Cited by 226DNA Repair MechanismsMitochondrial Function and PathologyEnzyme Structure and Function

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The Mitochondrial p55 Accessory Subunit of Human DNA Polymerase γ Enhances DNA Binding, Promotes Processive DNA Synthesis, and Confers N-Ethylmaleimide Resistance

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Lim, Susan E., Longley, Matthew J., Copeland, William C.·1999·Journal of Biological Chemistry

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Abstract

Human DNA polymerase gamma is composed of a 140-kDa catalytic subunit and a smaller accessory protein variously reported to be 43-54 kDa. Immunoblot analysis of the purified, heterodimeric native human polymerase gamma complex identified the accessory subunit as 55 kDa. We isolated the full-length cDNA encoding a 55-kDa polypeptide, expressed the cDNA in Escherichia coli and purified the 55-kDa protein to homogeneity. Recombinant Hp55 forms a high affinity, salt-stable complex with Hp140 during protein affinity chromatography. Immunoprecipitation, gel filtration, and sedimentation analyses revealed a 190-kDa complex indicative of a native heterodimer. Reconstitution of Hp140.Hp55 raises the salt optimum of Hp140, stimulates the polymerase and exonuclease activities, and increases the processivity of the enzyme by several 100-fold. Similar to Hp140, isolated Hp55 binds DNA with moderate strength and was a specificity for double-stranded primer-template DNA. However, Hp140.Hp55 has a surprisingly high affinity for DNA, and kinetic analyses indicate Hp55 enhances the affinity of Hp140 for primer termini by 2 orders of magnitude. Thus the enhanced DNA binding caused by Hp55 is the basis for the salt tolerance and high processivity characteristic of DNA polymerase gamma. Observation of native DNA polymerase gamma both as an Hp140 monomer and as a heterodimer with Hp55 supports the notion that the two forms act in mitochondrial DNA repair and replication. Additionally, association of Hp55 with Hp140 protects the polymerase from inhibition by N-ethylmaleimide.

