The Experts below are selected from a list of 6831 Experts worldwide ranked by ideXlab platform
Anindya Dutta - One of the best experts on this subject based on the ideXlab platform.
-
The Evolutionarily Conserved Zinc Finger Motif in the Largest Subunit of Human Replication Protein A Is Required for DNA
2013Co-Authors: Anindya DuttaAbstract:The largest subunit of the replication protein A (RPA) contains an evolutionarily conserved Zinc Finger Motif that lies outside of the domains required for binding to single-stranded DNA or forming the RPA holocomplex. In previous studies, we showed that a point mutation in this Motif (RPA m) cannot support SV40 DNA replication. We have now investigated the role of this Motif in several steps of DNA replication and in two DNA repair pathways. RPA m associates with T antigen, assists the unwinding of double-stranded DNA at an origin of replication, stimulates DNA polymerases � and �, and supports the formation of the initial short Okazaki fragments. However, the synthesis of a leading strand and later Okazaki fragments is impaired. In contrast, RPA m can function well during the incision step of nucleotid
-
the evolutionarily conserved Zinc Finger Motif in the largest subunit of human replication protein a is required for dna replication and mismatch repair but not for nucleotide excision repair
Journal of Biological Chemistry, 1998Co-Authors: Mahmud K K Shivji, Clark C Chen, Richard D Kolodner, Richard D Wood, Anindya DuttaAbstract:Abstract The largest subunit of the replication protein A (RPA) contains an evolutionarily conserved Zinc Finger Motif that lies outside of the domains required for binding to single-stranded DNA or forming the RPA holocomplex. In previous studies, we showed that a point mutation in this Motif (RPAm) cannot support SV40 DNA replication. We have now investigated the role of this Motif in several steps of DNA replication and in two DNA repair pathways. RPAm associates with T antigen, assists the unwinding of double-stranded DNA at an origin of replication, stimulates DNA polymerases α and δ, and supports the formation of the initial short Okazaki fragments. However, the synthesis of a leading strand and later Okazaki fragments is impaired. In contrast, RPAm can function well during the incision step of nucleotide excision repair and in a full repair synthesis reaction, with either UV-damaged or cisplatin-adducted DNA. Two deletion mutants of the Rpa1 subunit (eliminating amino acids 1–278 or 222–411) were not functional in nucleotide excision repair. We report for the first time that wild type RPA is required for a mismatch repair reaction in vitro. Neither the deletion mutants nor RPAm can support this reaction. Therefore, the Zinc Finger of the largest subunit of RPA is required for a function that is essential for DNA replication and mismatch repair but not for nucleotide excision repair.
-
the evolutionarily conserved Zinc Finger Motif in the largest subunit of human replication protein a is required for dna replication and mismatch repair but not for nucleotide excision repair
Journal of Biological Chemistry, 1998Co-Authors: Yiling Lin, Mahmud K K Shivji, Clark C Chen, Richard D Kolodner, Richard D Wood, Anindya DuttaAbstract:The largest subunit of the replication protein A (RPA) contains an evolutionarily conserved Zinc Finger Motif that lies outside of the domains required for binding to single-stranded DNA or forming the RPA holocomplex. In previous studies, we showed that a point mutation in this Motif (RPAm) cannot support SV40 DNA replication. We have now investigated the role of this Motif in several steps of DNA replication and in two DNA repair pathways. RPAm associates with T antigen, assists the unwinding of double-stranded DNA at an origin of replication, stimulates DNA polymerases alpha and delta, and supports the formation of the initial short Okazaki fragments. However, the synthesis of a leading strand and later Okazaki fragments is impaired. In contrast, RPAm can function well during the incision step of nucleotide excision repair and in a full repair synthesis reaction, with either UV-damaged or cisplatin-adducted DNA. Two deletion mutants of the Rpa1 subunit (eliminating amino acids 1-278 or 222-411) were not functional in nucleotide excision repair. We report for the first time that wild type RPA is required for a mismatch repair reaction in vitro. Neither the deletion mutants nor RPAm can support this reaction. Therefore, the Zinc Finger of the largest subunit of RPA is required for a function that is essential for DNA replication and mismatch repair but not for nucleotide excision repair.
