Defining Amino Acid Residues Involved in DNA−Protein Interactions and Revelation of 3‘-Exonuclease Activity in Endonuclease V†

Biochemistry ◽  
2005 ◽  
Vol 44 (34) ◽  
pp. 11486-11495 ◽  
Author(s):  
Hong Feng ◽  
Liang Dong ◽  
Athena M. Klutz ◽  
Nima Aghaebrahim ◽  
Weiguo Cao
Keyword(s):  
Amino Acid ◽  
Protein Interactions ◽  
Exonuclease Activity ◽  
Amino Acid Residues ◽  
Endonuclease V ◽  
Dna Protein Interactions

scholarly journals Mutation avoidance and DNA repair proficiency in Ustilago maydis are differentially lost with progressive truncation of the REC1 gene product.

1995 ◽  
Vol 15 (10) ◽  
pp. 5329-5338 ◽  
Author(s):  
K Onel ◽  
M P Thelen ◽  
D O Ferguson ◽  
R L Bennett ◽  
W K Holloman
Keyword(s):  
Amino Acid ◽  
Mutation Rate ◽  
Spontaneous Mutation ◽  
Ustilago Maydis ◽  
Radiation Sensitivity ◽  
Open Reading Frame ◽  
Exonuclease Activity ◽  
Amino Acid Residues ◽  
Spontaneous Mutation Rate ◽  
Reading Frame

The REC1 gene of Ustilago maydis has an uninterrupted open reading frame, predicted from the genomic sequence to encode a protein of 522 amino acid residues. Nevertheless, an intron is present, and functional activity of the gene in mitotic cells requires an RNA processing event to remove the intron. This results in a change in reading frame and production of a protein of 463 amino acid residues. The 3'-->5' exonuclease activity of proteins derived from the REC1 genomic open reading frame, the intronless open reading frame, and several mutants was investigated. The mutants included a series of deletions constructed by removing restriction fragments at the 3' end of the cloned REC1 gene and a set of mutant alleles previously isolated in screens for radiation sensitivity. All of these proteins were overproduced in Escherichia coli as N-terminal polyhistidine-tagged fusions that were subsequently purified by immobilized metal affinity chromatography and assayed for 3'-->5' exonuclease activity. The results indicated that elimination of the C-terminal third of the protein did not result in a serious reduction in 3'-->5' exonuclease activity, but deletion into the midsection caused a severe loss of activity. The biological activity of the rec1-1 allele, which encodes a truncated polypeptide with full 3'-->5' exonuclease activity, and the rec1-5 allele, which encodes a more severely truncated polypeptide with no exonuclease activity, was investigated. The two mutants were equally sensitive to the lethal effect of UV light, but the spontaneous mutation rate was elevated 10-fold over the wild-type rate in the rec1-1 mutant and 100-fold in the rec1-5 mutant. The elevated spontaneous mutation rate correlated with the ablation of exonuclease activity, but the radiation sensitivity did not. These results indicate that the C-terminal portion of the Rec1 protein is not essential for exonuclease activity but is crucial in the role of REC1 in DNA damage repair.

scholarly journals Peptide recognition and dephosphorylation by the vaccinia VH1 phosphatase

2020 ◽  
Author(s):  
Bryan M. Zhao ◽  
Megan Hogan ◽  
Michael S Lee ◽  
Beverly K. Dyas ◽  
Robert G. Ulrich
Keyword(s):  
Amino Acid ◽  
Protein Interactions ◽  
Catalytic Site ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Protein Protein Interactions ◽  
Host Cell Proteins ◽  
Enzyme Substrate ◽  
Dual Specificity ◽  
Contact Residues

ABSTRACTThe VH1 protein encoded by the highly conserved H1 locus of orthopoxviruses is a dual-specificity phosphatase (DUSPs) that hydrolyzes phosphate groups from phosphorylated tyrosine, serine, and threonine residues of viral and host cell proteins. Because the DUSP activities are required for virus replication, VH1 is a prime target for the development of therapeutic inhibitors. However, the presentation of a shallow catalytic site has thwarted all drug development efforts. As an alternative to direct targeting of catalytic pockets, we describe surface contacts between VH1 and substrates that are essential for full activity and provide a new pathway for developing inhibitors of protein-protein interactions. Critical amino acid residues were manipulated by site-directed mutagenesis of VH1, and perturbation of peptide substrate interactions based on these mutations were assessed by high-throughput assays that employed surface plasmon resonance and phosphatase activities. Two positively-charged residues (Lys-20 and Lys-22) and the hydrophobic side chain of Met-60 appear to orient the polarity of the pTyr peptide on the VH1 surface, while additional amino acid residues that flank the catalytic site contribute to substrate recognition and productive dephosphorylation. We propose that the enzyme-substrate contact residues described here may serve as molecular targets for the development of inhibitors that specifically block VH1 catalytic activity and thus poxvirus replication.

