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Annual report : National Institute of Environmental Health Sciences (Volume 1985) online

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which increase the fidelity of purified DNA polymerases.

Publications :

None



220



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT



PROJECT NUMBER



Z01 ES 61039-01 LG



PERIOD COVERED

October 1, 1984 to September 30, 1985



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)

Mechanism of DNA Recombination and Repair in Yeast Saccharomyces cerevisiae



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)

PI: A. Sugino Visiting Scientist LG NIEHS



Others:



F. W. Coleman
B.-Y. Ryu
T. Sugino
T. Chow



Senior Staff Fellow
Guest Worker
Guest Worker
Visiting Associate



LG NIEHS
LG NIEHS
LG NIEHS
CBTP NIEHS



COOPERATING UNITS (if any)



LAB/BRANCH

Laboratory of Genetics



SECTION

Mutagenesis Section



INSTITUTE AND LOCATION

NIEHS, NIH, Research Triangle Park. North Carolina



27709



TOTAL MAN-YEARS:

1.6



PROFESSIONAL:
1.1



OTHER:



0.5



CHECK APPROPRIATE BOX(ES)

□ (a) Human subjects
□ (a1) Minors
D (a2) Interviews



P (b) Human tissues ffi (c) Neither



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)

Proteins binding to single-stranded DNA are expected to participate in DNA
recombination and repair as well as DNA replication. Thus, three different
single-stranded-DNA-binding proteins have been purified from the yeast
Saccharomyces cerevisiae and antibodies have been raised against them. Using
the antibodies as probes, their genes have been identified and cloned from a
Agtll yeast DNA library. Deletions of these genes were then constructed, the
wild- type genes were replaced by the disrupted genes, and the resulting phenotypes
were studied. The RAD52 gene product is required for DNA recombination and
repair in yeast, The gene has been cloned and its nucleotide sequence determined
by other groups. However, this important gene product has not yet identified
and purified. By aid of a computer we identified several possible antigenic
regions in the RAD52 gene. The oligopeptides covering the antigenic regions
were chemically synthesized and conjugated to BSA and antibodies were raised
against the conjugates. In addition, several fusion plasmids of the RAD52 gene
and either the ^pL promoter or the yeast a -mating type pheromon leader sequence
or the yeast ADH promotor will be constructed in order to overproduce RAD52 pro-
tein in E_. col i and yeast. Finally, an in vitro DNA recombination system yeast
6-6 sequences has been developed. This system requires ATP, MgCl 2 , and super-
coiled plasmid DNA containing at least two delta sequences; it generates a
double-strand break near or at the one of delta sequences but does not cleave
plasmid DNA lacking delta sequences.



221



PHS 6040 (Rev. 1/84)



GPO 91 4-918



Z01 ES 61039-01 LG



Research Project:



Problem : The yeast Saccharomyces cerevisiae has many advantages for studying
DNA recombination and repair in eucaryotes. It has excellent genetics, is a
very simple organism, and is easily prepared in large amounts for biochemical
analysis. Furthermore, it has a good transformation system and it is very easy
to construct mutants by in vitro mutagenesis. A large number of radiation, UV-,
and some mutagen-sensitive mutants have been isolated and mapped on the yeast
chromosome. However, very little is known about what kind of enzymes par-
ticipate in DNA recombination and repair processes in yeast. Therefore, this
project focuses on identification and purification of some proteins which are
required for recombination and repair.

Objectives of Research Project: (1) An in vitro DNA recombination system which
mimics the in vivo reaction will be developed from yeast crude extracts. (2)
It will then be used for identifying and purifying the components necessary for
the DNA recombination reaction. (3) By analogy to procaryotic systems, it is
expected that DNA helicase, E. coli recA- like protein, nucleases, DNA topoiso-
merases, DNA ligase, and single-stranded DNA binding proteins are needed for
recombination and repair in yeast. These activities will be purified and their
genes will be isolated. (4) The isolated genes will be disrupted by insertion
of DNA fragments and integrated into the chromosome to replace the wild-type
gene; the resulting phenotype will then be studied. (5) Although several RAD
genes have been cloned and their nucleotide sequences have been determined, few
gene products has yet been isolated and purified. Therefore, we will try to
identify and purify some of the gene products produced in this project, using
ol i gopepti de-di rected anti bodi es .

