Molecular signature pathway of gene protein interaction in human mitochondrial DNA (mtDNA) metabolism linked disease

Accepted Manuscript Title: Molecular signature pathway of gene protein interaction in human mitochondrial DNA (mtDNA) metabolism linked disease Authors: Manojit Bhattacharya, Debabrata Senapati, Avijit Kar, Ramesh Chandra Malick, Bidhan Chandra Patra, Basanta Kumar PII: DOI: Reference:

S0976-2884(18)30082-1 https://doi.org/10.1016/j.injms.2018.05.001 INJMS 183

To appear in: Received date: Revised date: Accepted date:

21-4-2018 30-4-2018 2-5-2018

Please cite this article as: Bhattacharya M, Senapati D, Kar A, Malick RC, Patra BC, Kumar B, Molecular signature pathway of gene protein interaction in human mitochondrial DNA (mtDNA) metabolism linked disease, Indian Journal of Medical Specialities (2010), https://doi.org/10.1016/j.injms.2018.05.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Molecular signature pathway of gene protein interaction in human mitochondrial DNA (mtDNA) metabolism linked disease

Manojit Bhattacharya1, ∞Debabrata Senapati2, Avijit Kar2, Ramesh Chandra Malick1, Bidhan Chandra Patra2, Basanta Kumar Das1*

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ICAR-Central Inland Fisheries Research Institute, Barrackpore, Kolkata- 700 120, West Bengal, India. 2

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Centre For Aquaculture Research, Extension & Livelihood, Department of Aquaculture Management & Technology, Vidyasagar University, Midnapore- 721102, West Bengal, India.

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Authors contributed works equals.

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* Corresponding Authors:

Abstract

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Dr. Basanta Kumar Das, ICAR-Central Inland Fisheries Research Institute, Barrackpore, Kolkata- 700 120, West Bengal, India. Mail ID: [email protected]

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Mitochondrial DNA contains approximately 37 numbers genes which encoded functional proteins that are essential for cellular energy production and regulation of biochemical

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process within human. Therefore, any kind of mtDNA abnormalities can initiate an energy calamity and ensuing in crucial mitochondrial diseases. It is also depends on nuclear genes because its protein products properly help in mtDNA replication and maintenances of dNTP pools within cytosol and mitochondrial matrix part. Mitochondrial DNA (mtDNA) dNTP synthesis comes from two board categories: primarily mitochondrial salvage and cytosolic de

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novo pathways with the direct help towards some kind of factors and a variety of enzymes. However, defective mtDNA maintenance can lead to severe disease in human which may have poor curative therapeutic accessible approaches. Existing research supports that a vast number of drugs and inhibitor components have plays significant roles in regulating the mtDNA as well as dNTP pool alteration. The review will primarily focus on the protein

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regulatory pathways of mitochondrial deoxyribonucleotide (mtDNA) metabolism i.e. mtDNA synthesis, maintenances and its degradation with the connection of several mtDNA

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metabolism linked diseases.

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Keywords: mtDNA metabolism. de novo and salvage pathway. Mitochondrial disease. dNTP

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pool.

Molecular signature pathway of gene protein interaction in human mitochondrial DNA

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(mtDNA) metabolism linked disease

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1. Introduction

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The living cells are made up of different types of cell organelles but mitochondria are one of the most crucial parts of a cell. Mitochondria play many pivotal biological processes that are more essential for maintaining and regulating the mechanism of normal cellular physiological

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process [1]. As known, cells cannot survive without required energy and mitochondria do this main function as it considered the main power house of a cell [2]. Research findings also revealed that in aerobic cells, mitochondrial inner membrane actively takes a part to produce ATP component through a complex series of Electron Transport Chain (ETC) mechanism; where the coupled protein complex and proton carrier machineries generated ATP for the 2

maintenances of many biological functions of a cell. So, any mitochondrial components dysfunction is relating to the severe energy deficiencies within the cell [3]. It is the prime fact that mitochondria are the only cells organelle of human that contains its own DNA component (i.e.-mtDNA). In addition to humans nuclear genome (i.e.-haploid

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condition) is 200000 times length of its mitochondrial genome which builds with approximately total 20000 genes. Apart from this, in human mtDNA be 16,569 bp length

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circular doubled stranded molecules which can boast up only 37 genes that give up to 13 functional protein products for ETC complexes (i.e.-complex I, III, IV and V types), 22 peptides for tRNAs and rest 2 proteins are encoding rRNAs [4]. The entire component

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relating to mtDNA maintenances i.e. - replication and expression followed by transcription

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and further translation of its mt-mRNAs, which are also encoded by nuclear and require

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being directed to mitochondria after completion their protein translation mechanism.

