Functional Human Mitochondrial DNA Polymerase γ Forms a Heterotrimer

Mitochondrial DNA polymerase γ (pol γ) is responsible for replication and repair of mtDNA and is mutated in individuals with genetic disorders such as chronic external ophthalmoplegia and Alpers syndrome. pol γ is also an adventitious target for toxic side effects of several antiviral compounds, and mutation of its proofreading exonuclease leads to accelerated aging in mouse models. We have used a variety of physical and functional approaches to study the interaction of the human pol γ catalytic subunit with both the wild-type accessory factor, pol γB, and a deletion derivative that is unable to dimerize and consequently is impaired in its ability to stimulate processive DNA synthesis. Our studies clearly showed that the functional human holoenzyme contains two subunits of the processivity factor and one catalytic subunit, thereby forming a heterotrimer. The structure of pol γ seems to be variable, ranging from a single catalytic subunit in yeast to a heterodimer in Drosophila and a heterotrimer in mammals. Mitochondrial DNA polymerase γ (pol γ) is responsible for replication and repair of mtDNA and is mutated in individuals with genetic disorders such as chronic external ophthalmoplegia and Alpers syndrome. pol γ is also an adventitious target for toxic side effects of several antiviral compounds, and mutation of its proofreading exonuclease leads to accelerated aging in mouse models. We have used a variety of physical and functional approaches to study the interaction of the human pol γ catalytic subunit with both the wild-type accessory factor, pol γB, and a deletion derivative that is unable to dimerize and consequently is impaired in its ability to stimulate processive DNA synthesis. Our studies clearly showed that the functional human holoenzyme contains two subunits of the processivity factor and one catalytic subunit, thereby forming a heterotrimer. The structure of pol γ seems to be variable, ranging from a single catalytic subunit in yeast to a heterodimer in Drosophila and a heterotrimer in mammals. Mitochondria contain a single DNA polymerase, pol 4The abbreviations used are: polpolymeraseRUresonance unitsWTwild typePMSFphenylmethylsulfonyl fluorideSPRsurface plasmon resonanceITCisothermal titration calorimetryEMSAelectrophoretic mobility shift assays. γ, responsible for replication and repair of mtDNA (reviewed in Ref. 1Kaguni L.S. Annu. Rev. Biochem. 2004; 73: 293-320Crossref PubMed Scopus (334) Google Scholar). Human pol γ is isolated from mitochondria as a complex containing two subunits, a catalytic subunit, pol γA, of 139 kDa and an accessory subunit, pol γB, of 53 kDa (2Carrodeguas J.A. Kobayashi R. Lim S.E. Copeland W.C. Bogenhagen D.F. Mol. Cell. Biol. 1999; 19: 4039-4046Crossref PubMed Google Scholar, 3Gray H. Wong T.W. J. Biol. Chem. 1992; 267: 5835-5841Abstract Full Text PDF PubMed Google Scholar, 4Lim S.E. Longley M.J. Copeland W.C. J. Biol. Chem. 1999; 274: 38197-38203Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 5Johnson A. Tsai Y. Graves S. Johnson K. Biochemistry. 2000; 39: 1702-1708Crossref PubMed Scopus (123) Google Scholar). The catalytic subunit is a family A DNA polymerase with separate polymerase and 3′-5′ exonuclease domains. Two recent developments have stressed the importance of pol γ. Mutations in the catalytic subunit of human pol γ cause mitochondrial disorders (6Graziewicz M.A. Longley M.J. Bienstock R.J. Zeviani M. Copeland W.C. Nat. Struct. Mol. Biol. 2004; 11 (and references therein): 770-776Crossref PubMed Scopus (109) Google Scholar, 7Longley M.J. Graziewicz M.A. Bienstock R.J. Copeland W.C. Gene (Amst.). 2005; 354 (and references therein): 125-131Crossref PubMed Scopus (129) Google Scholar), and a transgenic mouse engineered to express an error-prone form of DNA pol γ lacking the 3′-5′ proofreading ability accumulates errors in mtDNA and undergoes accelerated aging (8Trifunovic A. Wredenberg A. Falkenberg M. Spelbrink J. Rovio A. Bruder C. Bohlooly-Y M. Gidlof S. Oldfors A. Wibom R. Tornell J. Jacobs H. Larsson N.