Experimental Approach and Scientific Justification : In order to develop an in
vitro DNA recombination system, we have slightly modified our in vitro replica-
tion system based on 2-M.m and ARS plasmid DNAs. Crude extracts were made from
either exponentially growing mitotic cells or synchronized meiotic cells in a
manner similar to that used for the crude extract for the in vitro DNA replica-
tion system. The crude extracts were incubated with buffer, magnesium chloride,
DTT, ATP and supertwisted plasmid DNA consisting of yeast delta sequences.
Recombination products produced in vitro were analyzed by agarose gel
electrophoresis. Delta sequences were chosen because six of them are located in
the cloned sup 4 sequence of yeast and the frequency of in vivo recombination
among these delta sequences is at least 100 times higher than among normal
genes. Also, this recombination requires the RAD52 gene product, as in the
general recombination reaction. If such an in vitro recombination system can be
developed, fractionation and reconstitution of the system will be employed to
identify and purify various recombination proteins and factors.

Several proteins which might be participate in DNA recombination and DNA repair
will be purified from yeast using assays of biochemical activity: single-
stranded DNA binding proteins, DNA topoisomerases, DNA helicases, nucleases, and
E_. coli recA -like proteins. Antibodies will be raised against the purified pro-
teins and the genes for each will be screened from a ^gtll yeast library using
the antibodies. Also, we will test whether or not the in vitro recombination
system is inhibited by the antibodies.



222



Z01 ES 61039-01 LG

In order to identify RAD52 and RAD3 gene products, ol igopepti de-directed anti-
body has been raised. This method involves computer analysis of the nucleotide
sequences to predict possible antigenic regions of the predicted gene product.
The oligopeptides covering the candidate antigenic regions have been chemically
synthesized and covalently linked to bovine serum albumin. Then the hybrid pro-
tein will be injected into rabbits to raise antibodies. These antibodies should
specifically interact with the intact gene product and should be useful tools to
identify and purify the gene products.

The second approach is to construct fusions between the yeast mating type alpha
pheromone leader peptide sequence and RAD52 or RAD3 gene, or between the bac-
teriophage \pL promotor and the RAD52 and RAD3 genes. The fused genes will be
introduced into either yeast or E. coli and expressed to overproduce the gene
product. The crucial feature of this approach is that expression of the gene
will be well controlled; therefore, the gene product will be detected even if it
rapidly turns over in vivo .

One radiation-sensitive mutant of yeast, rad!8-l , has high spontaneous mutability
(mutator phenotype). It is possible that the RAD18 gene product is one of the
accessory proteins of yeast DNA polymerase I (a subunit of the DNA polymerase I
holoenzyme). Thus, DNA polymerase I holoenzymes will be purified from both
wild-type and rad!8 cells to compare their subunit structures and accuracy of
the DNA polymerization reactions. In the meantime, the RAD18 gene will be
cloned.

Recent Accomplishments and Significance for Biomedical Research : By a slight
modification of our in vitro DNA replication system, we have developed an in
v i tro recombination system using yeast delta sequences from yeast crude extract.
This system has been extensively characterized. It requires ATP, magnesium
chloride and supercoiled plasmid DNA (which consists of at least two delta
sequences), and the main product of the reaction was a small circular DNA. The
reaction depends on the RAD52 and RAD3 products. Therefore, this system should
make possible the identification and purification of these two gene products.

Three different single-stranded DNA binding proteins (14,000, 20,000 and 38,000
daltons) have been purified to homogeneity and polyclonal antibodies have been
raised against each. To test whether the purified DNA-binding proteins are
required for recombination and/or repair, the genes have been identified and
cloned from a yeast genomic library in a ^gtll expression vector using the anti-
bodies. Then, deletions of the genes have been constructed and integrated into
the yeast chromosome and the wild-type genes have been replaced by the mutage-
nized genes. Although each gene has been disrupted, no phenotype changes have
been detected. Therefore, mutants carrying simultaneous deletions of two and
three of these genes are under construction.

