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RESEARCH INVESTIGATIONS
ASYMMETRIC BIDIRECTIONAL DNA REPLICATION IN MAMMALIAN CELLS
The objective of this research program is to understand the interactions between
cis-acting DNA elements and trans-acting factors that regulate the initiation of
DNA replication in mammalian cells. Faithful replication of the entire genome
once-per-cell cycle is essential for maintaining genetic stability in all living
organisms, and the origin of DNA replication (ori) is a key control point of this
regulation. Therefore, understanding the mechanism of ori activation is
fundamental to gaining insights into the regulation of genome duplication and
genetic stability. A short cis-acting genetic element is all that is required for ori
function in prokaryotes and simple eukaryotes. However, the chromatin context
and gene organizations are critically important in the regulation of mammalian
DNA replication. In particular, transcription and replication often interplay in the
regulation of mammalian oris, especially for those located at transcription
promoters.

Julia Romero and Hoyun Lee. 2008. One-way PCR-based mapping of a
replication initiation point (RIP). Nature Protocols 3, 1720-1735.
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Julia Romero and Hoyun Lee. 2008. Asymmetric bidirectional replication (ABR)
at the human Dbf4 locus. Nature Structural and Molecular Biology 15,722-729.
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
Xing Wu and Hoyun Lee. 2002. Human Dbf4/ASK promoter is activated through
the Sp1 and Mlu 1 cell-cycle box (MCB) transcription elements. Oncogene 21
(51), 7786-779621.
DEVELOPING EFFECTIVE AND SPECIFIC ANTICANCER MODALITIES
Effectively and selectively eliminating tumour cells without causing undesirable
side-effects to normal cells is one of the most difficult challenges of cancer
therapy. Since certain cellular signals are highly elevated in tumour cells (but not
in normal cells), blocking these “cancer-specific” signals can be an effective way
of controlling cancer. However, this approach is often not very efficient because
selectively blocking a signal can usually be achieved only at low concentrations
of the blockers, at which doses the efficacy of cancer-cell killing is low. We
postulate that this limitation can be largely overcome by combining with effective
and safe sensitizers.
We found that chloroquine, an inexpensive and safe anti-malarial and rheumatoid drug, can effectively sensitize cancer-cell killing by other agents.
Importantly, chloroquine does not, in most cases, sensitize normal-cell killing by
cancer drugs. We also take repurposing and repositioning approaches to
increase drug efficacy and specificity.
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
V. Raja Solomon and Hoyun Lee. 2010. Utilization of NMR-Based techniques,
In “Anticancer Drug Development, Structure-Activity Relationship Studies in
Drug Development by NMR Spectroscopy” (eds., A.U. Rahman and M.I.
Choudhary), Bentham Science Publishers (In press).

V. Raja Solomon, Changkun Hu, and Hoyun Lee. 2010. Design and synthesis of
anti-breast cancer agents from 4-piperazinylquinoline: A hybrid pharmacophore
approach. Bioorganic & Medicinal Chemistry 18, 1563-1572.

Changkun Hu, V. Raja Solomon, Pablo Cano, and Hoyun Lee. 2010. A 4aminoquinoline derivative that markedly sensitizes tumor cell killing by Akt
inhibitors with a minimum cytotoxicity to non-cancer cells. European Journal of
Medicinal Chemistry 45, 705-709.

V. Raja Solomon and Hoyun Lee. 2009. Chloroquine and its analogs: a new
promise of an old drug for effective and safe cancer therapies (invited review).
The European Journal of Pharmacology. 625, 220-233.

V. Raja Solomon, Changkun Hu, and Hoyun Lee. 2009. Hybrid Pharmacophore
Design and Synthesis of Isatin-Benzothiazole Analogs for their Anti-Breast
Cancer Activity. Bioorganic & Medicinal Chemistry 17, 7585-7592. * Identified
as a breaking paper by the LeadDiscovery’s DailyUpdates.

Changkun Hu, V. Raja Solomon, Gerardo Ulibarri, and Hoyun Lee. 2008. The
efficacy and selectivity of tumor cell killing by Akt inhibitors are substantially
increased by chloroquine. Bioorganic & Medicinal Chemistry 16, 7888-7893.