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Therefore, the maintenance of mitochondrial DNA as well as nuclear genes is an important

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key factor for governing the normal physiological processes of human being [5]. Mitochondrial DNA is not replicated independently, it must reliant on nucleus for

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maintenance and replication performance, so any defect in nuclear DNA and its by-products like proteins, enzymes can also produce error in mtDNA or simply mtDNA loss. However,

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in recent years, several important factors are identified which directly and indirectly involved mitochondrial DNA maintenance or in the homeostasis of the nucleotide precursor pool [6].

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In order that, here certain approach attempts to mtDNA metabolism by which mitochondrial Deoxyribonucleic acid (DNA) is maintained that are includes both as DNA synthesis and degradation pathway followed in mtDNA replication and repair mechanism, finally its regulation method as it key control of so many mtDNA linked diseases.

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2. Sources of dNTPs for mtDNA maintenance The mitochondria must have required a fruitful constant emergence of dNTPs for allowing its own DNA maintenances, continuous replication and finally its repair system. However, nuclear DNA replication occurs only in S phase of cell cycle but in mtDNA replication is not

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strictly dependant on regular cell cycle mechanism [7]. In fact, the mtDNA replication is continuous going on in every cell cycle events and more things are that it persists even in

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non-dividing cells [8]. So, a constant supply of dNTPs is so much required for mtDNA

replication and its maintenance [9]. In mammalian cells, it is known to be believed that the primary precursors of dNTPs within mitochondrion are two different metabolic sources:

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cytosolic de novo synthesis and salvage pathway (Figure 1).

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2.1 de novo synthesis

The origin of de novo synthesis is fully depends on the two cytosolic enzymes namely

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ribonucleotide reductase (RNR) and thymidylate synthase (TS), although RNR and TS both

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enzymatic pathway are also takes place for nuclear DNA metabolism, but current report has address that this pathway also identified within mitochondria [10]. Whereas, RNA catalyzes

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all the four ribonucleoside diphosphate (rNDPs) to its nearest to formed deoxyribonucleoside diphosphates (dNDPs) by only due to the reduction method. Immunogold electron microscopy, autoradiography, and immunohistochemistry methods shows that de novo dTMP

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synthesis pathways corresponding to involving essential enzymes TS (Thymidylate synthetase), serine hydroxymethyltransferase (SHMT), and dihydrofolatereductase (DHFR) has been attendant in mitochondria of rat cells [11]. However RNR activity has been noted many times in mitochondria isolated from mouse, pig, rat liver and Hela cells [12, 13]. On the contrary, additional literature shows that there is no RNR or TS activity existence in 4

mitochondria collected from mouse and rat tissues [14, 15]. Since, if there is evident the presence of de novo dNTP synthesis pathway in mitochondria, a Thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK) activity deficiencies should not lead to mtDNA alteration. Therefore, it prime need to more convincing evidences to prove the existence of

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mitochondrial de novo synthesis pathway. The RNR enzyme is a heterotetramer component having two copies of heavy subunit (R1)

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and two copies of light subunit denoting as the R2 or the p53R2 isoform. R2 expressed exponentially in dividing cells and it will be degraded in late mitosis by proteasome

components. By contrast, p53R2 is functionally found in every stages of cell cycle. When

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mitochondria replication is initiated and the cells demands DNA precursor are at highest

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level, the de novo synthesis fulfill the requirement couple the process of nuclear replication.

