The Oxidation State of [4Fe4S] Clusters Modulates the DNA-Binding Affinity of DNA Repair Proteins

Abstract

A central question important to understanding DNA repair is how certain proteins are able to search for, detect, and fix DNA damage on a biologically relevant timescale. A feature of many base excision repair proteins is that they contain [4Fe4S] clusters that may aid their search for lesions. In this report, we establish the importance of the oxidation state of the redox-active [4Fe4S] cluster in the DNA damage detection process. We utilize DNA-modified electrodes to generate repair proteins with [4Fe4S] clusters in the 2+ and 3+ states by bulk electrolysis under an O2-free atmosphere. Anaerobic microscale thermophoresis results indicate that proteins carrying [4Fe4S]3+ clusters bind to DNA 550 times more tightly than those with [4Fe4S]2+ clusters. The measured increase in DNA-binding affinity matches the calculated affinity change associated with the redox potential shift observed for [4Fe4S] cluster proteins upon binding to DNA. We further devise an electrostatic model that shows this change in DNA-binding affinity of these proteins can be fully explained by the differences in electrostatic interactions between DNA and the [4Fe4S] cluster in the reduced versus oxidized state. We then utilize atomic force microscopy (AFM) to demonstrate that the redox state of the [4Fe4S] clusters regulates the ability of two DNA repair proteins, Endonuclease III and DinG, to bind preferentially to DNA duplexes containing a single site of DNA damage (here a base mismatch) which inhibits DNA charge transport. Together, these results show that the reduction and oxidation of [4Fe4S] clusters through DNA-mediated charge transport facilitates long-range signaling between [4Fe4S] repair proteins. The redox-modulated change in DNA-binding affinity regulates the ability of [4Fe4S] repair proteins to collaborate in the lesion detection process.

A Redox Role for the [4Fe4S] Cluster of Yeast DNA Polymerase δ

“…In contrast, oxidized Pol δ leaves fewer primers unextended or fully extended, instead making more intermediate products between 150 bp and 1 kb. The greater proportion of extended primers is consistent with tighter DNA binding after cluster oxidation, as has been observed with both primase and DNA repair proteins (16, 27). However, the slower procession indicates that tighter binding impedes rapid procession, acting as a brake on PCNA-mediated DNA synthesis.…”

“…In this model, processive lagging strand replication proceeds until replications stress occurs and the Pol δ [4Fe4S] 2+ cluster is oxidized either directly or by an electron acceptor, possibly an oxidized [4Fe4S] protein or a guanine radical formed during oxidative stress. We have found, in the case of EndoIII, that cluster oxidation promotes a substantial increase in binding affinity (27). Here, given the already tight binding to DNA of Pol δ with PCNA, a still tighter binding causes Pol δ to slow its progression.…”

“…Given the retention of activity with decreased processivity, these results are consistent with an increase in DNA binding affinity upon oxidation, which would impede rapid sliding of PCNA-bound Pol δ. A significant increase in DNA binding is evident also with EndoIII upon oxidation (27). Critically, reduction by bulk electrolysis largely restores activity to native levels, confirming that cluster oxidation acts as a reversible switch.…”

Nitric Oxide Modulates Endonuclease III Redox Activity by a 800 mV Negative Shift upon [Fe4S4] Cluster Nitrosylation

“…If anything, the DNA affinity for the nitrosylated protein is higher, as would be expected given the more positive charge on the cluster site. 31 Interestingly, circular dichroism spectra indicate that [Fe 4 S 4 ] cluster nitrosylation decreases the ε 222 / ε 208 ellipticity ratio from 1.199 ± 0.005 to 0.90 ± 0.04 from native to nitrosylated EndoIII (Figure 5), which is consistent with disordering of α helices. 53 Despite this disordering, [Fe 4 S 4 ] cluster nitrosylation does not appear to lower the DNA-binding affinity.…”

Sensing DNA through DNA Charge Transport

“…Recently, experiments were conducted that systematically varied the oxidation state of the [4Fe4S] cluster and measured how the redox state of the metallocofactor influenced DNA binding affinity. 89 Electrophoretic mobility shift assays, isothermal titration calorimetry, and microscale thermophoresis were used to probe the nonspecific DNA binding of Endonuclease III, a base excision repair glycosylase that repairs oxidized pyrimidines in Escherichia coli . The protein with the oxidized cluster showed significantly stronger affinity for DNA.…”