The C-terminal domain is sufficient for endonuclease activity of Neisseria gonorrhoeae MutL

2009 ◽  
Vol 423 (2) ◽  
pp. 265-277 ◽  
Author(s):  
Viswanadham Duppatla ◽  
Chiranjeevi Bodda ◽  
Claus Urbanke ◽  
Peter Friedhoff ◽  
Desirazu N. Rao
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Metal Binding ◽  
Sedimentation Equilibrium ◽  
Velocity Sedimentation ◽  
Binding Motif ◽  
Endonuclease Activity ◽  
Dna Mismatch ◽  
Terminal Domain ◽  
Dependent Endonuclease

The mutL gene of Neisseria gonorrhoeae has been cloned and the gene product purified. We have found that the homodimeric N. gonorrhoeae MutL (NgoL) protein displays an endonuclease activity that incises covalently closed circular DNA in the presence of Mn2+, Mg2+ or Ca2+ ions, unlike human MutLα which shows endonuclease activity only in the presence of Mn2+. We report in the present paper that the C-terminal domain of N. gonorrhoeae MutL (NgoL-CTD) consisting of amino acids 460–658 exhibits Mn2+-dependent endonuclease activity. Sedimentation velocity, sedimentation equilibrium and dynamic light scattering experiments show NgoL-CTD to be a dimer. The probable endonucleolytic active site is localized to a metal-binding motif, DMHAX2EX4E, and the nicking endonuclease activity is dependent on the integrity of this motif. By in vitro comparison of wild-type and a mutant NgoL-CTD protein, we show that the latter protein exhibits highly reduced endonuclease activity. We therefore suggest that the mode of excision initiation in DNA mismatch repair may be different in organisms that lack MutH protein, but have MutL proteins that harbour the D[M/Q]HAX2EX4E motif.

scholarly journals Yeast PIAS-type Ull1/Siz1 Is Composed of SUMO Ligase and Regulatory Domains

2005 ◽  
Vol 280 (43) ◽  
pp. 35822-35828 ◽  
Author(s):  
Yoshimitsu Takahashi ◽  
Yoshiko Kikuchi
Keyword(s):  
Neck Region ◽  
Binding Motif ◽  
Conjugation System ◽  
Regulatory Domains ◽  
Terminal Domain ◽  
Sumo Ligase ◽  
Lysine Residues ◽  
Protein Instability ◽  
Two Hybrid

SUMO (small ubiquitin-like modifier)/Smt3 (suppressor of mif two) is a member of the ubiquitin-related protein family and is known to conjugate with many proteins. In the sumoylation pathway, SUMO/Smt3 is transferred to substrate lysine residues through the thioester cascade of E1 (activating enzyme) and E2 (conjugating enzyme), and E3 (SUMO ligase) functions as an adaptor between E2 and each substrate. Yeast Ull1 (ubiquitin-like protein ligase 1)/Siz1, a PIAS (protein inhibitor of activated STAT)-type SUMO ligase, modifies both cytoplasmic and nuclear proteins. In this paper, we performed a domain analysis of Ull1/Siz1 by constructing various deletion mutants. A novel conserved N-terminal domain, called PINIT, as well as the RING-like domain (SP-RING) were required for the SUMO ligase activity in the in vitro conjugation system and for interaction with Smt3 in an in vitro binding assay. The most distal N-terminal region, which contains a putative DNA-binding SAF-A/B, Acinus, and PIAS (SAP) motif, was not required for the ligase activity but was involved in nuclear localization. A strong SUMO-binding motif was identified, which interacted with Smt3 in the two-hybrid system but was not necessary for the ligase activity. The most distal C-terminal domain was important for stable localization at the bud neck region and thereby for the substrate recognition of septins. Furthermore, the C-terminal half conferred protein instability on Ull1/Siz1. Taken together, we conclude that the SP-RING and PINIT of Ull1/Siz1 are core domains of the SUMO ligase, and the other domains are regulatory for protein stability and subcellular localization.