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After that, the dNTPs pool is reasonably decreases once the replication process is over. In

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addition, the R2 and p53R2 subunits of RNR enzymes contribute a small but considerable

2.2 Salvage pathway

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amount dNTPs pool for mtDNA repair and replication specific to the non dividing cells.

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Mitochondria must import dNTPs from cytosol to matrix, which is fully dependent on dNTPs

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availability in cytosol might be predominantly synthesized by de novo via RNR otherwise it supplies via its own salvage pathway. The mitochondrial deoxynucleosides either phosphorylated or dephosphorylated by a distinct biochemical manner for maintaining the

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mitochondrial DNA poll balance and consequently it became triphosphorylated form for incorporation into mtDNA. In mitochondria, the first phosphorylation of deoxyribonucleoside step takes place by two irreversible enzymes, one is thymidine kinase 2 (TK2) for pyrimidine (dThd, dCyt and dUrd) and another is deoxyguanosine kinase (dGK) for purine (dGuo, dAdo, and deoxyinosine) [16]. Both TK2 and dGK is the rate limiting enzyme and it constitutive to

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express which depend on cell cycle [16-18]. On the other hand, one of the key enzymes mitochondrial deoxynucleotidase (mdN) remove the phosphate group from all the deoxynucleotide subunit and also an accessory subunit that bind DNA which ultimate increase the processivity of the other functional enzyme [19] [20] [21]. Then consequently it phosphorylated as a recycling manner by nucleotide monophosphate kinases (NMPK), and

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nucleotide diphosphate kinase (NDPK). The adenylate kinase (AK) 3 and 4 isoform belongs to NMPK group, but others members are not to be discovered where as MDPK is a

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multifunctional enzyme [22] and existing finding suggested that it play role like protein histidine kinase [23].

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The deoxynucleotide mono phosphate (DNMP) such as dTMP, dCMP, dAMP, and dGMP

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however undergo dephosphorylated to their corresponding nucleosides by 5" nucleotides. In

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contrast to, two 5" nucleotides prefer deoxyribonucleotides as a substrate via

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deoxyribonucleotidase (dNT). dNT belongs to two isoform, dNT-1 is located in the cytosol which have pronounced specificity for TK1 and dCK in cytosol where as dNT-2 is found in

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mitochondria, have counteracts to thymidine monophosphate and deoxyuridine

metabolism.

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monophosphate. So, it can easily say that dNT may play major role in regulating mtDNA

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3. Transport of mtDNA precursor Mitochondria have double membrane layered structure forming two separate spaces i.e.

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intermembrane space (IMS) stand between the outer and inner membrane, matrix within the inner membrane part. Mitochondrial matrix element play s significant role for a numbers of biochemical processes as it is composed the mtDNA molecule. Here, focus also consider basically to the inner membrane because it is not permeable to all nucleotides and nucleosides but intermembrane space are to be rested in equilibrium state with the cell cytoplasm [24].

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Therefore, the mitochondrial inner membrane must have presented a transporter which transport solutes for mtDNA metabolism. After synthesis dNTPs in cytosol it can be transported within mitochondria by several form (Figure 1); a) deoxyribonucleosides are transported into mitochondria through passive or facilitate diffusion and then it phosphorylated or b) directly deoxyribonucleotides undergo inward to mitochondria from the

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cytosol. On other side ribonucleotides, the precursor of DNA are move from cytosol or the

remaining ribonucleotides of mitochondrial matrix are phosphorylated to form dNTPs [25].

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Existing research support that mitochondria used all the three pathways, however the

pathways varies depends on different types of cells. In mammalian the known DNA carrier

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protein is DCN (i.e. - deoxyribonucleotide carrier) and equilibrative nucleoside transporter

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(ENT) [26]. The protein DNC is carry all the variety of dNTPs which is probable best

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substrates following by ribonucleotide diphosphates (rNDPs). For instance, the exact role of

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DNC are not clear however the experiments where mutation to DNC in normal cells lines, the mitochondrial dNTPs pool are standard but mitochondria of these cell lines are deficient of

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thyamine pyrophosphate. So, it is undoubtedly concluded that, the DNC primarily transport the vitamins and or phosphorylated form. Palmieri and his colleagues experiment show that