scholarly journals Insights on cross-species transmission of SARS-CoV-2 from structural modeling

2020 ◽  
Author(s):  
João PGLM Rodrigues ◽  
Susana Barrera-Vilarmau ◽  
João MC Teixeira ◽  
Elizabeth Seckel ◽  
Panagiotis Kastritis ◽  
...  
Keyword(s):  
Amino Acid ◽  
Severe Acute Respiratory Syndrome ◽  
Protein Interactions ◽  
Animal Species ◽  
Host Protein ◽  
Spike Protein ◽  
Computational Studies ◽  
Wild Animals ◽  
Amino Acid Residues ◽  
Global Pandemic

AbstractSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the ongoing global pandemic that has infected more than 14 million people in more than 180 countries worldwide. Like other coronaviruses, SARS-CoV-2 is thought to have been transmitted to humans from wild animals. Given the scale and widespread geographical distribution of the current pandemic, the question emerges whether human-to-animal transmission is possible and if so, which animal species are most at risk. Here, we investigated the structural properties of several ACE2 orthologs bound to the SARS-CoV-2 spike protein. We found that species known not to be susceptible to SARS-CoV-2 infection have non-conservative mutations in several ACE2 amino acid residues that disrupt key polar and charged contacts with the viral spike protein. Our models also predict affinity-enhancing mutations that could be used to design ACE2 variants for therapeutic purposes. Finally, our study provides a blueprint for modeling viral-host protein interactions and highlights several important considerations when designing these computational studies and analyzing their results.

Molecular Cloning of TSARG6 Gene Related to Apoptosis in Human Spermatogenic Cells

2004 ◽  
Vol 36 (2) ◽  
pp. 93-98 ◽  
Author(s):  
Gang Liu ◽  
Guang-Xiu Lu ◽  
Xiao-Wei Xing
Keyword(s):  
Amino Acid ◽  
Protein Interactions ◽  
Critical Role ◽  
Human Testis ◽  
Amino Acid Residues ◽  
Spermatogenic Cells ◽  
Protein Protein Interactions ◽  
New Gene ◽  
Pcr Technology ◽  
Dnak Protein

Abstract Beginning from a mouse EST (GenBank accession No. BE644537) which was significantly up-regulated in cryptorchidism and represented a novel gene, we cloned a new gene (GenBank accession No. AY138810) which is related to apoptosis in human spermatogenic cells by means of GeneScan program and PCR technology. The gene whose full cDNA length is 1875 bp containing 8 exons and 7 introns is located in human chromosome 11q13.3. Its protein containing 316 amino acid residues is a new member of HSP40 protein family because the sequence contains the highly conserved J domain which is present in all DnaJ-like proteins and is considered to have a critical role in DnaJ-DnaK protein-protein interactions. TSARG6 protein displays a 45% identity in a 348-amino acid overlap with DJB5_HUMAN protein. The result of RT-PCR and Northern blot analysis showed that TSARG6 is specifically expressed in adult testis and the transcript is 1.8 kb. Based upon all these observations, it is considered that we cloned a new gene which probably inhibited human testis spermatogenesis apoptosis.

Amino Acid Residues ofEscherichia coliAcyl Carrier Protein Involved in Heterologous Protein Interactions†

Biochemistry ◽  
2003 ◽  
Vol 42 (1) ◽  
pp. 167-176 ◽  
Author(s):  
Lesa M. S. Worsham ◽  
Laurie Earls ◽  
Carrie Jolly ◽  
Keisha Gordon Langston ◽  
M. Stephen Trent ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Interactions ◽  
Heterologous Protein ◽  
Carrier Protein ◽  
Amino Acid Residues

Theoretical affinity order among flavonoids and amino acid residues: An approach to understand flavonoid–protein interactions

2007 ◽  
Vol 819 (1-3) ◽  
pp. 121-129 ◽  
Author(s):  
Edelsys Codorniu-Hernández ◽  
Alberto Rolo-Naranjo ◽  
Luis A. Montero-Cabrera
Keyword(s):  
Amino Acid ◽  
Protein Interactions ◽  
Amino Acid Residues
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