Haiwen Zhang, Raja Solomon, Changkun Hu, Gerardo Ulibarri, and Hoyun Lee.
2008. Synthesis and in vitro cytotoxicity evaluation of 4-aminoquinoline
derivatives. Biomedicine and Pharmacotherapy 62, 65-69.
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RESEARCH INVESTIGATIONS
THE ROLE OF CDC7-DBF4 KINASE IN THE REGULATION OF DNA
REPLICATION AND CELL-CYCLE CONTROL
The Cdc7-Dbf4 serine/threonine kinase activates the initiation of DNA replication
by phosphorylating MCM proteins that are bound to the origins of DNA
replication. We reported previously that human Cdc7 nuclear import is mediated
directly by importin- through its binding to the Cdc7 nuclear localization
sequence (NLS). We also reported that human Cdc7 nuclear localization is
regulated by two additional elements: nuclear retention (NRS) and export
sequences (NES). Cdc7 proteins imported into the nucleus are retained in the
nucleus by associating with chromatin, for which NRS (306-326) is essential.
Importantly, this binding appears to be specific to the origin of DNA replication.
Furthermore, an NRS-defective Cdc7 mutant could not be retained in the
nucleus, although it was imported into the nucleus normally. Together, our data
suggest that NRS plays an important role in the activation of DNA replication by
Cdc7. The Cdc7 proteins unassociated with chromatin are bound by CRM1 via
two NES elements: NES1 at 458-467 within kinase insert III, and NES2 at 545554 within the kinase IX domain. The primary function of the Cdc7-CRM1
association may be to translocate nuclear Cdc7 to the cytoplasm. However, the
binding of CRM1 with Cdc7 at NES2 raises an interesting possibility that CRM1
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may also downregulate Cdc7 by masking its kinase domain. We currently focus
our research on the Cdc7 domain-function relation.
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James W. Knockleby, Julia Romero, Kathleen A. Kylie, and Hoyun Lee. 2010
The role of Dbf4/Drf1-dependent kinase Cdc7. Current Topics in Biochemistry
(In press)
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Byung Ju Kim and Hoyun Lee. 2008. Caspase-mediated cleavage of importin-
increases its affinity for MCM and downregulate DNA synthesis. BBA-Molecular
Cell Research 1783, 2287-2293.
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Byung Ju Kim, So-Young Kim, and Hoyun Lee. 2007. Identification and
characterization of Cdc7 nuclear retention and export sequences in the context of
chromatin binding. Journal of Biological Chemistry 282, 30029-30038.

Byung-Ju Kim and Hoyun Lee. 2006. Importin- mediates the nuclear
transportation of human Cdc7 by directly binding to its Kinase Insert II domain,
which can be antagonized by importin-. Journal of Biological Chemistry 281,
12041-12049. (Funding sources, NCIC, CIHR). * This paper is highlighted by the
Faculty of 1000 (Biology) in 2006.

Baoqing Guo, Julia Romero, Byung-Ju Kim and Hoyun Lee. 2005. High levels of
Cdc7 and Dbf4 proteins can arrest cell cycle progression. European Journal of
Cell Biology 84, 927-938.

Baoqing Guo and Hoyun Lee.2001.Cloning and characterization of Chinese
hamster CDC7 (ChCDC7). Somatic Cell and Molecular Genetics 25,159-171. *
PubMed cites this paper as published in 1999 by mistake.

Baoqing Guo and Hoyun Lee. 2001.Cloning and characterization of Chinese
hamster homologue of yeast DBF4 (ChDBF4). Gene 264, 249-256. (Funding
source, MRC/CIHR)
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RESEARCH INVESTIGATIONS
MOLECULAR MECHANISMS OF TUMOR CELL RADIATION- AND CHEMORESISTANCE
Despite the fact that radiation and chemotherapy have considerably improved
during the last several decades, the failure of these treatments is still
unacceptably high. This failure is often due to the selection of more radio- or
chemo-resistant subpopulations during cancer therapy. We are trying to
understand cellular response to radiation and chemotherapeutics at molecular
levels and, eventually, to develop effective combined modalities to control
cancers. Our research showed that the PI3K-Akt/PKB signal pathway plays a
critical role in tumor cell proliferation, survival and resistant to radiation and
chemotherapy. We currently examine the detailed mechanism as to how each
Akt/PKB isoform is involved in the regulation of tumor cell growth and survival.

