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Summer Student Research Program
Project Description
FACULTY SPONSOR’S NAME AND DEGREE: Andreas Ivessa, Ph.D.
PHONE: (973) 972 - 2015
DEPARTMENT AND INTERNAL MAILING ADDRESS:
Department of Cell Biology and Molecular Medicine
University of Medicine and Dentistry of New Jersey
185 South Orange Avenue, MSB G609
Newark, NJ 07101-1709
E-MAIL: [email protected]
PROJECT TITLE (200 Characters max):
Testing the functional interaction between the mitochondrial DNA polymerase gamma and the Pif1
DNA helicase in budding yeast
HYPOTHESIS:
We are studying the principles how mitochondrial DNA (mtDNA) is maintained and inherited using yeast
as a model system. Impaired duplication of mtDNA may lead to mutations or breakage in mtDNA.
Mutated mtDNA molecules might cause dysfunction of mitochondria. Several human diseases are known
that are either associated with or caused by mutant mtDNA molecules.
In particular, we are studying the Pif1 DNA helicase that is required to maintain mtDNA in the presence
of genotoxic stress. According to our results we hypothesize that the Pif1 DNA helicase governs the rate
of mtDNA replication by interacting with the mitochondrial DNA polymerase gamma during the mtDNA
replication process.
PROJECT DESCRIPTION (Include design, methodology, data collection, techniques, data analysis to be
employed and evaluation and interpretation methodology)
Mitochondrial function is essential for the viability of eukaryotic cells. Mitochondria provide cells mainly
with energy, but are also involved in other functions such as apoptosis. Failure in mitochondrial function
can be linked to the aging process and to several diseases including neurodegenerative disorders, heart
diseases, diabetes, and cancer.
We will use the model system bakers yeast (Saccharomyces cerevisiae) that is ideal to understand basic
principles of mtDNA replication. Yeast is also beneficial to study mtDNA mutations, since yeast can
survive without mitochondrial function and it is sufficient similar to human mtDNA. Multiple specific
mutations such as gene-knock-outs can be introduced into the nuclear genome in a reasonable short time
period.
MtDNA is mutated by toxic chemicals and reactive oxygen species that escape the respiratory chain
reaction. A network of mtDNA damage resistance pathways is proposed to prevent or repair mtDNA
damage. One member of this network is the DNA helicase Pif1 that unwinds both double-stranded DNA
and RNA/DNA hybrids. In bakers yeast, Pif1 is present in both the nucleus and mitochondria. Cells
lacking mitochondrial Pif1 exhibit higher mutation frequencies in mtDNA, and lose mtDNA more
frequently in the presence of fermentable carbon sources (glucose). MtDNA replication is impaired in
pif1 mutant cells resulting in increased mtDNA breakage. This breakage is increased in the presence of
mtDNA-damaging drugs.
According to our hypothesis the Pif1 DNA helicase may interact with the mitochondrial DNA
polymerase gamma (yeast: Mip1) that is essential for the replication of mtDNA. We will determine if Pif1
and Mip1 physically interact in vivo.
We will express Pif1 and Mip1 from their endogenous chromosomal locations as functional proteins that
contain C-terminal localized tags. These tags can then be recognized by commercially-available
antibodies. Pif1 protein will have a 3xHA tag at its C-terminus, and Mip1 will be expressed with a Cterminal YEGFP-tag. The strain expressing Mip1-YEGFP is available. We will basically construct two
strains: We will introduce the 3xHA tag into the C-terminal location of the PIF1 gene by homologous
recombination into the strain expressing the Mip1-YEGFP protein and into a strain that is lacking any tag.
Summer Student Research Program
Project Description
We will confirm the correctness of the integration of the tags into the yeast genome by PCR and Southern
technique. Four strains should then be available for the experiments: Yeast strains expressing only Mip1YEGFP, expressing only Pif1-HA, expressing both Mip1-YEGFP and Pif1-HA, and lacking any tag.
To test for the physical interactions between Pif1 and Mip1 we will grow the strain with the tagged
proteins to an early logarithmic growth phase in the presence of a non-fermentable carbon source
(glycerol). The cells will be lysed using glass beads in the presence of mild non-ionic detergents (e.g.
NP40, triton X-100, digitonin). Cell debris will be removed by high speed centrifugation. The resulting
clear supernatants will be pre-cleared with the same type of beads that are used to precipitate the
antibodies (e.g. magnetic beads coated with protein A or G).
In a typical experiment, we will precipitate one protein (Pif1 or Mip1) using antibodies against their tags
(anti-HA antibodies to precipitate Pif1-HA or anti-GFP antibodies to precipitate Mip1-YEGFP). The
antibody-protein complex will be precipitated using magnetic beads coated with protein A or G. The
interacting protein will be determined by western-blot analysis. For example, if Pif1-HA is precipitated
using anti-HA antibodies, Mip1-YEGFP will be detected in the precipitate using anti-GFP antibodies. As
control we will use strains that express either only one tagged protein (Pif1-HA or Mip1-YEGFP) or lack
any tag.
If we find an interaction between Pif1 and Mip1, we will also determine if the interaction is direct. For
this purpose, we will use DNase to digest DNA that might serve as a bridge between Pif1 and Mip1. If the
interaction between Pif1 and Mip1 is direct, Pif1 should precipitate with Mip1 also in the presence of
DNase and vice versa.
SPONSOR’S MOST RECENT PUBLICATIONS RELEVANT TO THIS RESEARCH:
Cheng, X., Dunaway, S., and Ivessa, A.S. 2007. The role of Pif1p, a DNA helicase in Saccharomyces
cerevisiae, in maintaining mitochondrial DNA. Mitochondrion, 7(3):211-22. (Epub 2006 Dec 9).
IS THIS PROJECT SUPPORTED BY EXTRAMURAL FUNDS?
Yes X or
No
(IF YES, PLEASE SUPPLY THE GRANTING AGENCY’S NAME)
Scientist Development Grant from the American Heart Association
THIS PROJECT IS:
Clinical
X Laboratory
THIS PROJECT EMPLOYS RADIOISOTOPES: X
Behavioral
Other
(license #: LIC 267; approved 03-28-2005)
THIS PROJECT INVOLVES THE USE OF ANIMALS
PENDING
APPROVED
IACUC PROTOCOL #
THIS PROJECT INVOLVES THE USE OF HUMAN SUBJECTS
PENDING
APPROVED
IRB PROTOCOL # M
WHAT WILL THE STUDENT LEARN FROM THIS EXPERIENCE?
The student should learn that diseases are generally caused by the failure of specific biological processes.
The student should further learn to work independently, draw conclusions on her/his own, and make
proposals for future experiments.