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Young, M. J., Longley, M. J., Li, F.-Y., Kasiviswanathan, R., Wong, L.-J., & Copeland, W. C. (2011) · Human Molecular Genetics · doi:10.1093/hmg/ddr209 · Cited by 66
“The salt-dependent stimulation of p140 by p55 has been demonstrated previously with the exonuclease proficient form of p140 on poly(rA)†oligo(dT)12 – 18 (10), and we saw almost identical stimulation of the exonuclease deficient form of pol g in this study (Fig.”
Influential
Mitochondrial DNA degradation: A quality control measure for mitochondrial genome maintenance and stress response
Zhao, L. (2019) · The Enzymes · doi:10.1016/bs.enz.2019.08.004 · Cited by 70
Global DNA methylation synergistically regulates the nuclear and mitochondrial genomes in glioblastoma cells
Sun, X., Johnson, J. L., & John, J. C. S. (2018) · Nucleic Acids Research · doi:10.1093/nar/gky339 · Cited by 62
“Taqman assays used in the Fluidigm qPCR arrays Gene name Taqman assay number Gene name Taqman assay number 18SrRNA Hs99999901_s1 MT-ND2 Hs02596874_g1 Actb Hs99999903_m1 MT-ND3 Hs02596875_s1 ADGRB1 Hs00181777_m1 MT-ND4 Hs02596876_g1 AGAP3 Hs01553103_g1 MT-ND4L Hs02596877_g1 AGPAT4 Hs01088853_m1 MT-ND5 Hs02596878_g1 ASL…”
BackgroundInfluential
Analysis of mutant DNA polymerase γ in patients with mitochondrial DNA depletion
Taanman, J., Rahman, S., Pagnamenta, A. T., Morris, A. A. M., Bitner-Glindzicz, M. A. K., Wolf, N. I., Leonard, J. V., Clayton, P. T., & Schapira, A. H. V. (2008) · Human Mutation · doi:10.1002/humu.20852 · Cited by 63
DNA Binding Properties of Human pol γB
Carrodeguas, J. A., Pinz, K. G., & Bogenhagen, D. F. (2002) · Journal of Biological Chemistry · doi:10.1074/jbc.m207030200 · Cited by 59
“Our results suggest that the presence of a 5 overhang in the primertemplate used by Lim et al. (1999) probably did not influence the binding. pol B has shown a similar ability to bind other duplex fragments longer than ds47 (data not shown), suggesting that DNAs must exceed a minimal size between…”
Background
Consequences of compromised mitochondrial genome integrity
Gustafson, M. A., Sullivan, E. D., & Copeland, W. C. (2020) · DNA repair · doi:10.1016/j.dnarep.2020.102916 · Cited by 57
Mitochondrial genetic variation is enriched in G-quadruplex regions that stall DNA synthesis in vitro.
Butler, T. J., Estep, K. N., Sommers, J. A., Maul, R., Moore, A., Bandinelli, S., Cucca, F., Tuke, M., Wood, A., Bharti, S., et al. (2020) · Human Molecular Genetics · doi:10.1093/hmg/ddaa043 · Cited by 54
“The observation that reactions with both polymerases produced DNA products resembling those produced by Pol γ alone is consistent with reports that Pol γ binds DNA with high affinity, whereas PrimPol exhibits weaker binding (40,41).”
Result
DNA base excision repair activities and pathway function in mitochondrial and cellular lysates from cells lacking mitochondrial DNA
Stuart, J. A. (2004) · Nucleic Acids Research · doi:10.1093/nar/gkh533 · Cited by 58
“Indeed, other factors including the polymerase g accessory subunit affect polymerase g’s interaction with oligonucleotides ( 22 ,23).”
Background
Progressive External Ophthalmoplegia and Vision and Hearing Loss in a Patient With Mutations in POLG2 and OPA1
Ferraris, S., Clark, S., Garelli, E., Davidzon, G., Moore, S. A., Kardon, R. H., Bienstock, R. J., Longley, M. J., Mancuso, M., Ríos, P. G., et al. (2008) · Archives of Neurology · doi:10.1001/archneurol.2007.9 · Cited by 57
“The G416A was grown, expressed, purified, and stored, as described previously.(10,14)”
MethodInfluential
Defects of Mitochondrial DNA Replication
Copeland, W. C. (2014) · Journal of Child Neurology · doi:10.1177/0883073814537380 · Cited by 54
“This also demonstrates that disruption of exonuclease activity is not embryonic lethal, as compared to the knockout of the entire POLG gene function.55 POLG2, the Gene for the p55 Accessory Subunit of Pol γ Using a partial human POLG2 clone identified during the cloning of the Drosophila accessory subunit,56 the full-…”
BackgroundMethodInfluential
Nuclear Transfer: Preservation of a Nuclear Genome at the Expense of Its Associated mtDNA Genome(s)
Bowles, E. J., Campbell, K., & John, J. C. S. (2007) · Current topics in developmental biology/Current Topics in Developmental Biology · doi:10.1016/s0070-2153(06)77010-7 · Cited by 54
The accessory subunit of DNA polymerase γ is essential for mitochondrial DNA maintenance and development in Drosophila melanogaster
Iyengar, B. G., Luo, N., Farr, C. L., Kaguni, L. S., & Campos, A. R. (2002) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.072664899 · Cited by 53
Nucleotide incorporation by human DNA polymerase opposite benzo[a]pyrene and benzo[c]phenanthrene diol epoxide adducts of deoxyguanosine and deoxyadenosine
Graziewicz, M. A. (2004) · Nucleic Acids Research · doi:10.1093/nar/gkh213 · Cited by 52
“The recombinant human accessory subunit (p55) was puri®ed to homogeneity from Escherichia coli and the heterodimeric forms of the polymerase were reconstituted as previously described (28).”
Method
A replication-linked mutational gradient drives somatic mutation accumulation and influences germline polymorphisms and genome composition in mitochondrial DNA