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most of the dNTPs comes into mitochondria from cytosol where it originated by direct transport in yeast and they have found a guanine nucleotide transporter with pyrimidine di-

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and tri-phosphates counter current exchanger. In humans, Human equilibrative nucleoside transporter 1 (hENT1) has been the known mitochondrial deoxynucleosides (dN) transporter

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[27], devoid or mutation of it has several dysfunction appear likewise the relapsing/refractory Hodgkin's disease (HD). Mitochondria not only used dNTPs from de novo synthesis pathways but some instances it attains dNTPs from salvage pathways. It is found that in human two out of four deoxyribonucleoside kinase (thymidine kinase 2 and deoxyguanosine kinase) are positioned 7

within mitochondria. These enzymes serves all the four canonical deoxyribonucleotides because of its have broad ribonucleotides substrate specificity. 4. Maintenance in replication The maturation and elongation of the daughter strand mtDNA is accomplished by DNA

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polymerase gamma (POLG) complex which composed by 140 kDa catalytic subunit and two 55 kDa accessory subunit of DNA polymerase POLG and POLG2 gene. Its larger catalytic

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subunit has both 5′ to 3′ polymerization ability and a 3′ to 5′ exonuclease activity, and both

functions are indispensable for mtDNA safeguarding [28]. The smaller accessory (β) subunit increases the affinity of POLG for DNA strand, confers processivity to the catalytic subunit,

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after binds double-stranded DNA, which is involved in the primer recognition other than

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POLG complex. The mtDNA replisome machinery is formed by TWINKLE (TWNK) protein

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and mitochondrial single stranded binding protein (mtSSB), TWINKLE act like helicase,

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which unwinds short stretches of dsDNA in the 5′ to 3′ direction to provide single-stranded

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templates for DNA polymerase and is also required for mtDNA maintenance [29, 30]. The mitochondrial SSB protein (mtSSB) specifically stimulated the rate of mtDNA synthesis by

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maintains the integrity of replicative intermediates in which large single-stranded DNA

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regions are present, preventing them from renaturation [31]. Although it has not yet identified that there must be additional factors which primary function to influences the modeling of mtDNA and its segregation, import of replication proteins or

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the expression level. In case of yeast, it have been identified 36 complementation groups of yeast mutants which exhibiting temperature-induced loss of mtDNA [32]. Furthermore, the existing research suggest some genetic backgrounds lead to a complete loss of mtDNA, although its relevant genes are yet not (directly or indirectly) required for mtDNA replication [33].

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5. Defective mtDNA metabolism leads disease A large numbers of proteins and enzymes (nuclear or mitochondrial DNA encoded) are requiring for replication and maintenance of mtDNA. Whereas a constant need of nucleotides is major importance mitochondrial nucleotide synthesis even in quiescent cells. Although

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defective mtDNA maintenance and synthesis primarily evident by multiple deletions or by depletion of the mitochondrial genome which leads a number of different defective (Table 1)

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and a significant common disease namely mitochondrial DNA depletion syndromes (MDDS). 5.1 mtDNA deletion

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Linkage analysis and recently next generation sequencing technology revolutionizing our

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ability to characterizing in several families abnormalities like progressive external

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ophthalmoplegia (PEO), exercise intolerance, ptosis, and multiple mtDNA deletions

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transmitted in either autosomal dominant (ad) or recessive manner. Autosomal dominant disorder progressive external ophthalmoplegia (adPEO) is caused by missense mutation in

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the polymerase domain of POLG, whereas similar disease also take in part in recessive manner (missense mutation) was caused by nonenzymatic coding regions of the POLG [34].

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Furthermore, mutation of the twinkle gene of t7 bacteriophage which inherited by one allele,

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and missense mutation encoding of ANT1 gene in heart and skeletal muscle were caused multiple mtDNA deletion and also adPEO disease [30, 35].

