Enzymatic Mechanism Controls Redox-mediated Protein-DNA Interactions at the Replication Origin of Kinetoplast DNA Minicircles

Abstract

Kinetoplast DNA (kDNA) is the mitochondrial DNA of trypanosomatids. Its major components are several thousand topologically interlocked DNA minicircles. Their replication origins are recognized by universal minicircle sequence-binding protein (UMSBP), a CCHC-type zinc finger protein, which has been implicated with minicircle replication initiation and kDNA segregation. Interactions of UMSBP with origin sequences in vitro have been found to be affected by the protein's redox state. Reduction of UMSBP activates its binding to the origin, whereas UMSBP oxidation impairs this activity. The role of redox in the regulation of UMSBP in vivo was studied here in synchronized cell cultures, monitoring both UMSBP origin binding activity and its redox state, throughout the trypanosomatid cell cycle. These studies indicated that UMSBP activity is regulated in vivo through the cell cycle dependent control of the protein's redox state. The hypothesis that UMSBP's redox state is controlled by an enzymatic mechanism, which mediates its direct reduction and oxidation, was challenged in a multienzyme reaction, reconstituted with pure enzymes of the trypanosomal major redox-regulating pathway. Coupling in vitro of this reaction with a UMSBP origin-binding reaction revealed the regulation of UMSBP activity through the opposing effects of tryparedoxin and tryparedoxin peroxidase. In the course of this reaction, tryparedoxin peroxidase directly oxidizes UMSBP, revealing a novel regulatory mechanism for the activation of an origin-binding protein, based on enzyme-mediated reversible modulation of the protein's redox state. This mode of regulation may represent a regulatory mechanism, functioning as an enzyme-mediated, redox-based biological switch.2 is a unique extrachromosomal DNA network, found in the single mitochondrion of trypanosomatids. It consists, in the species Crithidia fasciculata, of ϳ5,000 duplex DNA minicircles of 2.5 kbp and ϳ50 maxicircles of 37 kbp that are interlocked topologically to form a DNA network. Two short sequences, the dodecameric universal minicircle sequence (UMS), GGGGTTGGTGTA, and the hexameric sequence ACGCCC, which were located at the minicircle's replication origin and implicated with its replication initiation, were conserved in all trypanosomatid species studied (recently reviewed in Refs.

Leishmania Mitochondrial Peroxiredoxin Plays a Crucial Peroxidase-Unrelated Role during Infection: Insight into Its Novel Chaperone Activity

“…Most notably, no defects in kinetoplast morphology and division were noticed (Figure 1E). These observations argue against the involvement of mTXNPx in kDNA replication, a function previously attributed to mitochondrial TXNPxs [20], [22]. Even though these results indicate that mTXNPx is redundant in the insect form of L. infantum , they do not discard a relevant role for this enzyme during the amastigote stage.…”

“…UMSBP binds to the DNA when it is reduced and is released when oxidized. According to the proposed model, mTXNPx would oxidize UMSBP [20].…”

Low-Molecular-Mass Antioxidants in Parasites

“…UMSBP is a zinc finger protein that plays a key role in the replication of mitochondrial DNA in trypanosomatids. The binding of UMSBP to the kinetoplast DNA is significantly affected by the redox state and Tpx has been shown to act as the reducing agent (161,164,179,220). MSRs are involved in protein repair by catalyzing the reduction of methionine sulfoxide residues to methionine.…”

Interactions of a Replication Initiator with Histone H1-like Proteins Remodel the Condensed Mitochondrial Genome

“…Formaldehyde was added to a final concentration of 1%, and the reaction mixture was incubated for additional 5 min at 25°C, followed by its 5-fold dilution in triple-distilled water. Then, loading buffer was added, and the protein samples were electrophoresed in 16.5% polyacrylamide gel, as described previously (10).…”

“…Minicircle replication initiates at conserved origin sequences, which are bound by a mitochondrial singlestranded sequence-specific DNA-binding protein, named the universal minicircle sequence-binding protein (UMSBP) (4 -9). UMSBP is a CCHC-type zinc finger protein whose binding to origin sequences is regulated by the cell cycle control of the protein redox state (10,11). Cross-linking analysis has shown that UMSBP binds the kDNA network in vivo (8).…”