scholarly journals Structural insights into how GTP-dependent conformational changes in a metallochaperone UreG facilitate urease maturation

2017 ◽  
Vol 114 (51) ◽  
pp. E10890-E10898 ◽  
Author(s):  
Man Hon Yuen ◽  
Yu Hang Fong ◽  
Yap Shing Nim ◽  
Pak Ho Lau ◽  
Kam-Bo Wong
Keyword(s):  
Conformational Changes ◽  
Metal Binding ◽  
Sole Source ◽  
Planar Geometry ◽  
Binding Motif ◽  
Nickel Ion ◽  
Gtp Hydrolysis ◽  
Nickel Ions ◽  
Gtp Binding

The ability of metallochaperones to allosterically regulate the binding/release of metal ions and to switch protein-binding partners along the metal delivery pathway is essential to the metallation of the metalloenzymes. Urease, catalyzing the hydrolysis of urea into ammonia and carbon dioxide, contains two nickel ions bound by a carbamylated lysine in its active site. Delivery of nickel ions for urease maturation is dependent on GTP hydrolysis and is assisted by four urease accessory proteins UreE, UreF, UreG, and UreH(UreD). Here, we determined the crystal structure of the UreG dimer from Klebsiella pneumoniae in complex with nickel and GMPPNP, a nonhydrolyzable analog of GTP. Comparison with the structure of the GDP-bound Helicobacter pylori UreG (HpUreG) in the UreG2F2H2 complex reveals large conformational changes in the G2 region and residues near the 66CPH68 metal-binding motif. Upon GTP binding, the side chains of Cys66 and His68 from each of the UreG protomers rotate toward each other to coordinate a nickel ion in a square-planar geometry. Mutagenesis studies on HpUreG support the conformational changes induced by GTP binding as essential to dimerization of UreG, GTPase activity, in vitro urease activation, and the switching of UreG from the UreG2F2H2 complex to form the UreE2G2 complex with the UreE dimer. The nickel-charged UreE dimer, providing the sole source of nickel, and the UreG2F2H2 complex could activate urease in vitro in the presence of GTP. Based on our results, we propose a mechanism of how conformational changes of UreG during the GTP hydrolysis/binding cycle facilitate urease maturation.

scholarly journals Characterization of the RNA-Binding Domains in the Replicase Proteins of Tomato Bushy Stunt Virus

2003 ◽  
Vol 77 (17) ◽  
pp. 9244-9258 ◽  
Author(s):  
K. S. Rajendran ◽  
Peter D. Nagy
Keyword(s):  
Rna Binding ◽  
Tomato Bushy Stunt Virus ◽  
Binding Motif ◽  
Mobility Shift ◽  
Terminal Domain ◽  
Binding Domains ◽  
Replicase Proteins ◽  
Rna Binding Domains ◽  
Gel Mobility Shift

ABSTRACT Tomato bushy stunt virus (TBSV), a tombusvirus with a nonsegmented, plus-stranded RNA genome, codes for two essential replicase proteins. The sequence of one of the replicase proteins, namely p33, overlaps with the N-terminal domain of p92, which contains the signature motifs of RNA-dependent RNA polymerases (RdRps) in its nonoverlapping C-terminal portion. In this work, we demonstrate that both replicase proteins bind to RNA in vitro based on gel mobility shift and surface plasmon resonance measurements. We also show evidence that the binding of p33 to single-stranded RNA (ssRNA) is stronger than binding to double-stranded RNA (dsRNA), ssDNA, or dsDNA in vitro. Competition experiments with ssRNA revealed that p33 binds to a TBSV-derived sequence with higher affinity than to other nonviral ssRNA sequences. Additional studies revealed that p33 could bind to RNA in a cooperative manner. Using deletion derivatives of the Escherichia coli-expressed recombinant proteins in gel mobility shift and Northwestern assays, we demonstrate that p33 and the overlapping domain of p92, based on its sequence identity with p33, contain an arginine- and proline-rich RNA-binding motif (termed RPR, which has the sequence RPRRRP). This motif is highly conserved among tombusviruses and related carmoviruses, and it is similar to the arginine-rich motif present in the Tat trans-activator protein of human immunodeficiency virus type 1. We also find that the nonoverlapping C-terminal domain of p92 contains additional RNA-binding regions. Interestingly, the location of one of the RNA-binding domains in p92 is similar to the RNA-binding domain of the NS5B RdRp protein of hepatitis C virus.