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5.2 Mitochondrial dNTP pool Existing research showed that the mtDNA depletion syndromes are very likely dependent on mitochondrial nucleotide pools homeostasis. Here, two mitochondrial enzymes which involved deoxynucleoside salvage pathways encoded by nuclear genes are played very critical role. One tk2, that phosphorylated dThd and deoxycytidine, therefore, deficiency of

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TK2 protein is lead to reductions in deoxythymidine monophosphate (dTMP) and deoxycytidine monophosphate (dCMP) which subsequently dTTP and dCTP can form myopathic type of mtDNA depletion syndrome [36]. In addition to, another is decrease phosphorylation of deoxyadenosine and deoxyguanosine by dGK mutations can give raise a

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hepatocerebral form as followed low concentration of dATP and dGTP in mitochondria [37]. Furthermore, this asymmetric distribution of the dNTP pools is important for replication

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fidelity and failure to maintain appropriate dNTP concentrations can be detrimental, leading to DNA strand breaks, mutagenesis, and cell death [38, 39]. France scientist Jérôme Poli showed that a slow-replication mode followed 10-fold reduction in fork speed and 25-fold

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decrease in initiation rate [40]. However, Inonu University, School of Medicine scientist

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Ahmet Koc proved that Blocking dNTP synthesis by hydroxyurea stops DNA replication and

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causes cell cycle arrest [41].

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5.3 Targeting drugs

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Evidence suggests that in most cases a wide range of structurally diverse drugs and inhibitors selectively inhibit the mtDNA metabolism pathways (Table 1). DNA polymerase gamma is

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involved in the synthesis of mtDNA, which are inhibited by some of the Antiviral Nucleoside

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Analogue (ANA) drugs used to treat Human Immunodeficiency Virus (HIV) and Hepatitis B Virus (HBV) infections [42-44]. Some of these ANA drugs are Zidovudine (39-azido-39deoxythymidine), S tavudine (29, 39-didehydro-29, 39-dideoxythymidine), Zalcitabine (29,

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39-dideoxycytidine), Didanosine (29, 39-dideoxyinosine) which inhibit DNA polymerase gamma through two mechanism either direct inhibit or misincorporating into mtDNA becoming termination of DNA strand elongation. Ethidium bromide (EB) a intercalating drug was first identified in the 1950s in in vitro mammalian cells experiments that inhibit mtDNA

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metabolism where in vivo effect of mtDNA depletion are till currently unknown [45]. Another intercalating anticancer drug ditercalinium (DT) also perform same function [46] Non-intercalating DNA binding drugs such as methylglyoxalbis [guanylhydrazone] (MGBG), diethylspermine (DES), N1,N6-Bis(ethyl) spermidine (BESPD), and N, N9 Bis-

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[3(ethylamino)-propyl]1-7-heptane diamine (BEPH) reduce the copy number of mtDNA by interacting with negatively charged phosphate backbone through promoting conformational

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changes in DNA [47, 48]. Furthermore, dequalinium (DEQ) and MKT-077 a lipophilic cationic drugs induce depletion of mtDNA [49, 50].

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6. Damages and repairs

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Like any others genomes, mitochondrial DNA is continuously worked to its integrity from

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exogenous and endogenous sources. Earlier worker shown that, the repair mechanism of

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mtDNA having less information rather the nuclear DNA over a long time. As mtDNA comprises 1-3% of genetic material and its intimate relation to ETC (Electron Transfer

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Chain), so it persist higher mutation rates than nuclear DNA which might be accelerates by its own ROS (Reactive oxygen species) production. Whereas mtDNA posses all kind of DNA

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damage much like as nuclear DNA [51] and it is believed that it may contain numbers of

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common proteins required for DNA repair mechanism [52]. But, still scientists are failed to unmask their respective contribution in specific mtDNA repair process. Some evidences showed that, mitochondrial protein doesn't perform to recover the UV induce mtDNA

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damage [53]. Researcher also proved that after exposure to N-methy-N-nitro-Nnitrosoguanidine or 4-nitroquinoline- 1 oxide components mitochondrial DNA repair was very slower rate or might be absent [54]. These consequences discovered that damaged mtDNA must be destroyed and replaced with newly replicated undamaged DNA. Although, recent technical advances for the analysis of repair in particular sequences and treatment of

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the whole mtDNA sequence is possible to better understand support to some sorts of mtDNA damage repaired mechanism [55-58]. Presently, it is clearly understood that mitochondria possess DNA BER (base excision repair) mechanism, but it lacking the significant tools like Nucleotide Excision Repair (NER) facility [59, 60].

