©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A DNA Polymerase in the Mitochondrion of the Trypanosomatid Crithidia fasciculata(*)

(Received for publication, December 5, 1994)

Al F. Torri Paul T. Englund

From the Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We previously purified a Crithidia fasciculata mitochondrial DNA polymerase that has unusual properties. Unlike a conventional mitochondrial DNA polymerase , this enzyme is small, non-processive, deficient in 3`-exonuclease activity, and error prone (Torri, A. F., Kunkel, T. A., and Englund, P. T.(1994) J. Biol. Chem. 269, 8165-8171). In all of these characteristics, the enzyme resembles DNA polymerase beta, a nuclear enzyme thought to be involved in DNA repair. We have now cloned and sequenced the gene for this enzyme. The mitochondrial polymerase has significant homology, about 33% identity at the amino acid level, with human DNA polymerase beta. However, sequence analysis of the clone revealed the presence of a cleaved N-terminal presequence, presumably a mitochondrial import signal, which resembles presequences on other C. fasciculata mitochondrial proteins. The polymerase's function may be to repair the many gaps in newly replicated kinetoplast (mitochondrial) DNA minicircles in this parasite. This enzyme is the first example of a mitochondrial DNA polymerase beta.


INTRODUCTION

Crithidia fasciculata, a protozoan parasite related to the trypanosomes, has an unusual mitochondrial DNA known as kinetoplast DNA (kDNA). (^1)kDNA consists of 5000 minicircles and 25 maxicircles, all of which are topologically interlocked into one giant network. This kDNA network is condensed into a disc-like structure in the mitochondrial matrix. As part of our studies of kDNA replication, we previously purified a mitochondrial DNA polymerase from this parasite(1) . This enzyme differs from a conventional mitochondrial polymerase (DNA polymerase ), which typically is large in size, is highly processive, has a 3`-exonuclease activity, and has high fidelity (reviewed in (2) ). The C. fasciculata mitochondrial enzyme is small (43 kDa), poorly processive, deficient in exonuclease activity, and low in fidelity(3) . These properties are characteristic of a DNA polymerase beta (pol beta), a nuclear enzyme that efficiently fills small gaps in duplex DNA and is believed to function in DNA repair (reviewed in (2) ). Despite its similarity to a nuclear enzyme, the C. fasciculata polymerase is clearly mitochondrial. It was purified from a mitochondrial fraction, and by immunofluorescence it localized to two complexes of replication proteins situated on opposite sides of the kinetoplast disk(4) . These complexes, which are thought to be the site of minicircle replication, also contain topoisomerase II (5) and minicircles that are probably replication intermediates(4) .

To further characterize this mitochondrial polymerase, we cloned and sequenced its gene, allowing us to determine its relationship to other polymerases. Pol beta's sequence is highly conserved among vertebrates, although the yeast pol beta has lower homology, about 26% identity at the amino acid level. A comparison of the known pol betas with all other DNA polymerases, either prokaryotic or eukaryotic, indicates that they are related to only one other nucleotide polymerizing enzyme, terminal deoxynucleotidyltransferase. Pol beta and terminal deoxynucleotidyltransferase are so distinct from the other polymerases that they are the sole members of their own class of enzymes, the family X DNA polymerases(6, 7) . However, recent crystallographic analysis of rat pol beta revealed a DNA binding channel with some similarities to those found in other DNA polymerases(8, 9, 10) . The crystal structure suggests that pol beta utilizes a nucleotidyl transfer mechanism similar to that of other polymerases and implicates three invariant aspartic acid residues as essential for catalytic activity.

In sequencing the gene encoding the C. fasciculata mitochondrial polymerase, we found 33% identity, at the amino acid level, to human pol beta. In addition, the C. fasciculata enzyme has a cleaved N-terminal presequence that is similar to import signals on other C. fasciculata mitochondrial proteins. This enzyme is the first example of a mitochondrial pol beta identified in any organism.


EXPERIMENTAL PROCEDURES

Protein Sequencing

The C. fasciculata mitochondrial polymerase (Fraction 10) was purified as described previously(1) . Protein sequencing was performed by the Wistar Protein Microchemistry Laboratory (Philadelphia, PA). The homogeneous enzyme (0.5 nmol) was fractionated by SDS-polyacrylamide gel electrophoresis (12% resolving gel) and then electroblotted onto polyvinylidene difluoride membranes (Bio-Rad). The membrane was stained with 0.1% Amido Black in 10% acetic acid for 1 min, destained in 5% acetic acid for 1 min, and then washed in water for 15 min. The protein band was cut from the membrane and sent for analysis. The N-terminal sequence was obtained from intact protein. For internal sequences, the protein was digested with trypsin and the eluted tryptic fragments were purified by reverse phase high pressure liquid chromatography. Selected individual tryptic fragments were then sequenced.