scholarly journals Aquifex aeolicus PilT, Homologue of a Surface Motility Protein, Is a Thermostable Oligomeric NTPase

2002 ◽  
Vol 184 (23) ◽  
pp. 6465-6471 ◽  
Author(s):  
Timothy J. Herdendorf ◽  
Darrell R. McCaslin ◽  
Katrina T. Forest
Keyword(s):  
Specific Activity ◽  
Protein A ◽  
Large Family ◽  
Size Exclusion ◽  
Sedimentation Equilibrium ◽  
Binding Motif ◽  
Aquifex Aeolicus ◽  
Secretion Systems ◽  
Surface Motility

ABSTRACT Bacterial surface motility works by retraction of surface-attached type IV pili. This retraction requires the PilT protein, a member of a large family of putative NTPases from type II and IV secretion systems. In this study, the PilT homologue from the thermophilic eubacterium Aquifex aeolicus was cloned, overexpressed, and purified. A. aeolicus PilT was shown to be a thermostable ATPase with a specific activity of 15.7 nmol of ATP hydrolyzed/min/mg of protein. This activity was abolished when a conserved lysine in the nucleotide-binding motif was altered. The substrate specificity was low; UTP, CTP, ATP, GTP, dATP, and dGTP served as substrates, UTP having the highest activity of these in vitro. Based on sedimentation equilibrium and size exclusion chromatography, PilT was identified as a ≈5- to 6-subunit oligomer. Potential implications of the NTPase activity of PilT in pilus retraction are discussed.

scholarly journals Structural Features and Functional Dependency on β-Clamp Define Distinct Subfamilies of Bacterial Mismatch Repair Endonuclease MutL

2016 ◽  
Vol 291 (33) ◽  
pp. 16990-17000 ◽  
Author(s):  
Kenji Fukui ◽  
Seiki Baba ◽  
Takashi Kumasaka ◽  
Takato Yano
Keyword(s):  
Mismatch Repair ◽  
Metal Binding ◽  
Thermus Thermophilus ◽  
Direct Interaction ◽  
Structural Features ◽  
Aquifex Aeolicus ◽  
Bacterial Phyla ◽  
Dna Mismatch

In early reactions of DNA mismatch repair, MutS recognizes mismatched bases and activates MutL endonuclease to incise the error-containing strand of the duplex. DNA sliding clamp is responsible for directing the MutL-dependent nicking to the newly synthesized/error-containing strand. In Bacillus subtilis MutL, the β-clamp-interacting motif (β motif) of the C-terminal domain (CTD) is essential for both in vitro direct interaction with β-clamp and in vivo repair activity. A large cluster of negatively charged residues on the B. subtilis MutL CTD prevents nonspecific DNA binding until β clamp interaction neutralizes the negative charge. We found that there are some bacterial phyla whose MutL endonucleases lack the β motif. For example, the region corresponding to the β motif is completely missing in Aquifex aeolicus MutL, and critical amino acid residues in the β motif are not conserved in Thermus thermophilus MutL. We then revealed the 1.35 Å-resolution crystal structure of A. aeolicus MutL CTD, which lacks the β motif but retains the metal-binding site for the endonuclease activity. Importantly, there was no negatively charged cluster on its surface. It was confirmed that CTDs of β motif-lacking MutLs, A. aeolicus MutL and T. thermophilus MutL, efficiently incise DNA even in the absence of β-clamp and that β-clamp shows no detectable enhancing effect on their activity. In contrast, CTD of Streptococcus mutans, a β motif-containing MutL, required β-clamp for the digestion of DNA. We propose that MutL endonucleases are divided into three subfamilies on the basis of their structural features and dependence on β-clamp.