Debi P Nayak - One of the best experts on this subject based on the ideXlab platform.
-
mutations in influenza virus m1 cchh the putative Zinc Finger Motif cause attenuation in mice and protect mice against lethal influenza virus infection
Journal of Virology, 2006Co-Authors: Eric Kawai Hui, Donald F Smee, Minhui Wong, Debi P NayakAbstract:Mutations in CCHH, the putative Zinc Finger Motif, apparently do not play an important role in virus replication in MDCK cells in culture (E. K.-W. Hui, K. Ralston, A. K. Judd, and D. P. Nayak, J. Gen. Virol. 84:3105-3113, 2003). In this report, however, we demonstrate that the CCHH Motif plays a critical role in virulence in mice and that some CCHH mutants are highly attenuated in BALB/c mice. Some of the mutant viruses replicated the least in mice lungs, induced little or no lung lesions, and caused highly reduced morbidity and mortality. Furthermore, growth patterns of mutant viruses in different cell lines (MDCK, MLE12, 3LL, A549, and 293T) varied. Mutant viruses that were attenuated in mice also grew poorly in mouse and human cells in culture. However, wild-type (WT) and all mutant viruses replicated to the same titer in MDCK (canine) cells or embryonated chicken eggs. Attenuation in mice correlated with reduced growth in mouse cells in culture, suggesting that potential attenuation in a given host can be predicted from the growth characteristics of the virus in cultured cells (preferably lung cells) from the same species. In challenge experiments, mice immunized by infection with attenuated mutant viruses were fully protected from lethal challenge with WT virus. In summary, the replication and attenuating properties of these mutants suggest that the CCHH Motif provides a critical determinant for virulence in mouse and that mutations in the CCHH Motif yield potential vaccine candidates for the development of live species-specific attenuated influenza virus vaccines.
-
conserved cysteine and histidine residues in the putative Zinc Finger Motif of the influenza a virus m1 protein are not critical for influenza virus replication
Journal of General Virology, 2003Co-Authors: Eric Kawai Hui, Katherine S Ralston, Amrit K Judd, Debi P NayakAbstract:The influenza virus matrix protein (M1) possesses a cysteine and histidine (CCHH) Motif in the helix 9 (H9) and adjacent region (148 CATCEQIADSQHRSH 162). The CCHH Motif has been proposed as a putative Zinc Finger Motif and Zinc-binding activity has been implicated in virus uncoating as well as transcription inhibition and mRNA regulation. The function of the CCHH Motif in the influenza virus life cycle was investigated by site-directed mutagenesis (alanine replacement) and by rescuing mutant viruses by reverse genetics. Mutant viruses containing an alanine replacement of the cysteine and histidine residues, either individually or in combination, were seen to exhibit wt phenotype in multiple virus growth cycles and plaque morphology. In addition, synthetic peptides containing the putative Zinc Finger Motif did not inhibit virus replication in MDCK cells. However, mutation of Ala155 in H9 was lethal for rescuing infectious virus. These data show that the CCHH Motif does not provide a critical function in the influenza virus life cycle in cell culture and that the Zinc-binding function may not be involved in virus biology. However, the lethal phenotype of the Ala155 mutation shows that the H9 region of M1 provides some other critical function(s) in virus replication.
Ira G Wool - One of the best experts on this subject based on the ideXlab platform.