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7. Degradation It is found that the Endonuclease G (EndoG) is a vital enzyme but not only nuclease liable for

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selectable degradation of mtDNA [61]. It is believed that mtDNA must be proceed to

degradation by the initial studies show induce by UV-radiation to pyrimidine dimer in mammalian mitochondria [53], later experiment treatment with mutagenic agents such as N-

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methyl-N,-nitrosoguanidine, ethylmethanesulfonate, and benzo(a)pyrene from HeLa cells

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show excessive amounts of damage in mtDNA that are not replicated [62] and most recently

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investigation revealed that extensive DNA double-strand break (DSBs) that lead mtDNA

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degradation. However, Stalled DNA or RNA polymerases would generate a signal that

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triggers the mtDNA degradation process on damage DNA [63]. Furthermore, recent studies have suggested that mtDNA degraded fast and slow mode by oxidative stress. However, the

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inability of mtDNA damage repair have been proved by inhibition of abasic site processing by APE1 and treatment with methoxyamine that inhibit BER, influence degradation signal in

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response to oxidative damages [63]. Moreover, supportive evidences confirmed that, the function of Cyclooxygenase type II, ATPase subunit 6 and 8 enzymes leads to the

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spermatozoan activity within the human sperm mitochondrial part. Misbalancing or inhibition of such gene coded enzymes could show male infertility by blocking the Oxidative phosphorylation (OXPHOS) pathway for ATP synthesis during energy production [110,111]. 8. Future perspectives

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Alters in mtDNA metabolism can form severe disorder with no or limited prognosis in the majority of affected individuals, however there is no curative therapeutic approach is offered for any of the mention disease. Although mitochondrial DNA depletion syndromes (MDDS) are express heterogeneously with respect to different organs, so, current research finding should have a comprehensive evaluation to appraise the extent of association of different

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tissues systems [64]. Execution of alter mtDNA metabolism linked disease should engage a multidisciplinary group, which may together with different specialists and intend to offer

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supportive care along with the several symptomatic management. In spite of other therapeutic treatment opportunity for mtDNA metabolism comprise a special kind of dietary modulation [65], stem cell transplantation [66-68], cofactor supplementation [69, 70] may can lead the

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slow disease progression and improving the patient’s health.

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As conversed in this article, mtDNA aberrations and depletion is mainly caused by defective

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dNTP metabolism which leads to an imbalanced dNTP pool. So, possibly the major concern for a therapeutic approach on supply to increasing of deoxyribonucleoside accessibility is the

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risk of again imbalance of the mitochondrial as well as cytosolic dNTP pool composition, because it may result in mutagenesis of both the mitochondrial genomes and nuclear genomes

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[71]. So, the further supportive research is requisite to maintain the dNTP pool through

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pharmacological managing and evade the mutagenic side effects. In this present review, on the basic of result compiled it definitely conclude that several

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proteins and enzymes are tightly regulated wider range of mtDNA metabolism and makes error or inhibition of these regulator can influences a number of organ specific diseases. Furthermore, chromosomal locus of described regulatory proteins or enzymes is already identified (Table 1) which may recruit positional effect of gene expression that better explain tissue specific mitochondrial diseases in near future. To maintain and regulating the normal mtDNA metabolism and /or better treatment mtDNA metabolic linked disease, focus to 13

future research potentially mandatory to more in vitro studies and for the most part in vivo preclinical studies are obligatory. A number of available and in vitro generated animal models will be significant for evaluate the disease–phenotype reversion and yet to the

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secondary sound effects consequent from the disease linked treatment.

