Structural insights into the first incision reaction during nucleotide excision repair

Truglio, J.J.; Rhau, Benjamin; Croteau, Deborah L.; Wang, Liqun; Skorvaga, Milan; Karakaş, Erkan; DellaVecchia, Matthew J.; Wang, Hong; Houten, Bennett Van; Kisker, Caroline

doi:10.1038/sj.emboj.7600568

Cited by 86 publications

(135 citation statements)

References 48 publications

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“…Amino acids in EndoII (see Fig. 1) were selected for mutagenesis based on three criteria: (i) their importance for catalysis and their position on the putative catalytic surface (G49, R57, E118, and N130) (20,40,44); (ii) the loss of in vivo activity of EndoII (E91G, L84P and P127L) (3, 18, 43; K. Carlson and A. C. Nyström, unpublished data); and (iii) conservation of residues in regions unique to EndoII-the MR that is conserved in EndoII homologues in T4-like phage (http://phage.bioc.tulane.edu/; S72, K76, I80, A83, E86, and V90) and the less-conserved NTR (K12, L16, and I24). In addition, a glutamic acid next to E118 (E117) and a proline next to P127 (P128) were selected to test whether the two residues in these two neighboring sets had distinct roles.…”

Section: Resultsmentioning

confidence: 99%

“…The NTR and MR regions are boxed in the two EndoII sequences. Residues in Tma UvrC shown to be important for its activity (40) are marked with filled circles (F) below the Tma UvrC sequence. T4 EndoII residues where mutations (to alanine unless otherwise noted above the symbol) were introduced and analyzed in the present study are marked above the T4 EndoII sequence.…”

Section: Fig 1 (A)mentioning

confidence: 99%

“…Binding and cleavage by Eco29kI (19) and its isoschizomer Cfr421 (17) have been described recently, but otherwise these restriction endonucleases have not been extensively characterized (11,42,45,46). Structural analysis of the GIY-YIG-domains of UvrC (40) and I-TevI (41) show significant similarities in their suggested catalytic surface despite low sequence similarity (15% amino acid identity (40). EndoII shows 17% amino acid identity to UvrC from Fusobacterium nucleatum (EAA24392; 15% identity to Tma UvrC (Q9WYA3) (Fig.…”

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confidence: 99%

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Amino Acid Residues in the GIY-YIG Endonuclease II of Phage T4 Affecting Sequence Recognition and Binding as Well as Catalysis

Lagerbäck

Carlson

2008

J Bacteriol

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Phage T4 endonuclease II (EndoII), a GIY-YIG endonuclease lacking a carboxy-terminal DNA-binding domain, was subjected to site-directed mutagenesis to investigate roles of individual amino acids in substrate recognition, binding, and catalysis. The structure of EndoII was modeled on that of UvrC. We found catalytic roles for residues in the putative catalytic surface (G49, R57, E118, and N130) similar to those described for I-TevI and UvrC; in addition, these residues were found to be important for substrate recognition and binding. The conserved glycine (G49) and arginine (R57) were essential for normal sequence recognition. Our results are in agreement with a role for these residues in forming the DNA-binding surface and exposing the substrate scissile bond at the active site. The conserved asparagine (N130) and an adjacent proline (P127) likely contribute to positioning the catalytic domain correctly. Enzymes in the EndoII subfamily of GIY-YIG endonucleases share a strongly conserved middle region (MR, residues 72 to 93, likely helical and possibly substituting for heterologous helices in I-TevI and UvrC) and a less strongly conserved N-terminal region (residues 12 to 24). Most of the conserved residues in these two regions appeared to contribute to binding strength without affecting the mode of substrate binding at the catalytic surface. EndoII K76, part of a conserved NUMOD3 DNA-binding motif of homing endonucleases found to overlap the MR, affected both sequence recognition and catalysis, suggesting a more direct involvement in positioning the substrate. Our data thus suggest roles for the MR and residues conserved in GIY-YIG enzymes in recognizing and binding the substrate.Endonuclease II (EndoII) of coliphage T4, encoded by gene denA, catalyzes the initial step in host DNA degradation. It causes irreversible host shutoff and also initiates a nucleotide scavenge pathway that provides precursors for phage DNA synthesis (8). T4 phage DNA is protected from EndoII by the substitution of 5-hydroxymethyl deoxycytosine for dC during T4 DNA synthesis (9, 43). EndoII shares the sequence elements defining the GIY-YIG family of proteins (13,20) that includes the repair endonuclease UvrC and many homing endonucleases, such as the intron-encoded T4 endonuclease ITevI (20) and I-BmoI (10), as well as some type II restriction endonucleases (2,19). Like the homing endonucleases in the GIY-YIG family, EndoII cleaves a long, asymmetric, and ambiguous DNA sequence (4-6, 21). However, in contrast to what has been found for I-TevI (24), EndoII cleaves the two strands independently of each other (7), and only a CG dinucleotide 2 bp away from the scissile bond is strongly conserved (5). Double-strand cleavage by EndoII is the result of concerted singlestrand nicks (7), but many positions are only nicked, not cleaved.In UvrC (44), as well as in the GIY-YIG homing endonucleases (10, 12), the conserved motifs and catalytic activity reside in the amino-terminal domain, but the primary binding energy is conferred by a carboxy-termin...

