absence of calcium. The above observations seem to indicate that cyclic GMP acts as a tertiary link in the process of stimulus-secretion coupling in the tissue.

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.

Polar head groups are important for barrier-protective effects of oxidized phospholipids on pulmonary endothelium

2007 ◽  
Vol 292 (4) ◽  
pp. L924-L935 ◽  
Author(s):  
Anna A. Birukova ◽  
Panfeng Fu ◽  
Santipongse Chatchavalvanich ◽  
Dylan Burdette ◽  
Olga Oskolkova ◽  
...  
Keyword(s):  
Protective Effects ◽  
Oxidized Phospholipids ◽  
Barrier Dysfunction ◽  
Polar Head ◽  
Cell Counts ◽  
Head Groups ◽  
Stimulation Of

We have previously described protective effects of oxidized 1-palmitoyl-2-arachidonoyl- sn-glycero-3-phosphocholine (OxPAPC) on pulmonary endothelial cell (EC) barrier function and demonstrated the critical role of cyclopentenone-containing modifications of arachidonoyl moiety in OxPAPC protective effects. In this study we used oxidized phosphocholine (OxPAPC), phosphoserine (OxPAPS), and glycerophosphate (OxPAPA) to investigate the role of polar head groups in EC barrier-protective responses to oxidized phospholipids (OxPLs). OxPAPC and OxPAPS induced sustained barrier enhancement in pulmonary EC, whereas OxPAPA caused a transient protective response as judged by measurements of transendothelial electrical resistance (TER). Non-OxPLs showed no effects on TER levels. All three OxPLs caused enhancement of peripheral EC actin cytoskeleton. OxPAPC and OxPAPS completely abolished LPS-induced EC hyperpermeability in vitro, whereas OxPAPA showed only a partial protective effect. In vivo, intravenous injection of OxPAPS or OxPAPC (1.5 mg/kg) markedly attenuated increases in the protein content, cell counts, and myeloperoxidase activities detected in bronchoalveolar lavage fluid upon intratracheal LPS instillation in mice, although OxPAPC showed less potency. All three OxPLs partially attenuated EC barrier dysfunction induced by IL-6 and thrombin. Their protective effects against thrombin-induced EC barrier dysfunction were linked to the attenuation of the thrombin-induced Rho pathway of EC hyperpermeability and stimulation of Rac-mediated mechanisms of EC barrier recovery. These results demonstrate for the first time the essential role of polar OxPL groups in blunting the LPS-induced EC dysfunction in vitro and in vivo and suggest the mechanism of agonist-induced hyperpermeability attenuation by OxPLs via reduction of Rho and stimulation of Rac signaling.

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