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. 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. Kinetoplast DNA (kDNA) 2The abbreviations used are: kDNA, kinetoplast DNA; UMS, universal minicircle sequence; UMSBP, UMS-binding protein; TXN, tryparedoxin; TXNPx, tryparedoxin peroxidase; TR, trypanothione reductase; FACS, fluorescence-activated cell sorting; PBS, phosphate-buffered saline; EMSA, electrophoretic mobility shift assay; BSA, bovine serum albumin; DAPI, 4′,6-diamidino-2-phenylindole; PEG, polyethylene glycol. is a unique extrachromosomal DNA network, found in the single mitochondrion of trypanosomatids. It consists, in the species Crithidia fasciculata, of ∼5,000 duplex DNA minicircles of 2.5 kbp and ∼50 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. 1Liu B. Liu Y. Motyka S.A. Agbo E.E. Englund P.T. Trends Parasitol. 2005; 21: 363-369Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 2Lukes J. Hashimi H. Zikova A. Curr. Genet. 2005; 48: 277-299Crossref PubMed Scopus (170) Google Scholar, 3Shlomai J. Curr. Mol. Med. 2004; 4: 623-647Crossref PubMed Scopus (118) Google Scholar, 4Roy J. Faktorova D. Lukes J. Burger G. Protist. 2007; 158: 385-396Crossref PubMed Scopus (36) Google Scholar). An origin-binding protein, designated UMS-binding protein (UMSBP), was purified from C. fasciculata. The protein binds specifically the UMS dodecamer and a 14-mer sequence, containing the core hexamer, conserved at the H-strand of the minicircle replication origin (5Abeliovich H. Tzfati Y. Shlomai J. Mol. Cell Biol. 1993; 13: 7766-7773Crossref PubMed Scopus (31) Google Scholar, 6Abu-Elneel K. Kapeller I. Shlomai J. J. Biol. Chem. 1999; 274: 13419-13426Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 7Tzfati Y. Abeliovich H. Avrahami D. Shlomai J. J. Biol. Chem. 1995; 270: 21339-21345Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 8Tzfati Y. Abeliovich H. Kapeller I. Shlomai J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6891-6895Crossref PubMed Scopus (52) Google Scholar, 9Onn I. Kapeller I. Abu-Elneel K. Shlomai J. J. Biol. Chem. 2006; 281: 37468-37476Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 10Avrahami D. Tzfati Y. Shlomai J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10511-10515Crossref PubMed Scopus (24) Google Scholar). RNA interference analysis has recently revealed the function of UMSBP in minicircle replication initiation and in kDNA segregation, as well as in nuclear mitosis, implying a potential role for UMSBP in linking kDNA replication and segregation to nuclear M phase control during the trypanosome cell cycle (11Milman N. Motyka S.A. Englund P.T. Robinson D. Shlomai J. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 19250-19255Crossref PubMed Scopus (44) Google Scholar). UMSBP contains five CCHC-type zinc finger domains (5Abeliovich H. Tzfati Y. Shlomai J. Mol. Cell Biol. 1993; 13: 7766-7773Crossref PubMed Scopus (31) Google Scholar), which differ in their function. Although the C-terminal zinc its DNA binding the zinc finger is in that to the protein I. N. Shlomai J. 2004; PubMed Scopus (52) Google Scholar). and J. 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In the major enzymatic that the redox state is based on tryparedoxin and tryparedoxin peroxidase In this is an designated trypanothione A. PubMed Scopus Google Scholar, A. Mol. Parasitol. PubMed Scopus (118) Google Scholar). The form of which is by trypanothione in TXNPx, a of the with in the which contains the Biol. Med. 1999; PubMed Scopus Google Scholar). The function of is to the cell from H. PubMed Scopus Google Scholar, H. C. A. Biol. Med. PubMed Scopus Google Scholar). have that redox both the binding of UMSBP to the origin sequence and the protein have that C. tryparedoxin and and UMSBP DNA binding activity in vitro and that UMSBP activity may be regulated in the trypanosomatid cell through the control of the protein's redox state I. N. Shlomai J. 2004; PubMed Scopus (52) Google Scholar). the mechanism that mediates the oxidation of UMSBP in a regulatory has In a Motyka S.A. G. Englund P.T. J. Biol. Chem. 2006; 281: Full Text Full Text PDF PubMed Scopus (33) Google have that of UMSBP and of kDNA in TXNPx, have from the of mitochondrial TXN, the redox state of UMSBP through the oxidation of a that of UMSBP in the cell from its direct oxidation by and the of its through the oxidation of that oxidation of UMSBP is through an enzymatic reaction in which UMSBP is directly as a for oxidation by TXNPx, both the and the from a regulatory mechanism that the binding of UMSBP to the replication found that the of UMSBP to the origin sequence is controlled in vivo through a cell regulation of the protein redox state. for the on the direct oxidation