Date of Degree


Document Type


Degree Name





David Jeruzalmi

Committee Members

Ruth Stark

Wilma Saffran

Seth Darst

Manju Hingorani

Subject Categories

Biochemistry | Biophysics | Laboratory and Basic Science Research | Structural Biology


DNA damage repair, Nucleotide excision repair, electron microscopy, DNA damage recognition, insertion domain, DNA-damage recognition loop


Maintaining the cellular genome is paramount to survival by any organism. A mutated genome can have detrimental effects on different cellular processes, especially replication and transcription. Cells maintain their genome using different deoxyribonucleic acid (DNA) repair pathways. The nucleotide excision repair (NER) pathway has a unique capability of repairing the genome from several different mutations, deletions, and adducts. In bacteria, the NER pathway accomplishes repair through four important steps: damage recognition by UvrA, damage verification by UvrB, DNA incision by UvrC, and repair synthesis using various cellular machinery.

UvrA forms a head-to-head dimer (UvrA2) with two ATPase sites per monomer, named the proximal and distal sites. Prior work has suggested that UvrA performs DNA damage verification through a DNA-helix sensing mechanism. When damage is encountered, UvrA is unable to close around the DNA, thereby signaling that damage is present. Once damage is sensed by UvrA, UvrB is loaded in an adenosine triphosphate (ATP) dependent process. Structural and biochemical evidence has suggested that DNA can be interrogated through a UvrA2 complex with a UvrB bound to either monomer. Other biochemical studies have determined that UvrB rarely complexes with UvrA in solution. However, given a lack of evidence into this helix-sensing mechanism, more investigation is warranted to gain a full understanding into the UvrA-DNA damage sensing mechanism.

Presented here are several structural studies resulting in new UvrA structures, a truncated structure featuring the highest resolution to date, the first solution-based apo-UvrA2 structure from cryo-electron microscopy, a cryo-electron microscopy UvrA2UvrB2 structure, a first solution-based UvrA2-damaged DNA structure, and several nuclear magnetic resonance (NMR) spectra, which allows analysis of ATPase sites effects on UvrA in solution. Structural studies were further informed by mass spectrometry, ATPase, and binding studies.

The new crystal structure gives an intermediate view of a β-hairpin. Biochemical work has implicated a rotation in the β-hairpin in DNA damage verification. The high-resolution state of this new structure allows an examination into the ATPase sites. However, evidence from our novel cryo-electron microscopy structures suggests that the movements within the β-hairpin are far more minute. Moreover, our novel structures demonstrate that the β-hairpin interacts with a loop that has direct interaction with the center of the DNA duplex, rather than the DNA itself. Therefore, the loop has a secondary role in the DNA sensing mechanism rather than a primary role.

Our mass spectrometry data indicated that UvrB was rarely bound to UvrA whether ATP or DNA was present in solution. However, multiple methods have demonstrated that UvrA and UvrB do interact. Both the novel UvrA2UvrB2 and UvrA2 structures presented a caving-in of the insertion domains into the DNA binding cavity. The caving-in indicates that any DNA entering the binding cavity would require the insertion domains to rotate in to accept the DNA into the binding cavity. Further, the UvrA2UvrB2 demonstrates a complete collapse of the insertion domain. The insertion domain collapse in the UvrA2UvrB2 structure is postulated to be key for the one-dimensional search that UvrA2UvrB2 does along DNA, versus the three-dimensional search that UvrA2 does.

Finally, we present the first solution based UvrA structure bound to DNA. The structure indicates a loop under the damaged area that has a key function in detecting the damaged area, designated as the DNA-damage sensing loop. Additionally, the loop is coordinated by the β-hairpin of the third zinc module and is supported by a helix. Finally, the structure yielded clues into UvrB loading. Specifically, one UvrB binding domain rotates such that UvrB would be forced against the DNA in the case of the UvrB that would travel along the undamaged strand based off it 5’ to 3’ helicase activity. The second UvrB binding domain is rotated in such a way that UvrB would bind to UvrA high above the DNA and the UvrA dimer. The second UvrB would travel along the damaged DNA, but would require the collapse of the UvrA dimer or another pulling type motion to bring UvrB to the DNA duplex.

This work is embargoed and will be available for download on Thursday, June 02, 2022

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