-
the role of the Zinc Finger Motif and of the residues at the amino terminus in the function of yeast ribosomal protein yl37a
Journal of Molecular Biology, 2002Co-Authors: John Dresios, Yuenling Chan, Ira G WoolAbstract:Abstract YL37a is an essential yeast ribosomal protein that has a C2-C2 Zinc Finger Motif. Replacement of the cysteine residues had yielded variants that lacked the capacity to bind Zinc but still supported cell growth. In a continuation of an examination of the relation of the structure of YL37a to its function, the contribution of amino acid residues in the intervening sequence between the internal cysteine residues of the Motif was evaluated. Substitutions of alanine for the lysine residues at positions 44, 45, or 48, or for arginine 49 slowed cell growth. The most severe effect was caused by a double-mutation, K48A-R49A. A mutation of tryptophan 55 to alanine was lethal. Mutations to alanine of six conserved residues (K6, K7, K13, Y14, R17, and Y18) in the amino-terminal region decreased cell growth; the Y14 mutation was lethal. An in vitro assay for binding of YL37a to individual 26 S rRNA domains was developed. Binding of the recombinant fusion protein MBP-YL37a was to domains II and III; the Kd for binding to domain II was 79 nM; for domain III it was 198 nM. There was a close correspondence between the effect of mutations in YL37a on cell growth and on binding to 26 S rRNA. In the atomic structure of the 50 S subunit of Haloarcula marismortui, the archaebacteria homolog of yeast YL37a, L37ae, coordinates a Zinc atom and the Finger Motif is folded and interacts mainly with domain III of 23 S rRNA; whereas the amino-terminal region of L37ae interacts primarily with domain II. The biochemical and genetic experiments complement the three-dimensional structure and define for the first time the functional importance of a subset of the residues in close proximity to nucleotides.
-
the contribution of a Zinc Finger Motif to the function of yeast ribosomal protein yl37a
Journal of Molecular Biology, 1999Co-Authors: Anatoly A Rivlin, Yuenling Chan, Ira G WoolAbstract:Abstract Eukaryotic ribosomes have a large number of proteins but the exact nature of their contribution to the structure and to the function of the particle is not known. Of the 78 proteins in yeast ribosomes, six have Zinc Finger Motifs of the C2-C2 variety. Both genes encoding the essential yeast ribosomal protein YL37a, which has such a Zinc Finger Motif, were disrupted. The double deletion, which is lethal, can be rescued with a plasmid-encoded copy of a YL37a gene. Mutations were constructed in a plasmid-encoded copy of YL37a; the mutations caused the cysteine residues in the Motif (at positions 39, 42, 57 and 60) to be replaced, one at a time, with serine. The cysteine residue at position 39, the first of the four in the Motif, is essential for the function of YL37a, since a C39S mutation did not complement the null phenotype. However, plasmids encoding variants with C42S, C57S, or C60S mutations in the Zinc Finger Motif were able to rescue the null mutant. YL37a binds Zinc, but none of the mutant proteins, C39S, C42S, C57S, or C60S, was able to bind the metal. Thus, all four cysteine residues are essential for the binding of Zinc; only one, C39, is essential for the function of the ribosomal protein.
Tohru Marunouchi - One of the best experts on this subject based on the ideXlab platform.
-
NZF-2b is a novel predominant form of mouse NZF-2/MyT1, expressed in differentiated neurons especially at higher levels in newly generated ones.
Mechanisms of Development, 2002Co-Authors: Fumio Matsushita, Toshiki Kameyama, Tohru MarunouchiAbstract:NZF-2 (MyT1) is a member of C2HC-type Zinc Finger transcription factors. A novel form of mouse NZF-2 has been isolated. This novel form, NZF-2b, has an additional C2HC-type Zinc Finger Motif. The expression levels of NZF-2b are by far the more predominant than those of the already known form of NZF-2. In embryonic mouse nervous system, the expression of NZF-2b starts as early as at 9.5 days post-coitum (dpc) in newly differentiated neurons in the central nervous system (CNS) and the peripheral nervous system (PNS).
-
Gene expression pattern NZF-2b is a novel predominant form of mouse NZF-2/MyT1, expressed in differentiated neurons especially at higher levels in newly generated ones
2002Co-Authors: Fumio Matsushita, Toshiki Kameyama, Tohru MarunouchiAbstract:NZF-2 (MyT1) is a member of C2HC-type Zinc Finger transcription factors. A novel form of mouse NZF-2 has been isolated. This novel form, NZF-2b, has an additional C2HC-type Zinc Finger Motif. The expression levels of NZF-2b are by far the more predominant than those of the already known form of NZF-2. In embryonic mouse nervous system, the expression of NZF-2b starts as early as at 9.5 days post-coitum (dpc) in newly differentiated neurons in the central nervous system (CNS) and the peripheral nervous system (PNS). q 2002 Elsevier Science Ireland Ltd. All rights reserved.
Suk Hee Lee - One of the best experts on this subject based on the ideXlab platform.