-G. Nature. 2004; 429: 417-423Crossref PubMed Scopus (2061) Google Scholar, 9Kujoth G.C. Hiona A. Pugh T.D. Someya S. Panzer K. Wohlgemuth S.E. Hofer T. Seo A.Y. Sullivan R. Jobling W.A. Morrow J.D. Van Remmen H. Sedivy J.M. Yamasoba T. Tanokura M. Weindruch R. Leeuwenburgh C. Prolla T.A. Science. 2005; 309: 481-484Crossref PubMed Scopus (1627) Google Scholar). polymerase resonance units wild type phenylmethylsulfonyl fluoride surface plasmon resonance isothermal titration calorimetry electrophoretic mobility shift assays. The processivity and substrate binding properties of pol γA are enhanced by complex formation with the accessory subunit (2Carrodeguas J.A. Kobayashi R. Lim S.E. Copeland W.C. Bogenhagen D.F. Mol. Cell. Biol. 1999; 19: 4039-4046Crossref PubMed Google Scholar, 4Lim S.E. Longley M.J. Copeland W.C. J. Biol. Chem. 1999; 274: 38197-38203Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 5Johnson A. Tsai Y. Graves S. Johnson K. Biochemistry. 2000; 39: 1702-1708Crossref PubMed Scopus (123) Google Scholar). Most interestingly, the presence of the accessory subunit has been shown to decrease the fidelity of DNA synthesis by the catalytic subunit because it increases the ability of the enzyme to extend a mismatched primer (10Longley M.J. Nguyen D. Kunkel T.A. Copeland W.C. J. Biol. Chem. 2001; 276: 38555-38562Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). Initial characterizations of pol γ suggested that the enzyme forms a heterodimer containing one copy of each subunit. However, when we solved the crystal structure of mouse pol γB, it became apparent that this accessory factor is itself a homodimer with remarkable structural similarity to prokaryotic tRNA synthetases (Protein Data Bank code 1G5H (11Carrodeguas J.A. Theis K. Bogenhagen D.F. Kisker C. Mol. Cell. 2001; 7: 43-54Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar)). We considered it to be very unlikely that this dimerization is a crystal packing artifact, especially because we were able to show that wild-type pol γB sedimented more rapidly than a deletion derivative lacking a major portion of the dimerization interface (11Carrodeguas J.A. Theis K. Bogenhagen D.F. Kisker C. Mol. Cell. 2001; 7: 43-54Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). The incorporation of pol γB in the pol γ holoenzyme appears to be a relatively recent event in evolutionary terms. The protein has not been reported in yeast, where pol γ was first cloned as a product of the mip1 gene (12Foury F. J. Biol. Chem. 1989; 264: 20552-20560Abstract Full Text PDF PubMed Google Scholar), and efforts to find evidence for it have been unsuccessful (13Lucas P. Lasserre J.-P. Plissonneau J. Castroviejo M. Mitochondrion. 2004; 4: 13-20Crossref PubMed Scopus (20) Google Scholar). Drosophila pol γ differs from the enzyme in vertebrates as it is reported to contain only one subunit each of pol γA and pol γB (14Wernette C.M. Kaguni L.S. J. Biol. Chem. 1986; 261: 14764-14770Abstract Full Text PDF PubMed Google Scholar, 15Olson M. Wang Y. Elder R. Kaguni L. J. Biol. Chem. 1995; 270: 28932-28937Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), because the amino acids required for dimerization of human pol γB are absent in the Drosophila ortholog. Thus, pol γ provides an interesting example of a eukaryotic DNA polymerase with A of have the that pol pol γB have in mtDNA replication repair of the subunit A. Tsai Y. Graves S. Johnson K. Biochemistry. 2000; 39: 1702-1708Crossref PubMed Scopus (123) Google Scholar, M.J. Nguyen D. Kunkel T.A. Copeland W.C. J. Biol. Chem. 2001; 276: 38555-38562Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, J.A. Bogenhagen D.F. 2000; PubMed Scopus (39) Google Scholar, M. R. D. S. Copeland S. A. PubMed Scopus Google Scholar). we have the studies to the and for human pol γB by a of Our studies the first of the interaction subunits of human pol γ and show that the enzyme is a heterotrimer that contains one of the catalytic subunit and two of the processivity The binding we the two subunits that the subunits not in and that subunits not in mitochondrial DNA