9. Conclusion

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In medical science last few years the role of nuclear genes and their error protein expression that function in the maintenances of the mtDNA metabolism has gained increased more

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appreciation. Several disease related to the mtDNA metabolism are mostly involved for three

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component these are core protein involved in mtDNA replication, or within genes involved

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that supply the nucleotide precursors which needed for replication otherwise the inhibitor

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components of protein or enzymes that involved in mtDNA metabolism (Table 1).With recent awareness of mitochondrial patients due to inhibition or mutation of said components

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will helps to survive for better and cure easily as it is inherited homozygous or heterozygous condition. However, the varied polymorphic nature as well as the age of expression of these

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disease stumps our understanding and facing critical challenges to clinician and researchers.

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While in vitro yeast model and biochemical experiment have proven effective in understanding the pathological and biochemical defects, but animal model are very essential tool for better predicting the in vivo consequences of these disease. Hope present scenario of

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this research arena must be eliminating in this fatal area. May the mtDNA metabolism protein regulatory pathway will perform a new vista in molecular medical science for act to be a promising therapeutic agent.

Disclosure statement

The authors declare that they have no competing interests. 14

Acknowledgements This research was supported by the research grant from the University Grant Commission (UGC), Govt. of India and Science and Engineering Research Board, Department of Science and Technology, Govt. of India for

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financial assistance to carry out the research work (Project file No Ref. No.PDF/2016/001776).

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Figure caption

PT

ED

M

A

N

U

SC R

IP T

Fig 1: Gene-protein interaction pathway for mtDNA metabolism related diseases.

Synthesis and transport pathway of the deoxyribonucleotides for the mitochondrial DNA (mtDNA) metabolism.

CC E

The mitochondrial inner membrane is impermeable to charged molecules and the mitochondrial dNTP pool is maintained by either import of cytosolic deoxyribonucleotide diphosphates (dNDP) via a dedicated transporter

A

(DNC/ENT1/ANT1)) or by salvage of deoxyribonucleosides within the mitochondria.

30

ABBREV IATION

SYMB OL

Mitochondrial DNA polymerase catalytic subunit

polg1

POLG

CHROMO SOMAL LOCUS 15q25

DNA polymerase subunit gamma-2

MtDNA polymerase gamma 1 accessory subunit

Polg2

POLG2

17q23-24

Mitochondrial genome maintenance exonuclease 1 Twinkle

Maintenance mtDNA

Mgme1

MGME 1

20p11.23

Mitochondrial DNA ATPdependent helicase Pyrimidine salvage to recover nucleosides after DNA/RNA degradation. Formation of deoxyribonucl eotides from ribonucleotide s. Converting deoxynucleosi de triphosphates (dNTPs) to inorganic phosphate (iPPP) and a 2'deoxynucleosi de Phosphorylate d to thymidine

twinkle

TWNK

10q24.31

TYMP

22q13.33

Tp1

rnr

RRM1

11p15.4

Hydroxyurea and Gemcitabine

Samhd1

SAMH D1

Tk1

TK1

A

SAM domain and HD domaincontaining protein 1

Thymidine kinase 1

ASSOCIATED DISEASES

REFERE NCE

Dideoxynucl eotides and Nethylmaleimi desensitive[NE M], antiviral nucleoside analogue (ANA) drugs

Alpers' disease, ataxianeuropathy disorders, and dominant and recessive forms of progressive external ophthalmoplegi a. Progressive external ophthalmoplegi a with multiple mtDNA deletions and cytochrome c oxidase (COX)deficient muscle fibers Mitochondrial DNA depletion syndrome 11(MTDPS11) Progressive external ophthalmoplegi a Mitochondrial neurogastrointes tinalencephalom yopathy (MNGIE)

[21, 72]

Inhibit cellular division and also inhibits growth

[79, 80]

20q11.23

AicardiGoutieres syndrome

[81]

17q25.3

Mutagenesis

[82]

U

N

A

ED

CC E

Ribonucleotid e reductase

tp

PT

Thymidine phosphorylase

INHIBITOR

IP T

FUNCTION

[73, 74]

SC R

PROTEIN OR ENZYME NAME DNA polymerase subunit gamma-1

M

Table 1. Gene- proteins relating to mtDNA metabolism linked diseased, along their inhibitor components.