scholarly journals Stimulation of 3′→5′ Exonuclease and 3′-Phosphodiesterase Activities of Yeast Apn2 by Proliferating Cell Nuclear Antigen

2002 ◽  
Vol 22 (18) ◽  
pp. 6480-6486 ◽  
Author(s):  
Ildiko Unk ◽  
Lajos Haracska ◽  
Xavier V. Gomes ◽  
Peter M. J. Burgers ◽  
Louise Prakash ◽  
...  
Keyword(s):  
Proliferating Cell Nuclear Antigen ◽  
Gel Filtration ◽  
Nuclear Antigen ◽  
Binding Motif ◽  
Terminal Domain ◽  
Abasic Sites ◽  
Factor C ◽  
Proliferating Cell

ABSTRACT The Apn2 protein of Saccharomyces cerevisiae contains 3′→5′ exonuclease and 3′-phosphodiesterase activities, and these activities function in the repair of DNA strand breaks that have 3′-damaged termini and which are formed in DNA by the action of oxygen-free radicals. Apn2 also has an AP endonuclease activity and functions in the removal of abasic sites from DNA. Here, we provide evidence for the physical and functional interaction of Apn2 with proliferating cell nuclear antigen (PCNA). As indicated by gel filtration and two-hybrid studies, Apn2 interacts with PCNA both in vitro and in vivo and mutations in the consensus PCNA-binding motif of Apn2 abolish this interaction. Importantly, PCNA stimulates the 3′→5′ exonuclease and 3′-phosphodiesterase activities of Apn2. We have examined the involvement of the interdomain connector loop (IDCL) and of the carboxy-terminal domain of PCNA in Apn2 binding and found that Apn2 binds PCNA via distinct domains dependent upon whether the binding is in the absence or presence of DNA. In the absence of DNA, Apn2 binds PCNA through its IDCL domain, whereas in the presence of DNA, when PCNA has been loaded onto the template-primer junction by replication factor C, the C-terminal domain of PCNA mediates the binding.

scholarly journals Mutation of the Conserved Calcium-Binding Motif in Neisseria gonorrhoeae PilC1 Impacts Adhesion but Not Piliation

2013 ◽  
Vol 81 (11) ◽  
pp. 4280-4289 ◽  
Author(s):  
Yuan Cheng ◽  
Michael D. L. Johnson ◽  
Christine Burillo-Kirch ◽  
Jeffrey C. Mocny ◽  
James E. Anderson ◽  
...  
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Calcium Binding ◽  
Critical Role ◽  
Full Length ◽  
Binding Motif ◽  
Type Iv Pilus ◽  
Kingella Kingae ◽  
Content Type ◽  
Terminal Domain ◽  
Type Iv

ABSTRACTNeisseria gonorrhoeaePilC1 is a member of the PilC family of type IV pilus-associated adhesins found inNeisseriaspecies and other type IV pilus-producing genera. Previously, a calcium-binding domain was described in the C-terminal domains of PilY1 ofPseudomonas aeruginosaand in PilC1 and PilC2 ofKingella kingae. Genetic analysis ofN. gonorrhoeaerevealed a similar calcium-binding motif in PilC1. To evaluate the potential significance of this calcium-binding region inN. gonorrhoeae, we produced recombinant full-length PilC1 and a PilC1 C-terminal domain fragment. We show that, while alterations of the calcium-binding motif disrupted the ability of PilC1 to bind calcium, they did not grossly affect the secondary structure of the protein. Furthermore, we demonstrate that both full-length wild-type PilC1 and full-length calcium-binding-deficient PilC1 inhibited gonococcal adherence to cultured human cervical epithelial cells, unlike the truncated PilC1 C-terminal domain. Similar to PilC1 inK. kingae, but in contrast to the calcium-binding mutant ofP. aeruginosaPilY1, an equivalent mutation inN. gonorrhoeaePilC1 produced normal amounts of pili. However, theN. gonorrhoeaePilC1 calcium-binding mutant still had partial defects in gonococcal adhesion to ME180 cells and genetic transformation, which are both essential virulence factors in this human pathogen. Thus, we conclude that calcium binding to PilC1 plays a critical role in pilus function inN. gonorrhoeae.