of UMSBP by and the opposing regulatory effects of and on the of UMSBP with the conserved origin on an enzymatic mechanism, which the of UMSBP at the kDNA replication were by was from trypanothione was from and and were from trypanothione and Crithidia and were a of of of C. tryparedoxin peroxidase was a of H. and of mitochondrial tryparedoxin peroxidase were a of for and Cell and were a of from the UMSBP was as have I. N. Shlomai J. 2004; PubMed Scopus (52) Google Scholar), that from the was with the of by of and and of and C. were in containing and at with by was as J. Cell Sci. PubMed Google Scholar). a cell was to in containing by with at were by for at in and to for an of to as were at and with were in and at cell were by the of the C. cell a in as J. J. Biol. Chem. Full Text PDF PubMed Google Scholar), that was used Y. Abeliovich H. Avrahami D. Shlomai J. J. Biol. Chem. 1995; 270: 21339-21345Abstract Full Text Full Text PDF PubMed Scopus (43) Google and was from the at from C. cultures, were in PBS, in and at their the were for at in PBS, containing and for on In to cell the were through by their through a was to the of its to the was a The DNA of was an of the and are in of the was were as K. Kapeller I. Shlomai J. J. Biol. Chem. 1999; 274: 13419-13426Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 7Tzfati Y. Abeliovich H. Avrahami D. Shlomai J. J. Biol. 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PubMed Scopus Google with protein and The was containing the was were for as were was were by as were a were by for in in containing and for with a of as were for with a by as by the was were at 37 with in containing was by of was as have I. N. Shlomai J. 2004; PubMed Scopus (52) Google Scholar), that the and was used throughout the to was was a of the was on a protein with The was with of the and with of containing and was by a in the was in an monitoring protein a The was on a as activity was by the reduction of the of and Mol. Parasitol. PubMed Scopus Google Scholar). the reaction, in and in at The reaction was by as trypanothione was to its and was by The reaction was by the of and its reduction was by the of on UMSBP of on UMSBP binding to UMS was by the of UMSBP in the of the indicated of in an reaction containing BSA, and The were at for by the of of binding containing of BSA, and of The were at for and their were by and to a as as well as oxidation of UMSBP by TXNPx, UMSBP was in a reaction containing and for at The protein was by a and to and The reaction was by by to and analysis by of UMSBP by in this reaction was by monitoring the of UMSBP with as The reaction were on was to a of by for on The reaction were by as was at the indicated in a reaction containing BSA, of and were for at by the of at the indicated in a containing and The were for at UMS was in a binding containing of BSA, and The reaction were for an at and their were by and by C. were with PBS, on and with for This was by with a with in PBS, a with PBS, and with at The were for with PBS, by of with in at a was and the were for in a This was by with in and a with to at a The were for with for with in PBS, with PBS, and for was and the were The were a for the and for the Cell of the UMSBP Its to studies have that the state of UMSBP and protein are throughout the trypanosomatid cell cycle K. Robinson Englund P.T. Shlomai J. J. Cell Biol. PubMed Scopus Google Scholar), that regulation of UMSBP activity in vivo is of a The that binding of UMSBP to an the UMS, conserved at the minicircle replication origin, is I. N. Shlomai J. 2004; PubMed Scopus (52) Google was in with a the role of redox in the regulation of UMSBP activity in vivo has been this UMSBP activity was with the of the cell in cell UMSBP activity was in cell at from the synchronized and the in the cell cycle was by monitoring the of phase and by analysis of in These revealed that reduction of UMSBP is its UMS-binding activity in a cell of UMSBP activity was K. Robinson Englund P.T. Shlomai J. J. Cell Biol. PubMed Scopus Google Scholar), that the in the of UMSBP during the of the cell cycle. that UMSBP activity is regulated in vivo through a cell control of the protein redox state. in UMSBP activity during of the cell during kDNA replication and during nuclear kinetoplast DNA replication has the of a of UMSBP revealing an function of UMSBP during kDNA segregation and nuclear (11Milman N. Motyka S.A. Englund P.T. Robinson D. Shlomai J. