Attributes of the [4Fe4S] Cofactor Coordinated by UvrC, a DNA Repair Enzyme

Citation

Silva, Rebekah Miriam Brawer (2020) Attributes of the [4Fe4S] Cofactor Coordinated by UvrC, a DNA Repair Enzyme. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/r0j6-jk09. https://resolver.caltech.edu/CaltechTHESIS:06082020-163907557

Abstract

Protein-bound iron sulfur clusters are critical in cells and allow proteins to carry out many essential functions as electron carriers, catalysts for challenging organic reactions, and sensors of cellular environments. A wide range of protein families are known to coordinate iron sulfur clusters, and a growing category includes proteins involved in maintenance of the genome. Within the last three decades, iron sulfur clusters have been demonstrated to be important for enzymes that function in DNA repair, DNA replication, and transcription pathways. To date, iron sulfur clusters in the cubane [4Fe4S] geometry with all cysteine ligands have been exclusively reported for DNA repair and replication enzymes. In contrast to enzymes where the cofactor is necessary for active site chemistry or directly-linked to protein function, the [4Fe4S] cluster in the overwhelming majority of repair and replication enzymes is not involved in the catalytic modification of DNA substrates. Rather, the role of the cofactor appears to vary in function from protein to protein, and has been demonstrated to be important for protein stability, in the assembly of multisubunit proteins, and for substrate recognition, among other roles. Through investigations of the redox chemistry of the cofactor, our group has found that these enzymes participate in DNA-mediated charge transport chemistry, the process through which electrons rapidly migrate through well-stacked, duplex DNA. Long-range, DNA-mediated redox signaling provides a means of rapid communication among DNA-processing proteins for organizing repair and replication activities across the nucleus.

Notably, the first observations of the [4Fe4S] cofactor associated with repair and replications enzymes has consistently occurred well after the first biochemical studies of these enzymes. In some cases, the demonstration of a [4Fe4S] center has taken place decades later after initial work. Some proteins have required use of anaerobic methods in order to detect the cofactor, perhaps explaining why in some cases the metal center had eluded observation. Analysis of protein sequences might be expected to help accelerate identification of new iron sulfur centers in repair and replication enzymes. However, even with the abundance of sequencing data available in the post-genomic era, prediction of a metal center based on sequences alone has been challenging. This is in large part because the spacing of the coordinating cysteine residues can be quite irregular, leading to a weak bioinformatic signature.

Identifying proteins with overlooked [4Fe4S] cofactors poses an exciting challenge, and there are some elegant examples in the literature where data from genetics assays has been used in combination with careful sequence analysis to predict and discover iron sulfur centers in repair and replication enzymes. Described here is the evolution of our studies on one well-known repair enzyme from Escherichia coli, UvrC. UvrC is part of the nucleotide excision repair pathway in the Bacteria domain which is responsible for addressing the wide class of bulky, helix-distorting lesions that can form after exposure to sources such as ultraviolet light, cigarette smoke, chemotherapeutics, and protein-DNA crosslinks. UvrC, an excision nuclease with two distinct active sites that incise the phosphodiester backbone on either side of the site of damage, has been historically challenging to study. Given how essential UvrC is in repairing damaged substrates, new insight has been greatly needed.

Through integration of several key reports from the literature regarding the sequence of UvrC and evidence that pointed to a cofactor from genetics assays, our group predicted that UvrC is a [4Fe4S] protein. Development of a new overexpression system and an anaerobic purification method allowed for isolation of UvrC in holo form. We used spectroscopic techniques to confirm that the cluster type was [4Fe4S], and a combination of spectroscopy and chromatography to demonstrate that the UvrC-bound cofactor is susceptible to oxidative degradation. We also found that loss of the cofactor, either through aerobic degradation or mutation of coordinating cysteines, is associated with aggregation of apoprotein. Importantly, in its holo form with the cofactor bound, UvrC forms high affinity complexes with duplexed DNA substrates; the apparent dissociation constants to well-matched and damaged duplex substrates are 100 ± 20 nM and 80 ± 30 nM, respectively. This high affinity DNA binding contrasts reports made for isolated protein lacking the cofactor. Moreover, using DNA electrochemistry, we find that the cluster coordinated by UvrC is redox-active and participates in DNA-mediated charge transport chemistry with DNA-bound midpoint potential of 90 mV vs. NHE.

The work detailed in this dissertation has highlighted how critical the [4Fe4S] center is for UvrC, where the cofactor has been implicated in protein stabilization, substrate binding, and redox signaling on DNA. Handling an apo form of UvrC may have led to the previous challenges catalogued by researchers. Through the development of entirely new methods to study UvrC under anaerobic conditions, many opportunities are now available to study UvrC and the NER pathway anew in vitro and in vivo. Such work will contribute additional insight on how iron sulfur clusters are essential for enzymes that maintain genomic integrity.

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