[75]

[76, 77]

[78]

31

dCTD

DCTD

4q35.1

dTTP

Mutagenesis

[83]

TS

TYMS

18p11.32

5fluorouracil (5-FU)

Thymineless Death

[84, 85]

cdN

NT5C

17p11.2

ATP

Smith–Magenis syndrome

dCK

DCK

4q13.3

Important role in purine metabolism and in adenosine homeostasis atalyzes the irreversible hydrolytic deamination of cytidine and deoxycytidine to uridine and deoxyuridine Metabolizes inosine into hypoxanthine and guanosine into guanine

ADA

ADA

20q13.12

Thymidine kinase 2

Deoxyguanosi ne kinase

SC R

Chronic Lymphocytic Leukemia

[87-89]

Severe combined immunodeficien cy (SCID)

[90]

CDA

CDA

1p36.12

Pentostatin

Tumorigenic

[91, 92]

PNP

PNP

14q11.2

Severe combined immunodeficien cy (SCID)

[93, 94]

Enzyme of mitochondrial dNTP salvage pathway

tk2

TK2

16q21

Infantile fatal encephalomyop athy

[95, 96]

hosphorylatio n of purine deoxyribonucl eosides phosphates of uracil and thymine

dGK

DGUO K

2p13.1

Forodesine,8 -mercaptoacyclovir, 8bromo-9(3,4hydroxybuty l)guanine, 3´hexanoylami no-3´deoxythymid ine dGTP

Hepatic disease and neurologic manifestations

[97, 98]

mdN

NT5M

17p11.2

(S)-1-[2′deoxy-3′,5′O-(1-

Myoneurogastro intestinalenceph

[99]

A

CC E

Purine nucleoside phosphorylase

PT

ED

Cytidine deaminase

[20, 86]

18F-L-1-(2′deoxy-2′FluoroArabi nofuranosyl) Cytosine (18F-LFAC) Sulfhydryl reagents

U

Adenosine deaminase

N

Deoxycytidine kinase

A

Cytosolic deoxyribonucl eotidase

M

Thymidylate synthetase

IP T

Supplies the nucleotide substrate for thymidylate synthetase. Conversion of deoxyuridine monophospha te (dUMP) to deoxythymidi ne monophospha te (dTMP) Phosphates of deoxyribonucl eotides, with a preference for dUMP and dTMP Phosphorylate s deoxycytidine

Deoxycytidyla te deaminase

Mitochondrial deoxyribonucl eotidase

32

deoxyribonucl eotides

Equilibrative nucleoside transporter (ENT)

-

-

NDPK

NME2

17q21.33

AMPactivated protein kinase (AMPK)

Cardiovascular disease

RRM2

RRM2

2p24-p25

OXPHOS pathway for ATP synthesis

SC R

IP T

-

Hydroxyurea

[101103]

[104, 105]

U

-

17q25.1

N

dcn

SLC29 A1

10q22.1

Dipyridamol e and dilazep

COX-2, ATPase6

COX-2, ATPase 6

1q31.1

Rofecoxib, Atpif1

MDDS, congenital microcephaly

[26, 107]

H syndrome, pigmented hypertrichosis with insulindependent diabetes, and Faisalabad histiocytosis Deficiency in spermatocytes respiration, male infertility

[108, 109]

[110, 111]

A

CC E

Cyclooxygena se type II, ATPase subunit 6 and 8

-

A

Deoxyribonucl eotide carrier (DCN )

[100]

M

Ribonucleosid e diphosphate reductase

alomyopathy syndrome

NMPK

ED

Nucleotide diphosphate kinase

catalyzes the reaction of ATP and NMP to yield ADP and nucleoside diphosphate (NDP) Major role in the synthesis of nucleoside triphosphates other than ATP catalyzes the formation of deoxyribonucl eotides from ribonucleotide s Transport dNTPs from cytosol into mitochondria Mitochondrial deoxynucleosi de (dN) transporter

PT

Nucleoside mono phosphate kinase

phosphono) benzylideneβ-d-threopentofuranos yl]thymine (DPB-T

33