scholarly journals Types of nuclear endonuclease activity capable of inducing internucleosomal DNA fragmentation are completely different between human CD34+ cells and their granulocytic descendants

Blood ◽  
1995 ◽  
Vol 86 (3) ◽  
pp. 917-923 ◽  
Author(s):  
N Anzai ◽  
H Kawabata ◽  
T Hirama ◽  
H Masutani ◽  
Y Ueda ◽  
...  
Keyword(s):  
Dna Fragmentation ◽  
Myeloid Cells ◽  
Leukemic Cell ◽  
Endonuclease Activity ◽  
Cell Nuclei ◽  
Granulocytic Differentiation ◽  
Normal Peripheral Blood ◽  
Dependent Endonuclease ◽  
Cd34 Cells

Abstract A hallmark of apoptosis is internucleosomal DNA fragmentation resulting from the activation of endonucleases. We characterized the endonuclease activity of human myeloid cell nuclei that cleaved their own nuclear chromatin to oligonucleosomal length fragments. Polymorphonuclear leukocytes (PMNs) of normal peripheral blood contained both Ca2+/Mg(2+)- dependent and DNase II-like acidic endonuclease activities in their nuclei. Immature myeloid cells of normal bone marrow at various stages of granulocytic maturation had similar nuclease activities. In contrast, a clear difference was shown in the circulating CD34+ cells, in that only Mg(2+)-dependent, Ca(2+)-independent endonuclease activity was detected. Consistent with these findings is the emergence of the Ca2+/Mg(2+)-dependent and acidic endonuclease concomitantly with the disappearance of the Mg(2+)-dependent endonuclease when CD34+ cells were induced to differentiate in vitro toward granulocytes. Leukemic cell lines of all lineages also had Mg(2+)-dependent nuclease activity. Our results suggest an association of the Mg(2+)-dependent endonuclease with hematopoietic progenitor cells and that the relative activities of the nuclear nuclease in human myeloid cells change substantially during granulocytic differentiation.

scholarly journals The carboxyl terminus of phage HK022 Nun includes a novel zinc-binding motif and a tryptophan required for transcription termination

2000 ◽  
Vol 14 (6) ◽  
pp. 731-739
Author(s):  
Randolph S. Watnick ◽  
Stephanie Chiyoko Herring ◽  
Arthur G. Palmer ◽  
Max E. Gottesman
Keyword(s):  
Rna Polymerase ◽  
Rna Binding ◽  
Transcription Termination ◽  
Tryptophan Residue ◽  
Binding Motif ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal

The amino-terminal arginine-rich motif of the phage HK022 Nun protein binds phage λ nascent mRNA transcripts while the carboxy-terminal domain binds RNA polymerase and arrests transcription. The role of specific residues in the carboxy-terminal domain in transcription termination were investigated by mutagenesis, in vitro and in vivo functional assays, and NMR spectroscopy. Coordination of zinc to three histidine residues in the carboxy-terminus inhibited RNA binding by the amino-terminal domain; however, only two of these histidines were required for transcription arrest. These results suggest that additional zinc-coordinating residues are supplied by RNA polymerase in the context of the Nun–RNA polymerase complex. Substitution of the penultimate carboxy-terminal tryptophan residue with alanine or leucine blocks transcription arrest, whereas a tyrosine substitution is innocuous. Wild-type Nun fails to arrest transcription on single-stranded templates. These results suggest that Nun inhibition of transcription elongation is due in part to interactions between the carboxy-terminal tryptophan of Nun and double-stranded DNA, possibly by intercalation. A model for the termination activity of Nun is developed on the basis of these data.

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