replication and of pol γA was from with the engineered to express pol γA as by Longley M.J. Lim S.E. Copeland W.C. Biochemistry. PubMed Scopus Google Scholar). were in in with and of were the of of were by in of with and The was for The was with and for to the The was to contain and and in with and with of the protein was with a of in with of the containing pol γA were with to the to The enzyme was to a in with with the protein was with a of to in containing were a in containing The pol γA from this were The enzyme was to a The of the protein was The pol γB and pol were in with as by (11Carrodeguas J.A. Theis K. Bogenhagen D.F. Kisker C. Mol. Cell. 2001; 7: 43-54Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). were a the was by a the of the from of pol γB were in of with and and were in a The was for The was with and the to the The was to contain and and the protein was by and as for pol γA The enzyme was to a The of pol γB and pol were and pol γ holoenzyme was by of containing pol γA and containing pol γB a The used for this from one to the protein of the as by The were for a and to contain and The holoenzyme was and the as for pol The used to protein were and S. P. Biochem. 1989; PubMed Scopus Google Scholar). The for pol γA was and that used for both pol γB and pol was because the deletion not that to The were protein and were the used in the that were when the were with were a and a with were with of protein and were studies were and for wild-type pol γB and and for pol The were by two the of and the and were was as J. P. PubMed Scopus Google and the of protein were from amino for wild-type pol γB and for pol plasmon resonance to binding was a pol γA was a the in with of resonance units pol γB and pol were ranging from to the with pol γA for by of and of and were The of pol γB and pol interaction with pol γA was in the A was it to with The a where to A by a of the complex to The is from the as a with of pol γA was (pol γB and pol were to pol γA for by of and of the was for pol γB pol were and the was the where is the resonance a The of the for DNA was the A of a in was used to an of and Two an were used as with was used the of for DNA (pol γA, pol and pol in with for and for were for by of with was titration calorimetry were with a with a of The pol γB pol were as the containing pol were by a containing The of of both with were and to Data were the with the to the and models. electrophoretic mobility shift were as (11Carrodeguas J.A. Theis K. Bogenhagen D.F. Kisker C. Mol. Cell. 2001; 7: 43-54Abstract Full Text Full Text PDF PubMed Scopus (110) Google the the of the J.A. Bogenhagen D.F. 2000; PubMed Scopus (39) Google Scholar), that the were as to the of the were in containing pol γA, and where pol γB pol forms of pol γB as a for this were as a binding and of were as a to protein that from the A was the was the and was considered a the The binding and were the of a were and in of the were and to a to the of and DNA polymerase were as (2Carrodeguas J.A. Kobayashi R. Lim S.E. Copeland W.C. Bogenhagen D.F. Mol. Cell. Biol. 1999; 19: 4039-4046Crossref PubMed Google with of pol γA and of pol pol in DNA as The polymerase was processivity were as by (2Carrodeguas J.A. Kobayashi R. Lim S.E. Copeland W.C. Bogenhagen D.F. Mol. Cell. Biol. 1999; 19: 4039-4046Crossref PubMed Google and in the of of of Human pol γ and of the and have been unable to express pol γA in in an of enzyme for we the protein in from a it is to express the holoenzyme in by the A and subunits, we to express the subunit in the to the of a of pol γB an for each protein of this we the binding of wild-type pol γB to a deletion derivative that we to as pol contains a in of of pol γB, was first reported by (11Carrodeguas J.A. Theis K. Bogenhagen D.F. Kisker C. Mol. Cell. 2001; 7: 43-54Abstract Full Text Full Text PDF PubMed Scopus (110) Google to the of the in the mouse pol γB crystal structure in dimerization of the accessory derivative has ability to to with pol γA has ability to stimulate of the catalytic subunit. Thus, we to the of wild-type pol γB and pol to pol γA in