Sánchez-Contreras, M., Sweetwyne, M. T., Kohrn, B. F., Tsantilas, K. A., Hipp, M. J., Schmidt, E. K., Fredrickson, J., Whitson, J. A., Campbell, M. D., Rabinovitch, P. S., et al. (2021) · Nucleic Acids Research · doi:10.1093/nar/gkab901 · Cited by 54
Disease Mutations in the Human Mitochondrial DNA Polymerase Thumb Subdomain Impart Severe Defects in Mitochondrial DNA Replication
Kasiviswanathan, R., Longley, M. J., Chan, S. S. L., & Copeland, W. C. (2009) · Journal of Biological Chemistry · doi:10.1074/jbc.m109.011940 · Cited by 55
Mutations in DNA polymerase gamma cause error prone DNA synthesis in human mitochondrial disorders.
Copeland, W. C., Ponamarev, M. V., Nguyen, D., Kunkel, T. A., & Longley, M. J. (2003) · Acta Biochimica Polonica · doi:10.18388/abp.2003_3723 · Cited by 51
“nents, where subsequent to actions of uracil-DNA glycosylase and AP endonuclease, pol can fill a single nucleotide gap in the presence of a 5 terminal deoxyribose phosphate (dRP) flap (Longley et al., 1998).”
BackgroundMethodInfluential
Binding parameters and thermodynamics of the interaction of the human cytomegalovirus DNA polymerase accessory protein, UL44, with DNA: implications for the processivity mechanism
Loregian, A., Sinigalia, E., Mercorelli, B., Palù, G., & Coen, D. M. (2007) · Nucleic Acids Research · doi:10.1093/nar/gkm506 · Cited by 50
“cognate catalytic subunit in the absence of DNA and stimulates its polymerizing activity on activated DNA templates (23, 43 ).”
Background
Aging-associated accumulation of mitochondrial DNA mutations in tumor origin
Kong, M., Guo, L., Xu, W., He, C., Jia, X., Zhao, Z., & Gu, Z. (2022) · Life Medicine · doi:10.1093/lifemedi/lnac014 · Cited by 51
mip1 containing mutations associated with mitochondrial disease causes mutagenesis and depletion of mtDNA in Saccharomyces cerevisiae
Stumpf, J. D., Bailey, C. M., Spell, D., Stillwagon, M., Anderson, K. S., & Copeland, W. C. (2010) · Human Molecular Genetics · doi:10.1093/hmg/ddq089 · Cited by 47
Biolayer Interferometry: A Novel Method to Elucidate Protein–Protein and Protein–DNA Interactions in the Mitochondrial DNA Replisome
Ciesielski, G. L., Hytönen, V. P., & Kaguni, L. S. (2015) · Methods in molecular biology · doi:10.1007/978-1-4939-3040-1_17 · Cited by 47
Clustering of Alpers disease mutations and catalytic defects in biochemical variants reveal new features of molecular mechanism of the human mitochondrial replicase, Pol γ
Euro, L., Farnum, G. A., Palin, E., Suomalainen, A., & Kaguni, L. S. (2011) · Nucleic Acids Research · doi:10.1093/nar/gkr618 · Cited by 48
“The accessory subunit, Pol gB, serves as a processivity factor, enhancing the DNA-binding affinity and catalytic activities of Pol gA (5,6).”
Human Mitochondrial DNA Polymerase γ Exhibits Potential for Bypass and Mutagenesis at UV-induced Cyclobutane Thymine Dimers
Kasiviswanathan, R., Gustafson, M. A., Copeland, W. C., & Meyer, J. N. (2011) · Journal of Biological Chemistry · doi:10.1074/jbc.m111.306852 · Cited by 45
“The accessory subunit is a 55-kDa protein (p55) required for tight DNA binding and processive DNA synthesis (24).”
Background
The interface of transcription and DNA replication in the mitochondria
Kasiviswanathan, R., Collins, T., & Copeland, W. C. (2011) · Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms · doi:10.1016/j.bbagrm.2011.12.005 · Cited by 45
“In addition, the processivity of the catalytic subunit is enhanced as much as 50-fold upon interaction with the accessory subunit [30].”
BackgroundInfluential
Increase in mitochondrial DNA mutations impairs retinal function and renders the retina vulnerable to injury
Kong, Y. X. G., Bergen, N. V., Trounce, I. A., Bui, B. V., Chrysostomou, V., Waugh, H. S., Vingrys, A. J., & Crowston, J. G. (2011) · Aging Cell · doi:10.1111/j.1474-9726.2011.00690.x · Cited by 43
“One enzyme central to the process of mtDNA replication is polymerase c (PolG; Kaguni, 2004; Graziewicz et al., 2006), which consists of a catalytic subunit with polymerase and exonuclease activity and a small accessory subunit that enhances binding (Lim et al., 1999).”
Background
R964C Mutation of DNA Polymerase γ Imparts Increased Stavudine Toxicity by Decreasing Nucleoside Analog Discrimination and Impairing Polymerase Activity
Bailey, C. M., Kasiviswanathan, R., Copeland, W. C., & Anderson, K. S. (2009) · Antimicrobial Agents and Chemotherapy · doi:10.1128/aac.01659-08 · Cited by 44
“As such, WT and R964C Pol catalytic subunits (exonuclease deficient) and the accessory subunit were expressed, purified, and reconstituted as described previously (10, 15 , 17).”
BackgroundMethod
POLB: A new role of DNA polymerase beta in mitochondrial base excision repair
Kaufman, B. A., & Houten, B. V. (2017) · DNA repair · doi:10.1016/j.dnarep.2017.11.002 · Cited by 43
Functional Roles of the N- and C-Terminal Regions of the Human Mitochondrial Single-Stranded DNA-Binding Protein
Oliveira, M. T., & Kaguni, L. S. (2010) · PLoS ONE · doi:10.1371/journal.pone.0015379 · Cited by 43
“pol γ activity is stimulated ∼2 fold as the concentration of KCl in the reaction increases from 20 to 100 mM, in agreement with previously published data”
Result
Mitochondrial DNA replication, nucleoside reverse-transcriptase inhibitors, and AIDS cardiomyopathy
Lewis, W. (2003) · Progress in Cardiovascular Diseases · doi:10.1053/pcad.2003.3b · Cited by 43