Preparation of DNA

C. fasciculata cells (11) were sedimented at 5000 times g for 10 min at room temperature and resuspended (10^9 cells/ml) in lysis buffer (0.1 M NaCl, 0.1 M Tris, pH 8.0, 0.1 M EDTA, 1% SDS). The lysate was incubated at 37 °C for 1 h with RNase A (0.2 mg/ml) and then incubated at 37 °C for 1 h with Proteinase K (0.5 mg/ml). The sample was extracted (with equal volumes of phenol and phenol/CHCl(3) (1:1 ratio) and CHCl(3)), and the DNA was precipitated with isopropyl alcohol and then resuspended in 10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA at a concentration of 0.1 mg/ml.

Isolation of a Gene Fragment by PCR

Degenerate oligonucleotides (26-mers) were based on the sequence of two of the enzyme's peptides. Each oligonucleotide was constructed with an EcoRI restriction site at its 5` end. The upstream oligonucleotide, derived from the N-terminal protein sequence (residues 19-24 in Fig. 1), was GGGAATTCTTYCARGARATGGCNGAY. The downstream oligonucleotide, based on one of the internal peptide sequences (residues 219-224 in Fig. 1), was GGGAATTCRTGYTCNCRTARTTNCC. PCR was performed using Perkin-Elmer reagents in a 100-µl volume overlaid with 100 µl of PCR oil. The reaction contained 250 pmol of each primer, 0.2 mM of each dNTP, 0.2 µg of C. fasciculata DNA, 1 times PCR Buffer II, 2.5 mM MgCl(2), and 0.5 unit of AmpliTaq DNA polymerase. The reaction was heated at 94 °C for 5 min and then subjected to 50 cycles of amplification (1.5 min at 94 °C; 2 min at 55 °C; 4 min at 72 °C) using a DNA thermal cycler (Perkin-Elmer). The PCR product was digested with EcoRI and ligated into the EcoRI site of Bluescript SK+ (Stratagene). Transformations were performed in DH5alpha cells (Life Technologies, Inc.), and the clones were sequenced using Sequenase (U. S. Biochemical Corp.). DNA sequence analysis was performed with MacVector software (Kodak).


Figure 1: Comparison of C. fasciculata mitochondrial polymerase (top) with human pol beta (bottom). The protein sequence alignments were performed using the Gap program in the Wisconsin Package (Genetics Computer Group). Verticallines between the residues indicate shared identity. The boxedregion at the N terminus of the protein designates the presequence, which is encoded by the gene but not present on the purified enzyme. The underlinedregions of the mitochondrial polymerase sequence indicate the peptides, which were sequenced directly. In order to distinguish between the individual peptides sequenced, Arg and Lys (which were sequenced in the peptides spanning residues 189-201 and 202-227, respectively) were not underlined. The DNA sequence encoding this protein has been submitted to GenBank (accession no. U19912).



Isolation of a Genomic Clone

Southern blot analysis of C. fasciculata DNA digested with various restriction enzymes and probed with the PCR clone indicated that the entire polymerase gene was contained within a 5-kilobase SalI/NotI restriction fragment. A genomic SalI/NotI library was constructed in gt22A (Life Technologies, Inc.) and packaged using the Gigapack II Gold System (Stratagene). The library was plated, transferred onto Nytran membranes (Schleicher & Schuell), and probed with the PCR clone using the Genius Detection System (Boehringer Mannheim) according to the manufacturer's instructions. Two positive clones were obtained from the 4 times 10^5 phage screened. One of the clones was selected, subcloned into Bluescript KS+, and transformed into DH5alpha cells. The DNA was sequenced using Sequenase (U. S. Biochemical Corp.) according to the manufacturer's instructions. Both strands were sequenced using various oligonucleotide primers.


RESULTS AND DISCUSSION

In an analysis of the amino acid sequence of the C. fasciculata mitochondrial DNA polymerase we identified 29 residues at the N terminus and an additional 72 residues of internal sequence from tryptic peptides. The six tryptic peptides ranged in size from 8 to 26 residues, and one was contained within the N-terminal sequence. From these sequences, we designed two PCR primers to amplify and clone a region of the polymerase gene from C. fasciculata genomic DNA. The resulting 612-base pair clone encoded an open reading frame that contained five of the six sequenced regions of the protein. We used this PCR clone to screen a C. fasciculata genomic library, isolating a genomic clone that contained the entire coding region of the polymerase. Fig. 1shows the deduced sequence of the protein (uppersequence). The six regions identified by peptide sequencing are underlined, and all agree with the deduced sequence.