this a of containing pol γA with a of pol γB of the subunits and of pol γ holoenzyme by a this of a by and a with of pol γA and of pol γB, we a of of pol γ pol γA is the of pol have a of enzyme for the studies of the of the polymerase subunits reported The of is shown in is apparent that the of the subunit to pol γA is by when the is with pol than with wild-type pol We the properties of pol γB and pol by in a (11Carrodeguas J.A. Theis K. Bogenhagen D.F. Kisker C. Mol. Cell. 2001; 7: 43-54Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). this we in more by as shown in wild-type pol γB, were to a single the was as the in the an of was with the of for pol to the to a to is with that the wild-type protein is a homodimer with We that the pol γB is by a than of for the was the were a the apparent by the was a a and a the that the was a to the not A was a for a The was and the was The that pol a ability to dimerize is with the from the crystal structure of mouse pol this we that is in pol to the dimerization interface of Thus, it is that pol a ability to dimerize protein as a protein the as in in in pol γ. the in is a we this as an to study the of the the of a titration in of pol γA and pol γB, as the were to a The of pol γA and pol γB are shown in the of each pol γB was to pol γA was a of A to the of pol γB was a of pol γB was is with the that pol γB is a A binding titration with pol γA and pol showed a of not the of A and and the of the titration calorimetry was used to the of holoenzyme of the and the The of the was that was not was in with both wild-type pol γB and pol for of pol γB and pol subunits with pol γA were and A in of the by pol γB the pol in binding to pol γA was is a of the of the with that the of the complex pol γA and pol γB is of pol γA with the pol γB is as an with of and a of of pol γA with the pol protein is as as it is with the wild-type this was binding with a pol γA in of the we considered that this the for the interaction the for pol γB and pol binding to pol of from titration were pol errors shown in are the errors shown in are the of from titration were The errors shown in are the in a of the pol γB was to study the interaction of pol γA with pol γB pol in pol γA was the surface to the protein to the to the of resonance units to of to the of binding of pol γB to pol γA was in because of the pol γA and the were also for and the interaction of pol γB with pol γA are shown in and were as in the in a a was to and as as are in with the is unable to form a protein and The for the wild-type protein is The pol and pol γA is by a factor of are with the apparent of for the pol γA interaction from polymerase by Johnson A. Tsai Y. Graves S. Johnson K. Biochemistry. 2000; 39: 1702-1708Crossref PubMed Scopus (123) Google for pol γB and pol binding to the pol γA in and the were from of the and a and the were from of the and a and the were from of the and a and the were from of the and a is the from the by the pol errors shown in are the and the were from of the and a is the from the by the The errors shown in are the in a The the of pol γA with DNA as as pol were the relatively in of pol γA for binding pol as with the wild-type subunit. we to the ability of to the interaction of pol γA with DNA of the used to study DNA is to with an electrophoretic mobility shift We have used this in the to study the interaction of pol γB with pol γA in (2Carrodeguas J.A. Kobayashi R. Lim S.E. Copeland W.C. Bogenhagen D.F. Mol. Cell. Biol. 1999; 19: 4039-4046Crossref PubMed Google Scholar, J.A. Theis K. Bogenhagen D.F. Kisker C. Mol. Cell. 2001; 7: 43-54Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, J.A. Bogenhagen D.F. 2000; PubMed Scopus (39) Google Scholar). We have shown that the with pol γA in binding we have not the of this We a binding in containing forms of pol γ to were and with a of to the of of a of polymerase from the complex be more to the than the The of the be by from the of and a of to study has been the of this M. PubMed Scopus Google Scholar). that pol γA from a The pol holoenzyme complex more the as and with a of A