Stacey Santi and Hoyun Lee. 2010. The Akt isoforms occupy a distinct
subcellular localization within cells. Am J Physiology-Cell physiology (Published
on-line, December 16, 2009) (doi:10.1152/ajpcell.00375.2009).

Helen Zhao, Yong Cai, Stacy Santi, Robert Lafrenie and Hoyun Lee. 2005.
Chloroquine-mediated radiosensitization is due to the destabilization of the
lysosomal membrane and subsequent induction of necrotic cell death. Radiation
Research 164, 250-257.
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
Andrew G. Pearce, Tamika M. Segura, Anne C. Rintala, Nina Rintala-Maki, and
Hoyun Lee. 2001. The generation and characterization of radioresistant model
system to study radioresistance in human breast cancer cells. Radiation Research
156, 739-750. (Funding sources, CRS, NSERC)
THE STRUCTURE-FUNCTION OF PCNA
Proliferating cell nuclear antigen (PCNA), a DNA replication processivity factor, is
an essential component for eukaryotic chromosome replication and is often used
as a cancer diagnostic and prognostic marker. PCNA is also involved in a wide
range of other cellular activities, including DNA damage repair, cell-cycle control,
apoptosis and epigenetic inheritance. The diverse function of PCNA may be
regulated largely by post-translational modifications and association with different
protein partners at a given time and space. We have previously shown by highresolutional two-dimensional gel electrophoresis that there are three distinct
PCNA isoforms that differ in their acetylation status. The Acidic (A), Main (M) and
Basic (B) forms are 34 kDa-pI4.52, -pI4.57 and -pI4.62 proteins, respectively. We
also found, in contrast to the currently accepted single homotrimer-ring model,
that mammalian PCNA trimers exist in a cell as a back-to-back doublet complex.
Since the double-trimer complex exposes two front sides, this structure can
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provide great flexibility in coordinating DNA replication with diverse cellular
functions.

Byung Ju Kim and Hoyun Lee. 2008. Lys-110 is essential for targeting PCNA to
replication and repair foci, and the K110A mutant activates apoptosis. Biology of
Cell 100, 675-686.
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Stanislav N. Naryzhny and Hoyun Lee. 2007. Characterization of proliferating
cell nuclear antigen (PCNA) isoforms in normal and cancer cells: There is no
cancer-associated form of PCNA. FEBS Letter 581, 4917-4920.

Stanislav N. Naryzhny, Leroi V. DeSouza, K. W. Michael Siu and Hoyun Lee.
2006. Characterization of human proliferating cell nuclear antigen physicochemical properties: aspects of double trimer stability. Biochemistry and Cell
Biology 84, 669-676.

Stanislav N. Naryzhny, Helen Zhao and Hoyun Lee. 2005. Proliferating cell
nuclear antigen (PCNA) may function as a double-homotrimer complex in the
mammalian cell. Journal of Biological Chemistry 280, 13888-13894.

Stanislav N. Naryzhny and Hoyun Lee. 2004. The post-translational
modifications of proliferating cell nuclear antigen (PCNA): acetylation, not
phosphorylation, plays an important role in the regulation of its function. Journal
of Biological Chemistry 279, 20194-20199.

Stanislav N. Naryzhny and Hoyun Lee. 2003. Observation of multiple isoforms
and specific proteolysis patterns of PCNA in the context of cell-cycle
compartments and sample preparations. Proteomics 3, 930-936.

Hoyun Lee and Stanislav N. Naryzhny. 2006. Cooperation of multiple cellular
functions by PCNA: Implication of the PCNA double trimer model, p.163-180. In
H. Lee (ed.), Proliferating Nuclear Antigen (PCNA), Research Signpost, Kerala.
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