Ongoing
Research
DNA replication is a fundamental process in the life
cycle of every living organism and its regulation is essential for the normal
development of cells and their regulated proliferation. Regulation of chromosome
replication is mediated by interactions of replication origins and other control
elements in the DNA with their trans-acting proteins. Our studies focus on the
mechanisms that control these interactions, and thereby, regulate the initiation
of DNA replication in eukaryotes, using the replication of the kinetoplast DNA
(kDNA) of trypanosomatids as an experimental model. Although kDNA is an
extrachromosomal genome, its replication provides an attractive model for the
study of several major aspects of chromosomal DNA replication, as the two
systems share several major features in common, including the control of
duplication of multiple replicons, and restriction of their replication to the
S-phase of the cell cycle.
kDNA is a DNA network
found in the single mitochondrion of trypanosomatids. The network consists of
~5,000 duplex DNA minicircles and ~25 maxicircles, which are interlocked into a
disk-shape catenane. Two short sequences, a 12-mer "universal minicircle
sequence" (UMS) and a hexamer, were conserved at the origins of the minicircle L
and H strands (oriL and oriH), respectively. The minicircle origin binding
protein, designated the universal minicircle sequence binding protein (UMSBP),
which binds these sequences, is one of the major subjects of our recent
research.
In earlier studies, we
have described in C. fasciculata the single-stranded DNA binding protein UMSBP,
that interacts specifically with the conserved UMS sequence at the oriL and with
a 14-mer sequence (H14, including the conserved hexamer) at the oriH. UMSBP's
encoding gene and genomic locus were cloned and analyzed and were found to be
conserved in other trypanosomatid species. UMSBP is a 13.7 kDa protein,
containing five CCHC-type zinc fingers, which oligomerize in solution. The
protein was immunolocalized to two neighboring sites, in the kineto-flagellar
zone (KFZ), in the same region as DNA primase, Pol I-type DNA polymerases and a
center of minicircle replication intermediates. Structure-function analysis has
revealed that UMSBP binds the DNA ligand as a monomer. The protein N-terminal
domain, mediates its dimerization, while the C-terminal domain is involved in
UMSBP-DNA interactions. Regulation of UMSBP binding at the minicircle
replication origin seems to be mediated through two post translational
modification pathways, redox signaling and protein phosphorylation. Both DNA
binding and dimerization of UMSBP are sensitive to redox. Oxidation results in
UMSBP dimerization, with concomitant inhibition of its DNA binding activity,
while reduction yields monomers that are active in the binding of DNA. C.
fasciculata tryparedoxin activates the binding of UMSBP to UMS DNA in vitro. An
N-terminally truncated UMSBP mutant was apparently released from the redox
effect, suggesting that UMSBP N-terminal domain plays a major role in the
redox-mediated regulation of UMSBP. In addition to its control through redox
signaling, UMSBP is phosphorylated in the cell at serine residues, through the
action of a protein kinase C-type protein kinase.
Other aspects of our
current research focus on the recognition of the minicircle replication origin
by the replication machinery and the parameters that determined the choice of a
functional origin; the assembly of the replication initiation complex; DNA
helicases that are involved in kDNA replication and the parameters that
characterize the movement of the minicircle replication fork.
Assigning functions to genes is one of the major
challenges of the post-genomic era. Usually, functions are assigned based on
similarity of the coding sequences to sequences of known genes, or by
identification of transcriptional cis-regulatory elements that are known to be
associated with specific pathways or conditions. In trypanosomatids, where
regulation of gene expression takes place mainly at the post-transcriptional
level, new approaches for function assignment are needed. In a recent study,
that included a whole genome analysis of Leishmania major, we demonstrated the
identification of novel S-phase expressed genes in Leishmania major, based on a
post-transcriptional control element that was recognized in Crithidia
fasciculata as involved in the cell cycle-dependent expression of several
nuclear and mitochondrial S-phase expressed genes. Hypothesizing that a similar
regulatory mechanism is manifested in L. major, we have applied a computational
search for similar control elements in the genome of L. major. Our computational
scan yielded 132 genes, 33% of which are homologues of known DNA metabolism
genes and 63% lack any annotation. Experimental testing of seven of these genes
revealed that their mRNAs cycle throughout the cell cycle, reaching a maximum
level during S-phase or just prior to it. It is suggested that screening for
post-transcriptional control elements associated with a specific function
provides an efficient method for assigning functions to trypanosomatid genes.