with pol γ with the protein that this complex was as as that with wild-type pol γB of the to study is that this provides ability to because in the are from as the We used to study the binding of pol γA and of pol γA with wild-type pol γB pol of and in the of titration to the of protein to the a The are to the resonance of the binding of pol γA to the the presence of wild-type pol γB in the pol γ holoenzyme the binding of the polymerase to pol an of The that pol pol γA with and the polymerase were in of that this derivative very ability to stimulate DNA synthesis by pol γA (11Carrodeguas J.A. Theis K. Bogenhagen D.F. Kisker C. Mol. Cell. 2001; 7: 43-54Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). polymerase with this used only of pol and used only as the we the ability of this derivative to wild-type pol γB to stimulate pol γA protein and DNA as as the be in in of pol γA and a DNA synthesis is by of pol γB of of pol were required to stimulate pol γA, and the was with wild-type pol ability to stimulate pol γA was when DNA was used as substrate as a pol γB was only as reported (2Carrodeguas J.A. Kobayashi R. Lim S.E. Copeland W.C. Bogenhagen D.F. Mol. Cell. Biol. 1999; 19: 4039-4046Crossref PubMed Google Scholar, 4Lim S.E. Longley M.J. Copeland W.C. J. Biol. Chem. 1999; 274: 38197-38203Abstract Full Text Full Text PDF PubMed Scopus (183) Google the pol ability to stimulate pol γA, the was We to study polymerase processivity in pol γ was with a of of by pol γ was by of with a to as a binding to polymerase that from the as the first and is of with only and of show that wild-type pol γ to as as the first The of not to when the was and the in and of that the an to of Thus, we that the of product shown in and of the a single polymerase binding pol was used in the were by than that the pol was impaired as a processivity of the primer was pol γA pol γ the first of the of the pol γ Our show that pol γB forms a in and isothermal titration calorimetry that the pol γB to the pol γA to form a heterotrimer with the structure is the first study of the of subunits in the of the of for the subunit we by with the binding in the presence of DNA in the enzyme studies of Johnson A. Tsai Y. Graves S. Johnson K. Biochemistry. 2000; 39: 1702-1708Crossref PubMed Scopus (123) Google Scholar). The pol complex have a of appears to be than of the of the polymerase isolated from mitochondria H. Wong T.W. J. Biol. Chem. 1992; 267: 5835-5841Abstract Full Text PDF PubMed Google Scholar, 4Lim S.E. Longley M.J. Copeland W.C. J. Biol. Chem. 1999; 274: 38197-38203Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). We the that in of the of the structure of the However, the of the we it is more that the apparent of the enzyme from mitochondria be to the that the in studies used protein is that the holoenzyme be in with A and subunits of protein to an of the is to the of polymerase subunits in The interaction the pol γA and subunits is to of the holoenzyme to the subunits in of pol γA pol γB subunits when the holoenzyme was from mitochondrial (2Carrodeguas J.A. Kobayashi R. Lim S.E. Copeland W.C. Bogenhagen D.F. Mol. Cell. Biol. 1999; 19: 4039-4046Crossref PubMed Google Scholar, J.A. Bogenhagen D.F. 2000; PubMed Scopus (39) Google Scholar). we it unlikely that for the of pol γ in mtDNA replication and repair for the pol γ subunits to this from the that two subunits are and mitochondria is the by single subunits are a holoenzyme in the we the that single subunits be to in of subunits has been We also the of wild-type pol γB with that of a form that a for showed that dimerization of this protein is with an apparent of for the pol the ability to to pol γA in a complex with an with that of the wild-type pol γB the in both are with the that of the that pol γA forms with pol γB are when it pol has been suggested for the Drosophila pol γ subunits, it is that human pol γA and pol γB an interaction interface L. Kaguni L.S. Biochemistry. 