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. 2019 Aug 28:2019:5641746.
doi: 10.34133/2019/5641746. eCollection 2019.

Mechanism of DNA Lesion Homing and Recognition by the Uvr Nucleotide Excision Repair System

Seung-Joo Lee  1 , Rou-Jia Sung  2 , Gregory L Verdine  1   2   3
Affiliations

Mechanism of DNA Lesion Homing and Recognition by the Uvr Nucleotide Excision Repair System

Seung-Joo Lee et al. Research (Wash D C). .

Abstract

Nucleotide excision repair (NER) is an essential DNA repair system distinguished from other such systems by its extraordinary versatility. NER removes a wide variety of structurally dissimilar lesions having only their bulkiness in common. NER can also repair several less bulky nucleobase lesions, such as 8-oxoguanine. Thus, how a single DNA repair system distinguishes such a diverse array of structurally divergent lesions from undamaged DNA has been one of the great unsolved mysteries in the field of genome maintenance. Here we employ a synthetic crystallography approach to obtain crystal structures of the pivotal NER enzyme UvrB in complex with duplex DNA, trapped at the stage of lesion-recognition. These structures coupled with biochemical studies suggest that UvrB integrates the ATPase-dependent helicase/translocase and lesion-recognition activities. Our work also conclusively establishes the identity of the lesion-containing strand and provides a compelling insight to how UvrB recognizes a diverse array of DNA lesions.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1
Figure 1
Schematic of Uvr nucleotide excision repair pathway.
Figure 2
Figure 2
Entrapment strategy to form a preincision complex. (a) Schematic describing the disulfide crosslinking (DXL) and stepwise assembly strategy. (b) Details of the trapping chemistry. A cysteine residue engineered into UvrB attacks a disulfide-bearing tether attached to the N4-position of a cytosine. Curved arrows denote electron flow in the crosslinking reaction. (c) UvrC incision assays on the crosslinked UvrB-dsDNA complexes were analyzed on 15% denaturing polyacrylamide gel. 5′ ends of both inner and outer strands are radioactively labeled. Lanes 1-4 were cropped from the same experiment. Lanes 5 and 6 (with DNA ladder) were run on 23.5% denaturing polyacrylamide gels for better resolution. (d) DNA sequence used for this study and experimentally determined 5′ and inferred 3′ incision sites. The lesion-mimetic cytosine, located 8-nucleotide downstream on the inner strand, is shown in red.
Figure 3
Figure 3
Overall structure of the preincision complex. (a, b, c) Structure of the UvrB-dsDNA-ADP·Pi complex in three views. UvrB domains 1a, 1b, 2, and 3 and the β-hairpin are colored in green, pink, grey, cyan, and blue, respectively. The crosslinked cytosine on the inner strand (gold) is highlighted in red. ADP and Pi are shown in sticks. Experimentally determined UvrC incision sites on the inner strand are shown as red spheres. (d) Schematic illustrating the interactions between UvrB and DNA. Colour-coding for UvrB residues is as in (a-c). (e) 2Fo – Fc electron density (grey mesh), contoured at 1.5s, of ADP and Pi. (f) Close-up view of the extrahelical cytosine embedded inside the narrow hydrophobic pocket.
Figure 4
Figure 4
Proposed translocation mechanism of UvrB. (a) Proposed rigid body movement of RecA-like domains upon ATP-binding and ATP-hydrolysis. (b) Interactions between the inner strand and two nucleic acid binding sites in domains 1a and 3. For clarity, only 8-nucleotide stretch of the inner strand is shown. Nucleotides in contact with helicase motifs are shown in colors while others are shown in white. The Interactions are represented in dashed lines in all panels. (c) F527 (red) lodged deeply in the minor groove. Helicase motifs and F-loop are colored in blue and pink, respectively. (d) DNA bubble (green) flanked by F527 on one end and the β-hairpin on the other in two orthogonal views. Domains 1a, 1b, and 2 are omitted for clarity. (e) Superposition of domain 3 of UvrB in the absence (grey; PDB ID, 1D9Z) [16] and presence of dsDNA substrate (cyan). Motif V, which undergoes conformational transition upon DNA binding, is highlighted in yellow. (f) The ATPase site of UvrB bound to ATP in the absence of dsDNA (PDB ID, 1D9Z) [16]. (g) The activated ATPase site in the UvrB-dsDNA-ADP·Pi structure.
Figure 5
Figure 5
Biochemical assays identifying the lesion-containing strand. (a) Schematic illustrating the UvrB translocation assay. (b) The reactions analyzed by native PAGE. (c) 50-mer DNA substrates used for UvrB crosslinking assay. A single fluorescein-adducted thymine (Flu-dT) is located at various positions either on the same (Set I) or opposite (Set II) strand to a disulfide-bearing tether (XL). (d) Crosslinked products analyzed by SDS-PAGE. Control reactions contain the unmodified duplex DNA (U/D) and duplex DNA containing either fluorescein (Flu) or disulfide-bearing tether (XL) alone. (e) The average and standard deviation of three independent experiments is shown for each DNA substrate shown in Figure 6(c).
Figure 6
Figure 6
Proposed lesion recognition mechanism and biochemical studies. (a) The cross-section view of UvrB-dsDNA-ADP·Pi complex in surface representation. Proposed trajectories of undamaged and damaged “bulky” bases are shown in green and red arrows, respectively. Green and blue dashed lines delineate the boundaries of the lesion-selectivity filter and β-hairpin. (b) The cross-section view of the UvrB lesion-selectivity filter.
Figure 7
Figure 7
Proposed DNA repair mechanism in the Uvr nucleotide excision repair system.
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References

    1. Hoeijmakers J. H. J. DNA damage, aging, and cancer. The New England Journal of Medicine. 2009;361(15):1475–1485. doi: 10.1056/nejmra0804615. - DOI - PubMed
    1. Lindahl T., Wood R. D. Quality control by DNA repair. Science. 1999;286(5446):1897–1905. doi: 10.1126/science.286.5446.1897. - DOI - PubMed
    1. Setlow R. B., Carrier W. L. The disappearance of thymine dimers from dna: an error-correcting mechanism. Proceedings of the National Acadamy of Sciences of the United States of America. 1964;51(2):226–231. doi: 10.1073/pnas.51.2.226. - DOI - PMC - PubMed
    1. Boyce R. P., Howard-flanders P. Release of ultraviolet light-induced thymine dimers from dna in e. coli k-12. Proceedings of the National Academy of Sciences of the United States of America. 1964 - PMC - PubMed
    1. Howard-Flanders P., Boyce R. P., Theriot L. Three loci in Escherichia coli K-12 that control the excision of pyrimidine dimers and certain other mutagen products from DNA. Genetics. 1966;53(6):1119–1136. - PMC - PubMed

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