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. 2007 Jan 24;26(2):613-22.
doi: 10.1038/sj.emboj.7601497.

Structure of the C-terminal half of UvrC reveals an RNase H endonuclease domain with an Argonaute-like catalytic triad

Erkan Karakas  1 , James J Truglio, Deborah Croteau, Benjamin Rhau, Liqun Wang, Bennett Van Houten, Caroline Kisker
Affiliations

Structure of the C-terminal half of UvrC reveals an RNase H endonuclease domain with an Argonaute-like catalytic triad

Erkan Karakas et al. EMBO J. .

Abstract

Removal and repair of DNA damage by the nucleotide excision repair pathway requires two sequential incision reactions, which are achieved by the endonuclease UvrC in eubacteria. Here, we describe the crystal structure of the C-terminal half of UvrC, which contains the catalytic domain responsible for 5' incision and a helix-hairpin-helix-domain that is implicated in DNA binding. Surprisingly, the 5' catalytic domain shares structural homology with RNase H despite the lack of sequence homology and contains an uncommon DDH triad. The structure also reveals two highly conserved patches on the surface of the protein, which are not related to the active site. Mutations of residues in one of these patches led to the inability of the enzyme to bind DNA and severely compromised both incision reactions. Based on our results, we suggest a model of how UvrC forms a productive protein-DNA complex to excise the damage from DNA.

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Figures

Figure 1
Figure 1
Overall structure of the 5′ endonuclease and (HhH)2 domain of TmUvrC. (A) Domain architecture of TmUvrC. (B) Structure of UvrCC-term. The endonuclease and (HhH)2 domain are shown in yellow and cyan, respectively. Selected residues are displayed in all-bonds representation. Secondary structure elements and the N- and C-termini are labeled.
Figure 2
Figure 2
Comparison of RNase H family members. Domain architecture and active site of T. maritima UvrC endonuclease domain, PIWI domain of P. furiosus Argonaute (PDB ID: 1Z25), B. halodurans RNase HI (PDB ID: 1ZBI) and E. coli Tn5 transposase (PDB ID: 1MUS). The DNA/RNA was omitted from the structures of Tn5 transposase and RNase HI for clarity. Structurally related elements are colored similarly and unrelated elements are shown in yellow. Side chains of selected active site residues are shown in all-bonds representation. Manganese ions and water molecules are shown as green and red spheres, respectively.
Figure 3
Figure 3
Incision activity of TmUvrC mutants. (A) The 5′ end-labeled F2650/NDB duplex was incubated with 20 nM Bca UvrA, 100 nM Bca UvrB and 50 nM of the indicated TmUvrC protein for 30 min (A, B) or 5 min (C, D) at 55°C. The reactions were terminated with stop buffer and the incision products were resolved on a 10% denaturing polyacrylamide gel. (A, C) Dotted lines indicate that these lanes were not originally next to one another in the original gel. (B, D) Graphical representation of the data in (A) and (C), mean±s.d., n=3 refers to experiments performed on separate days. It is important to note here that UvrC does not turnover, and the incision kinetics represent a single binding event to a UvrB–DNA complex.
Figure 4
Figure 4
Electrostatic surface potential and sequence conservation of the C-terminal half of UvrC. (A) Electrostatic surface potential was calculated with PyMol/APBS and contoured at ±10 kBT. The top panel features the active site of the protein and the bottom view is a 180° rotation. (B) Sequence conservation using the same orientations as in (A). The degree of conservation was obtained by alignment of 47 UvrC sequences with ClustalX. Strictly conserved (red), very highly conserved (blue), highly conserved (green) and moderately conserved (black) amino acids are highlighted. The remainder of the protein is colored in gray. Bound sulfate molecules are shown in all-bonds representation. Selected amino acids are labeled.
Figure 5
Figure 5
Amino acids R394, R395, H495 and R496 are important for incision. (A) Incision activity. The incubation time was 30 min at 55°C. (B) Graphical representation of the data in (A), mean±s. d., n=3 refers to experiments performed on separate days. (C) The duplex, F19,30/NDB, contains a nick where the normal 3′ incision would occur. It was labeled on the 5′ end of the F19,30 strand so that incision by 20 nM Bca UvrA, 100 nM Bca UvrB and 50 nM of the indicated TmUvrC protein for 30 min at 55°C could be monitored. A representative gel is shown. (D) Graphical representation of data in (C), mean±s.d., n=3 refers to experiments performed on separate days. (E) A representative gel of the electrophoretic mobility shift assay containing 200 nM UvrC and 2 nM NDT/NDB duplex is shown. The indicated protein and DNA were allowed to incubate with DNA for 15 min at room temperature and were then loaded onto a 4% polyacrylamide gel. (F) Quantitation of the electrophoretic mobility shift assay in (E). These data are reported as the mean±s.d. (n=3 refers to experiments performed on separate days).
Figure 6
Figure 6
DNA binding model. (A) Side-by-side comparison of the (HhH)2 domain of RuvA (left), TmUvrC (center) and EcUvrC (right) after superposition. The DNA backbone of the RuvA/DNA complex (left panel) is shown as an orange worm. Selected residues are shown in all-bonds representation and are labeled. The N- and C-termini are indicated. The HhHI and HhHII motifs are colored yellow and green, respectively. The helical linker between the two motifs is colored blue. (B) The endonuclease domains of eight TmUvrCC-term structures are superimposed to show the orientation of the (HhH)2 domains relative to the endonuclease domain in the different crystal forms. (C) Model of TmUvrC interacting with DNA based on a superposition with the Tn5 transposase–DNA complex. The endonuclease and (HhH)2 domain of TmUvrC are colored yellow and cyan, respectively. The DNA is orange and drawn with spokes for clarity. The side chains of the catalytic triad and D405 are depicted as all-bonds. The bound magnesium is shown as a green sphere. In the left panel, the (HhH)2 domain is depicted in the position found in the crystal structure. The DNA-interacting region of the (HhH)2 domain is shown in red. In the right panel, the (HhH)2 domain has been rotated to form a productive UvrC/DNA complex. A dashed line indicates the connection point between the endonuclease domain and the (HhH)2 domain.
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References

    1. Aravind L, Walker DR, Koonin EV (1999) Conserved domains in DNA repair proteins and evolution of repair systems. Nucleic Acids Res 27: 1223–1242 - PMC - PubMed
    1. Ariyoshi M, Vassylyev DG, Iwasaki H, Nakamura H, Shinagawa H, Morikawa K (1994) Atomic structure of the RuvC resolvase: a Holliday junction-specific endonuclease from E. coli. Cell 78: 1063–1072 - PubMed
    1. Caron PR, Kushner SR, Grossman L (1985) Involvement of helicase-II (UvrD gene product) and DNA Polymerase-I in excision mediated by the UvrABC protein complex. Proc Natl Acad Sci USA 82: 4925–4929 - PMC - PubMed
    1. Ceschini S, Keeley A, McAlister MS, Oram M, Phelan J, Pearl LH, Tsaneva IR, Barrett TE (2001) Crystal structure of the fission yeast mitochondrial Holliday junction resolvase Ydc2. EMBO J 20: 6601–6611 - PMC - PubMed
    1. Davies DR, Goryshin IY, Reznikoff WS, Rayment I (2000) Three-dimensional structure of the Tn5 synaptic complex transposition intermediate. Science 289: 77–85 - PubMed

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