2001; PubMed Scopus Google Scholar). for the structure of the holoenzyme that a pol γB two for the interaction with pol a be with the of an is not with clearly the formation of a We an for the interaction pol γA and pol to this pol γA with one pol γB subunit in the pol γB this interaction interaction of a catalytic subunit with the pol γB subunit. pol γA to the pol it is that pol γA forms with pol γB that are in the pol binding of this complex to are with and it be interesting to a crystal structure of the pol γ holoenzyme to this processivity factor to stimulate the catalytic subunit. of pol γA DNA and have of the wild-type pol γB and pol to the of the accessory subunit to the interaction of the polymerase with DNA both electrophoretic mobility shift and we showed that the enzyme by of pol to pol γA to and in the presence of The apparent for the binding of wild-type holoenzyme to a is pol γA and the pol complex the substrate with of and binding are than reported by Johnson A. Tsai Y. Graves S. Johnson K. Biochemistry. 2000; 39: 1702-1708Crossref PubMed Scopus (123) Google polymerase a have shown that of pol γA is is by pol γB (2Carrodeguas J.A. Kobayashi R. Lim S.E. Copeland W.C. Bogenhagen D.F. Mol. Cell. Biol. 1999; 19: 4039-4046Crossref PubMed Google Scholar, 4Lim S.E. Longley M.J. Copeland W.C. J. Biol. Chem. 1999; 274: 38197-38203Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). Our show both pol γB and pol binding of pol γA to pol γA binding to DNA M.J. P. T. Biochemistry. PubMed Scopus Google Scholar). pol γB to a pol to the interaction of pol γA with DNA to it to by pol γB clearly increases the processivity of pol γA, it of the catalytic subunit as Johnson A. Tsai Y. Graves S. Johnson K. Biochemistry. 2000; 39: 1702-1708Crossref PubMed Scopus (123) Google have shown that the accessory subunit the for binding and increases the Longley (10Longley M.J. Nguyen D. Kunkel T.A. Copeland W.C. J. Biol. Chem. 2001; 276: 38555-38562Abstract Full Text Full Text PDF PubMed Scopus (197) Google have that effects in polymerase Our physical studies the interaction of pol γB and pol with pol γA have not the of of polymerase However, we that the binding of pol to pol γA and the of the enzyme not to of polymerase and the pol have a ability to as a form of pol γB, because it for binding to pol γA is very in to this are of pol γB to that the interaction pol γB and pol γA is with that of of processivity with pol γB is of the processivity such as pol and that polymerase binding to DNA by the DNA T. S. P. J. Cell. Full Text PDF PubMed Scopus Google Scholar). as as in the binding of to DNA polymerase, the interaction of the accessory subunit with the polymerase is by a interface that be to a S. S. T. Nature. PubMed Scopus Google Scholar, S. S. A. PubMed Scopus Google Scholar). is also for A. J.M. Mol. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, A. J.M. J. 2004; PubMed Scopus Google and J.M. Mol. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google that not form and W.A. 2001; PubMed Scopus Google have suggested that processivity be in of two by as by the of a interaction We that pol γ this in the pol γ provides a example of an DNA polymerase that has a structure in The pol γB and prokaryotic tRNA synthetases (11Carrodeguas J.A. Theis K. Bogenhagen D.F. Kisker C. Mol. Cell. 2001; 7: 43-54Abstract Full Text Full Text PDF PubMed Scopus (110) Google has been as an of gene Y. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). is by Drosophila pol γ, is a heterodimer containing one copy each of the A and subunits L.S. Annu. Rev. Biochem. 2004; 73: 293-320Crossref PubMed Scopus (334) Google Scholar, 15Olson M. Wang Y. Elder R. Kaguni L. J. Biol. Chem. 1995; 270: 28932-28937Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Drosophila pol γB only with human pol γB, the only deletion in the of the two that the Drosophila protein contains a deletion of of the containing the a deletion to that used to human pol We that the Drosophila protein have a deletion of this from a with the Our that pol is able to pol γ that this protein have to Drosophila pol γB to stimulate its catalytic have deletion of this be of to the of Drosophila and pol γ We for with protein The used a by to D.