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 19250-19255Crossref PubMed Scopus (44) Google Scholar). The that the redox state of UMSBP throughout the cell cycle in the protein reduction which with the of its UMS-binding activity. directly the in the UMSBP redox state throughout the trypanosomatid cell have the of during the of the cell cycle. this of from were and with which in the protein that are The which were and in the reaction, were by a of the of the the was with a which with the in the The binding of in UMSBP by the its by A. S. A. J. PubMed Scopus Google Scholar). This shift in the of the protein is by and by and the which were and were for with the are that were in the were for the with UMSBP in the protein represent UMSBP that at the in the cell cycle UMSBP oxidation was in with the of the cell as by monitoring the in of phase and by analysis of cell at from the synchronized These revealed that the of UMSBP in a cell indicated that of the UMSBP redox state during the trypanosomatid cell cycle with the of its UMS-binding activity the here that UMSBP activity is regulated in vivo through a cell control of the protein's redox state. Interactions UMSBP redox state in vivo in a cell of reduction during and and of oxidation during of the cell cycle implying that both reduction and oxidation of UMSBP are regulated in the trypanosomatid have that an UMSBP and activates its binding to UMS I. N. 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Shlomai J. 2004; PubMed Scopus (52) Google Scholar). the the of on the of UMSBP, an the which may have affected the redox state of the reaction, an protein is by the that of the UMSBP on the with of in the of a and protein is by the in the the of UMSBP The for the of the protein as the of the reaction, is The that this may represent a has to be the of UMSBP to UMS DNA through the for the UMSBP and TXNPx, the of UMSBP by the reaction, and the of UMSBP in this the was that UMSBP, which contains the as a for direct oxidation by have this for the of purified on the binding of UMSBP to as is in of on UMSBP its to the UMS DNA that UMSBP as a for the of analysis an of UMSBP binding to UMS DNA with of in the reaction, to in the of of the with an of the DNA binding reaction, in the of The for this of is may be that the phase of the of with and in the This may be in the phase by a in the of the and the of located the protein as for oxidation by the of on the DNA binding activity of UMSBP from the oxidation of UMSBP by the this the which with in the protein, and UMSBP, which was with UMSBP is a protein which contains its with the is to a for in the protein the of UMSBP with the the and of In the in UMSBP was with and with and to analysis that UMSBP a electrophoretic mobility its with the with UMSBP that was with a and analysis of the revealed the shift in mobility of of the UMSBP the of for the with the as a of the In with this is an that of UMSBP with in by at several in the UMSBP sequence, as indicated by N. and J. which be to in the protein as a of its oxidation by of and on the potential of on the binding of UMSBP to the origin sequence, the opposing effects of and in the binding of UMSBP to this have reconstituted the tryparedoxin redox of from the purified enzymes TR, TXN, and TXNPx, and this reaction in vitro to the binding reaction In this reaction, is through the of to in which in UMSBP was to the reconstituted enzymatic which was of TXN, of DNA binding the of of UMSBP, be in the analysis and The of the binding of UMSBP to DNA and and as have I. 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PubMed Scopus Google Scholar). The in the the of UMSBP redox state and its DNA binding during the in the cell the cell regulation of UMSBP redox state to its during the of the trypanosomatid cell cycle. In a RNA interference analysis (11Milman N. Motyka S.A. Englund P.T. Robinson D. Shlomai J. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 19250-19255Crossref PubMed Scopus (44) Google Scholar), have found that the minicircle origin-binding protein, UMSBP, during the phase of the cell a role in the initiation of minicircles this has revealed an function for this protein, during kDNA segregation and nuclear (11Milman N. Motyka S.A. Englund P.T. Robinson D. Shlomai J. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 19250-19255Crossref PubMed Scopus (44) Google Scholar). These the of UMSBP in the control of the trypanosomal cell cycle. 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Med. 1999; PubMed Scopus Google Scholar). is a of the which are by in are conserved throughout enzymatic which are of the redox state of the may be in the of the that the mechanism in this may represent a mode of regulation of protein through enzyme-mediated of their redox state. In a the reversible of by their and their to the well regulation of activity through their enzyme-mediated reversible for the of trypanothione and C. and H. and for the of a the C. tryparedoxin and for a of tryparedoxin peroxidase and for a of tryparedoxin peroxidase