(Received for publication, December 5, 1994)
From the
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
, 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
. 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
.
Crithidia fasciculata, a protozoan parasite related to
the trypanosomes, has an unusual mitochondrial DNA known as kinetoplast
DNA (kDNA). ()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
(pol
), 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 's
sequence is highly conserved among vertebrates, although the yeast pol
has lower homology, about 26% identity at the amino acid level. A
comparison of the known pol
s with all other DNA polymerases,
either prokaryotic or eukaryotic, indicates that they are related to
only one other nucleotide polymerizing enzyme, terminal
deoxynucleotidyltransferase. Pol
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
revealed a DNA binding
channel with some similarities to those found in other DNA
polymerases(8, 9, 10) . The crystal structure
suggests that pol
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 . 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
identified in any
organism.
Figure 1:
Comparison of C. fasciculata mitochondrial polymerase (top) with human pol (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).
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
. Fig. 1shows a sequence comparison with human pol
.
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
from yeast and human shares 26% identity and 51% similarity.
A comparison of the C. fasciculata enzyme and yeast pol
reveals 24% identity and 50% similarity (not shown). Although the C. fasciculata enzyme is slightly larger than the human pol
(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
(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
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
and human
terminal deoxynucleotidyltransferase (25% identity). As mentioned
above, pol
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 (MitoPol).
The enzymatic properties of the C.
fasciculata pol , 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
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
s from the same organism.
What
is the function of the pol 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
) 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
is ideally positioned to carry out this
repair. It is possible that a pol
was specially imported into the C. fasciculata mitochondrion for the purpose of repairing
these gaps. Alternatively, it is possible that a pol
is present,
perhaps at low levels, in all eukaryotic mitochondria.