Authors: Lim, S. E., Longley, M. J., & Copeland, W. C.
Affiliations: National Institutes of Health (US)
Corresponding author: Copeland, William C.
ORCID: Longley, Matthew J.
Copeland, William C.
Venue: Journal of Biological Chemistry
Publisher: Elsevier BV
Published: 1999-12
Volume: 274
Issue: 53
Pages: 38197-38203
ISSN: 0021-9258 (ISSN-L)
DOI: 10.1074/jbc.274.53.38197
OpenAlex: W2001256046
Cited by: 226
References: 30
Citations by year: 2026: 3 · 2025: 3 · 2024: 5 · 2023: 6 · 2022: 3 · 2021: 9 · 2020: 10 · 2019: 3 · 2018: 4 · 2017: 9 · 2016: 6 · 2015: 7 · 2014: 5 · 2013: 7 · 2012: 13
FWCI: 2.97
Type: journal article
Language: en
Open access: hybrid
doi.org
Licence: cc-by
www.elsevier.com/tdm/userlicense/1.0/ (tdm)
creativecommons.org/licenses/by/4.0/ (vor, 7617 d)
Locations: Journal of Biological Chemistry (publishedVersion, cc-by)
PubMed (publishedVersion)
Funders: National Institute of Environmental Health Sciences
Topics: Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Mitochondrial Function and Pathology
Life Sciences › Biochemistry, Genetics and Molecular Biology › Structural Biology › Enzyme Structure and Function
Keywords: DNA polymerase gamma, heterodimer, DNA binding, processivity, exonuclease activity, salt tolerance, mitochondrial DNA repair, mitochondrial DNA replication, recombinant protein purification
MeSH terms: DNA Polymerase gamma, Base Sequence, DNA, DNA-Directed DNA Polymerase, DNA Replication, Escherichia coli, Ethylmaleimide, Humans, Mitochondria, Molecular Sequence Data, Protein Binding, Recombinant Proteins, Sodium Chloride, DNA Primers, DNA, Complementary, Dimerization, Catalytic Domain

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The Mitochondrial p55 Accessory Subunit of Human DNA Polymerase γ Enhances DNA Binding, Promotes Processive DNA Synthesis, and Confers N-Ethylmaleimide Resistance

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