A Structural Model for the Damage-sensing Complex in Bacterial Nucleotide
Excision Repair |
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Authors: | Danaya Pakotiprapha Yi Liu Gregory L Verdine and David Jeruzalmi |
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Institution: | Departments of ‡Molecular and Cellular Biology and §Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138 and the ¶Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115 |
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Abstract: | Nucleotide excision repair is distinguished from other DNA repair pathways
by its ability to process a wide range of structurally unrelated DNA lesions.
In bacteria, damage recognition is achieved by the UvrA·UvrB ensemble.
Here, we report the structure of the complex between the interaction domains
of UvrA and UvrB. These domains are necessary and sufficient for full-length
UvrA and UvrB to associate and thereby form the DNA damage-sensing complex of
bacterial nucleotide excision repair. The crystal structure and accompanying
biochemical analyses suggest a model for the complete damage-sensing
complex.Nucleotide excision repair is distinguished from other DNA repair pathways
by its ability to process a diverse set of lesions. In bacteria, the initial
steps are carried out by three proteins: UvrA, UvrB, and UvrC. The
UvrA·UvrB complex conducts surveillance of DNA and recognizes damage.
Having located a lesion, UvrA “loads” UvrB onto the DNA at the
damaged sites and then dissociates. Damage searching, formation of the
UvrB·DNA “preincision” complex, and dissociation of UvrA
are regulated by ATP (1). UvrB
subsequently recruits the endonuclease UvrC, which catalyzes incisions on
either side of the lesion (2,
3). Following incision, UvrC
and the damage-containing oligonucleotide are removed by UvrD (helicase II),
whereas UvrB remains bound to the gapped DNA and recruits DNA polymerase I for
repair synthesis. Sealing of the single-stranded nick completes the repair
process and restores the original DNA sequence
(4).Since its discovery more than 40 years ago, bacterial nucleotide excision
repair has been extensively studied, resulting in a large body of work that
describes the protein components and the details of how they operate.
Notwithstanding the trove of genetic and biochemical data, several key
questions remain unanswered. For example, how does the same set of proteins
handle a diverse set of lesions while maintaining specificity? How do UvrA and
UvrB cooperate during damage recognition, and what is the precise role of ATP?
Ongoing studies in the field, including those described below, aim to address
these issues.Recently, we reported the structure of Geobacillus
stearothermophilus UvrA and the identification of binding sites for DNA
and UvrB (5). We also
established that the identified UvrB-binding domain is necessary and
sufficient to mediate the UvrA-UvrB interaction and that the isolated
interaction domains of UvrA (5)
and UvrB (6) bind to each other
in solution.To understand the interaction between UvrA and UvrB, we have determined the
crystal structure of the complex between the two isolated interaction domains.
The structure revealed that UvrA-UvrB interaction interface is largely polar,
mediated by several highly conserved charged residues. Site-directed
mutagenesis and biochemical characterization of the mutant proteins confirmed
the importance of the observed interactions. Based on the interaction domain
complex structure, we have constructed a structural model for the full-length
UvrA·UvrB ensemble and propose two models for lesion recognition that
will serve as a basis for future experiments. |
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