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Section: Resultsmentioning

confidence: 99%

Section: Fig 1 (A)mentioning

confidence: 99%

mentioning

confidence: 99%

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Amino Acid Residues in the GIY-YIG Endonuclease II of Phage T4 Affecting Sequence Recognition and Binding as Well as Catalysis

Lagerbäck

Carlson

2008

J Bacteriol

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“…While we have made great strides towards understanding both UvrB and UvrC due to recent crystal structures [16,18,[39][40][41][42][43][44][45], there is limited knowledge of UvrA's structure and how it initiates the NER reaction. We have begun to define UvrA's functions by deletion and site directed mutagenesis.…”

Section: Discussionmentioning

confidence: 99%

“…UvrC contains two nuclease centers and each site is responsible for a single strand cut [15]. The first cut is mediated by the GIY-YIG type-nuclease in the N-terminus of UvrC [16]. It produces a single strand break four to five nucleotides 3′ to the DNA lesion.…”

Section: Introductionmentioning

confidence: 99%

Cooperative damage recognition by UvrA and UvrB: Identification of UvrA residues that mediate DNA binding

et al. 2008

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Nucleotide excision repair (NER) is responsible for the recognition and removal of numerous structurally unrelated DNA lesions. In prokaryotes, the proteins UvrA, UvrB and UvrC orchestrate the recognition and excision of aberrant lesions from DNA. Despite the progress we have made in understanding the NER pathway it remains unclear how the UvrA dimer interacts with DNA to facilitate DNA damage recognition. The purpose of this study was to define amino acids residues in UvrA that provide binding energy to DNA. Based on conservation among ~300 UvrA sequences and 3D-modeling, two positively charged residues, Lys680 and Arg691, were predicted to be important for DNA binding. Mutagenesis and biochemical analysis of Bacillus caldontenax UvrA variant proteins containing site directed mutations at these residues demonstrate that Lys680 and Arg691 make a significant contribution toward the DNA binding affinity of UvrA. Replacing these side chains with alanine or negatively charged residues decreased UvrA binding 3-37-fold. Survival studies indicated that these mutant proteins complemented a WP2 uvrA − strain of bacteria 10% to 100% of WT UvrA levels. Further analysis by DNase I footprinting of the double UvrA mutant revealed that the UvrA DNA binding defects caused a slower rate of transfer of DNA to UvrB. Consequently, the mutants initiated the oligonucleotide incision assay nearly as well as WT UvrA thus explaining the observed mild phenotype in the survival assay. Based on our findings we propose a model of how UvrA binds to DNA.

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A Gold Nanoparticle Nanonuclease Relying on a Zn(II) Mononuclear Complex

et al. 2020

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Similarly to enzymes,f unctionalized gold nanoparticles efficiently catalyze chemical reactions,hence the term nanozymes.H erein, we present our results showing how surface-passivated gold nanoparticles behave as synthetic nanonucleases,a ble to cleave pBR322 plasmid DNAwith the highest efficiency reported so far for catalysts based on asingle metal ion mechanism. Experimental and computational data indicate that we have been successful in creating acatalytic site precisely mimicking that suggested for natural metallonucleases relying on as ingle metal ion for their activity.I tc omprises one Zn(II) ion to which ap hosphate diester of DNAi s coordinated. Importantly,a si nn ucleic acids-processing enzymes,apositively charged arginine playsakey role by assisting with transition state stabilization and by reducing the pK a of the nucleophilic alcohol of as erine.O ur results also show howd esigning ac atalyst for am odel substrate (bis-pnitrophenylphosphate) may provide wrong indications as for its efficiency when it is tested against the real target (plasmid DNA).

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Structural insights into the first incision reaction during nucleotide excision repair

Cited by 86 publications

References 48 publications

Amino Acid Residues in the GIY-YIG Endonuclease II of Phage T4 Affecting Sequence Recognition and Binding as Well as Catalysis

Amino Acid Residues in the GIY-YIG Endonuclease II of Phage T4 Affecting Sequence Recognition and Binding as Well as Catalysis

Cooperative damage recognition by UvrA and UvrB: Identification of UvrA residues that mediate DNA binding

A Gold Nanoparticle Nanonuclease Relying on a Zn(II) Mononuclear Complex

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