-
role of Zinc Finger Motif in redox regulation of human replication protein a
Antioxidants & Redox Signaling, 2001Co-Authors: Mu Wang, Jinsam You, Suk Hee LeeAbstract:Replication protein A (RPA) is a heterotrimeric Zinc-Finger protein complex involved in DNA replication, repair, and genetic recombination. Unlike other Zinc-Finger proteins, RPA's Zinc-Finger Motif is not essential for its single-stranded DNA (ssDNA) binding activity, but is involved in redox regulation of its single-stranded DNA (ssDNA) binding activity. To get an insight into the regulation of RPA-ssDNA interaction, wild-type RPA (wt-RPA) and Zinc-Finger mutant were examined for ssDNA binding activity using surface plasmon resonance technique. Interaction of wt-RPA with ssDNA under nonreducing conditions was very weak (KD × 2.33 × 10-8 M) compared with that under reducing conditions (KD = 7.35 × 10-11 M), whereas ssDNA binding affinity of the Zinc-Finger mutant was not affected by redox. The divalent ion chelator, o-phenanthroline, significantly reduced wt-RPA-ssDNA interaction, but had no effect on the Zinc-Finger mutant. The inhibitory effect of o-phenanthroline on RPA-ssDNA interaction was reversed ...
-
functional characterization of Zinc Finger Motif in redox regulation of rpa ssdna interaction
Biochemistry, 2000Co-Authors: Jinsam You, Mu Wang, Suk Hee LeeAbstract:The 70-kDa subunit of eukaryotic replication protein A (RPA) contains a conserved four cysteine-type Zinc-Finger Motif that has been implicated in regulation of DNA replication and repair. Unlike other Zinc-Finger proteins, RPA Zinc-Finger Motif is not a DNA-binding component, and deletion of the Zinc-Finger had very little effect on its ssDNA binding activity. Recently, we described a novel function for the Zinc-Finger Motif in regulation of RPA's ssDNA binding activity through reduction−oxidation (redox). In this study, we carried out a detailed analysis of wild-type RPA and Zinc-Finger mutants in redox regulation of their ssDNA binding activity. Any mutation at a Zinc-Finger cysteine abolished its redox role in regulation of RPA−ssDNA interaction, suggesting that all four Zinc-Finger cysteines are required for redox regulation. Reactivity of cysteine residues to 5,5‘-dithiobis(2-nitrobenzoic acid) (DTNB) indicated that wild-type RPA contained 8.2 reactive thiols/molecule including all four cysteines in...
-
in vitro analysis of the Zinc Finger Motif in human replication protein a
Biochemical Journal, 1999Co-Authors: Jiaowang Dong, Jangsu Park, Suk Hee LeeAbstract:Human replication protein A (RPA) is composed of 70, 34 and 11 kDa subunits (p70, p34 and p11 respectively) and functions in all three major DNA metabolic processes: replication, repair and recombination. Recent deletion analysis demonstrated that the large subunit of RPA, p70, has multiple functional domains, including a DNA polymerase alpha-stimulation domain and a single-stranded DNA-binding domain. It also contains a putative metal-binding domain of the 4-cysteine type (Cys-Xaa4-Cys-Xaa13-Cys-Xaa2-Cys) that is highly conserved among eukaryotes. To study the role of this domain in DNA metabolism, we created various p70 mutants that lack the Zinc-Finger Motif (by Cys-->Ala substitutions). Mutation at the Zinc-Finger domain (ZFM) abolished RPA's function in nucleotide excision repair (NER), but had very little impact on DNA replication. The failure of Zinc-Finger mutant RPA in NER may be explained by the observation that wild-type RPA significantly stimulated DNA polymerase delta activity, whereas only marginal stimulation was observed with Zinc-Finger mutant RPA. We also observed that ZFM reduced RPA's single-stranded DNA-binding activity by 2-3-fold in the presence of low amounts of RPA. Interestingly, the ZFM abolished phosphorylation of the p34 subunit by DNA-dependent protein kinase, but not that by cyclin-dependent kinase. Taker together, our results strongly suggest a positive role for RPA's Zinc Finger domain in its function.