We searched the sequence data base to determine if the C. fasciculata mitochondrial polymerase is related to any other known polymerase. This search revealed that the mitochondrial enzyme shared the most significant homology with pol beta. Fig. 1shows a sequence comparison with human pol beta. The two enzymes share 33% identity and 54% similarity at the amino acid level. This level of homology is significant in light of the fact that pol beta from yeast and human shares 26% identity and 51% similarity. A comparison of the C. fasciculata enzyme and yeast pol beta reveals 24% identity and 50% similarity (not shown). Although the C. fasciculata enzyme is slightly larger than the human pol beta (376 versus 318 amino acids, respectively), it is actually smaller than the yeast enzyme (582 amino acids). A comparison of the C. fasciculata and human sequences reveals that the parasite polymerase has a 17-residue extension at the N terminus, as well as insertions of 8 and 26 residues near the C terminus (see Fig. 1). According to the crystal structure of the rat pol beta(8) , these insertions occur in external loops that connect regions of secondary structure. The three conserved aspartic acid residues (designated by * below the human pol beta sequence in Fig. 1), thought to be essential for catalytic activity, maintain their relative positions in the C. fasciculata protein. Finally, the homology between the C. fasciculata enzyme and human terminal deoxynucleotidyltransferase (21% identity) is similar to the homology between human pol beta and human terminal deoxynucleotidyltransferase (25% identity). As mentioned above, pol beta and terminal deoxynucleotidyltransferase are the only members of the family X DNA polymerases(6, 7) .

A 9-amino acid presequence, beginning with the initiating methionine residue, is not present on the mature protein (boxed in Fig. 1). Our N-terminal sequencing indicated that the glycine residue immediately downstream from this presequence is the first amino acid residue of the mature protein. The presequence is similar to three other cleaved presequences, thought to be import signals, reported for C. fasciculata mitochondrial proteins (Fig. 2). These other presequences are from DNA binding proteins, p16, p17, and p18, which had been isolated by in vivo chemical cross-linking to C. fasciculata kDNA (12) . All four of these presequences are 9 amino acids in length, arginine-rich, and positively charged. The only other known C. fasciculata cleaved presequence, from a mitochondrial hsp70 protein, is 20 residues in length(13) . The C. fasciculata mitochondrial topoisomerase II does not appear to have a cleaved N-terminal presequence(14) .


Figure 2: Comparison of C. fasciculata mitochondrial presequences. The previously identified 9 amino acid presequences from the C. fasciculata mitochondrial proteins p16, p17, and p18 (12) are aligned with the presequence from the mitochondrial polymerase (MitoPolbeta).



The enzymatic properties of the C. fasciculata pol beta, such as low processivity, suggest that it may not be the major mitochondrial replicative enzyme. Therefore, it is possible that another enzyme, not yet discovered but perhaps related to DNA polymerase , is also present in the C. fasciculata mitochondrion. It is also possible that there is another pol beta in the nucleus of C. fasciculata. If so, it will be of great interest to compare the enzymatic properties and amino acid sequences of mitochondrial and nuclear pol betas from the same organism.

What is the function of the pol beta in the mitochondrion? It must play a role in the replication or maintenance of kDNA minicircles or maxicircles. For the purpose of replication, minicircles are released from the network by a topoisomerase II (reviewed in (15, 16, 17) ). The free minicircles are thought to replicate in one of the two complexes of replication proteins (which includes pol beta) mentioned above. After replication the progeny minicircles are reattached to the network adjacent to these complexes(4, 18) . The progeny minicircles that contain the lagging strand are heavily gapped, with one small gap positioned approximately every 100 nucleotides(19) . Many of these gaps are repaired just prior to attachment of the minicircle to the network, and the pol beta is ideally positioned to carry out this repair. It is possible that a pol beta was specially imported into the C. fasciculata mitochondrion for the purpose of repairing these gaps. Alternatively, it is possible that a pol beta is present, perhaps at low levels, in all eukaryotic mitochondria.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM 27608 and by the MacArthur Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
The abbreviations used are: kDNA, kinetoplast DNA; pol, polymerase; PCR, polymerase chain reaction.


ACKNOWLEDGEMENTS

We thank Viiu Klein for technical assistance. We also thank Laura Rocco, Catharine Johnson, and Kuo-Yuan Hwa for many enlightening discussions.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.