Header

UZH-Logo

Maintenance Infos

Inducible Gene Replacement of DNA Polymerase Delta


Kehl, P. Inducible Gene Replacement of DNA Polymerase Delta. 2009, University of Zurich, Faculty of Science.

Abstract

Deoxyribonucleic acid (DNA) polymerases ε and δ are responsible for leading
and lagging strand synthesis during eukaryotic DNA replication. Because these
enzymes have intrinsic error rates of 1.1 x10-5 and 3.7 x10-5 per base pair, error-free
replication of genomes of these organisms can only be achieved with the help of
auxiliary mechanisms that remove misincorporated nucleotides from newlysynthesized
DNA prior to cell division. In all organisms studied to date, the replicative
polymerases possess intrinsic proofreading exonucleases, which remove mispaired
nucleotides from the 3’ termini of newly-synthesized DNA and thus improve
replication fidelity by ~2 orders of magnitude. Postreplicative mismatch repair (MMR)
then mediates the removal of mispairs that escaped proofreading. This process
improves the fidelity of DNA replication by up to 3 orders of magnitude, such that
even genomes of higher eukaryotes can be replicated without errors. The
consequences of missing MMR have been studied in great detail during the past
decade, primarily because of a link to cancer. However, to date it is unclear whether
malignancy arises as a result of the mutator phenotype on MMR-deficient cells, or
whether it is linked to an as yet unknown function of the MMR proteins. In order to
address this question, we set out to generate a cell system in which the mutator
phenotype would not be induced by defective MMR. We wanted to influence the
fidelity of DNA replication by introducing mutations into the polymerase and
exonuclease domains of the replicating polymerases, but this approach is
complicated by the fact that these enzymes are essential. The aim of my study was
to develop a system that would overcome these difficulties and allow us to regulate
replication fidelity by altering the base selectivity and/or the proofreading
exonuclease activity of polymerase δ.
I focused on mutations that were already characterised in yeast and mice.
However, in contrast to these studies, I wanted to devise an isogenic system, in
which the variant enzymes would replace the endogenous activity by the combination
of inducible expression and ribonucleic acid (RNA) interference mediated knockdown
technologies. In this way, the wild type and mutator phenotypes could be
studied in one and the same human cell line. Moreover, the flexibility of the inducible
system should ensure that the exogenous variant is expressed in similar amounts to
the endogenous wild type protein. I refer to the system as “gene replacement”.

Abstract

Deoxyribonucleic acid (DNA) polymerases ε and δ are responsible for leading
and lagging strand synthesis during eukaryotic DNA replication. Because these
enzymes have intrinsic error rates of 1.1 x10-5 and 3.7 x10-5 per base pair, error-free
replication of genomes of these organisms can only be achieved with the help of
auxiliary mechanisms that remove misincorporated nucleotides from newlysynthesized
DNA prior to cell division. In all organisms studied to date, the replicative
polymerases possess intrinsic proofreading exonucleases, which remove mispaired
nucleotides from the 3’ termini of newly-synthesized DNA and thus improve
replication fidelity by ~2 orders of magnitude. Postreplicative mismatch repair (MMR)
then mediates the removal of mispairs that escaped proofreading. This process
improves the fidelity of DNA replication by up to 3 orders of magnitude, such that
even genomes of higher eukaryotes can be replicated without errors. The
consequences of missing MMR have been studied in great detail during the past
decade, primarily because of a link to cancer. However, to date it is unclear whether
malignancy arises as a result of the mutator phenotype on MMR-deficient cells, or
whether it is linked to an as yet unknown function of the MMR proteins. In order to
address this question, we set out to generate a cell system in which the mutator
phenotype would not be induced by defective MMR. We wanted to influence the
fidelity of DNA replication by introducing mutations into the polymerase and
exonuclease domains of the replicating polymerases, but this approach is
complicated by the fact that these enzymes are essential. The aim of my study was
to develop a system that would overcome these difficulties and allow us to regulate
replication fidelity by altering the base selectivity and/or the proofreading
exonuclease activity of polymerase δ.
I focused on mutations that were already characterised in yeast and mice.
However, in contrast to these studies, I wanted to devise an isogenic system, in
which the variant enzymes would replace the endogenous activity by the combination
of inducible expression and ribonucleic acid (RNA) interference mediated knockdown
technologies. In this way, the wild type and mutator phenotypes could be
studied in one and the same human cell line. Moreover, the flexibility of the inducible
system should ensure that the exogenous variant is expressed in similar amounts to
the endogenous wild type protein. I refer to the system as “gene replacement”.

Statistics

Additional indexing

Item Type:Dissertation
Referees:Jiricny J, Thoma F
Communities & Collections:04 Faculty of Medicine > Institute of Molecular Cancer Research
07 Faculty of Science > Institute of Molecular Cancer Research
Dewey Decimal Classification:570 Life sciences; biology
Date:2009
Deposited On:12 Feb 2010 14:14
Last Modified:11 Aug 2017 14:50
Free access at:Official URL. An embargo period may apply.
Official URL:http://e-collection.ethbib.ethz.ch/show?type=diss&nr=18246
Related URLs:http://opac.nebis.ch/F/?local_base=NEBIS&con_lng=GER&func=find-b&find_code=SYS&request=005810247

Download

Full text not available from this repository.

TrendTerms

TrendTerms displays relevant terms of the abstract of this publication and related documents on a map. The terms and their relations were extracted from ZORA using word statistics. Their timelines are taken from ZORA as well. The bubble size of a term is proportional to the number of documents where the term occurs. Red, orange, yellow and green colors are used for terms that occur in the current document; red indicates high interlinkedness of a term with other terms, orange, yellow and green decreasing interlinkedness. Blue is used for terms that have a relation with the terms in this document, but occur in other documents.
You can navigate and zoom the map. Mouse-hovering a term displays its timeline, clicking it yields the associated documents.

Author Collaborations