It is well known that the RAD52 gene product is required for DNA recombination
and repair in yeast. Although this important gene has recently been cloned and
its nucleotide sequence has been determined, the gene product has not yet been
identified and purified. To facilitate the identification and purification of
this gene product, we have been pursuing the following approaches. (1) Based
on computer analysis of the RAD52 gene sequence, several possible antigenic



223



Z01 ES 61039-01 LG



oligopeptide regions in the predicted RAD52 protein have been determined. Such
oligopeptides have been synthesized chemically, covalently linked to bovine
serum albumin and injected into a rabbit to raise antibodies. (2) The second
approach was to construct the fusion between the yeast mating type alpha phero-
mon leader sequence and the RAD52 gene (pMF8- RD52 ) or between the bacteriophage
\pl_ promotor and the RAD52 gene (pK333- RAD52 ). During the expression experi-
ments, we found that the RAD52 gene product is extremely unstable and is rapidly
degraded in vivo . This is consistent with the prediction, by computer analysis,
of the predicted gene product from the nucleotide sequence.

Plans for Future :

Our in vitro recombination system using yeast delta sequences is very promising.
Therefore, we will continue to characterize and fractionate the system to iden-
tify and purify various components. Once purified, the components will be
characterized and their genes will be identified.

Since we have found that the RAD52 gene product rapidly turns over in both E.
coli and yeast, we are going to try to isolate E_. coli and yeast mutants whTch
stably maintain the RAD52 gene product. We will also try to clone the RAD18
gene, which may code for one of the subunits of yeast DNA polymerase I.

Publications:



Resnick, M. A., Sugino, A., Nitiss, J., and Chow, T.: DNA polymerases, deoxyri
bonucleases, and recombination during meiosis in Saccharomyces cerevisiae . Mol
Cell. Biol. 12: 2811-2817, 1984. " "



v^S?



224



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT



PROJECT NUMBER



Z01 ES 61040-01 LG



PERIOD COVERED

October 1, 1984 to September 30, 1985



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)

Genetic and Biochemical Analysis of Yeast DNA Polymerase I



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)

PI: R. K. Hamatake Staff Fellow LG NIEHS

A. Sugino Visiting Scientist LG NIEHS



Others: A. B. Clark



Biologist



LG NIEHS



COOPERATING UNITS (if any)



LAB/BRANCH

Laboratory of Genetics



SECTION

Mutagenesis Section



INSTITUTE AND LOCATION

NIEHS, NIH, Research



TOTAL MAN-YEARS:

1.25



ri angle Park, North Carolina 27709



PROFESSIONAL:

0.75



OTHER:



0.5



CHECK APPROPRIATE BOX(ES)

□ (a) Human subjects
□' (a1) Minors
□ (a2) Interviews



□ (b) Human tissues ED (c) Neither



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)

A detailed analysis of the yeast replicative DNA polymerase, DNA pol I, is being
undertaken at the molecular and genetic level. The objectives of this project
are to map and clone the gene for DNA pol I, to identify subunits and accessory
proteins that influence DNA pol I activity and, ultimately, to identify the pro-
teins that regulate its activity on native DNA templates and to determine the
mechanism of their interactions.

Using purified DNA pol I, we have identified several proteins that stimulate its
synthetic activity. These include three different RNase H proteins, three dif-
ferent single-stranded DNA binding proteins and a DNA-dependent ATPase (ATPase
III) that possesses a helicase activity.

An aphidicolin-sensitive (aph s ) strain of yeast has been used to identify
several possible DNA pol I clones. Acquisition of the pol I gene in a high copy
number plasmid would presumably confer an aphidi col in-resistant phenotype to the
transformed aph cell. Analysis of aphidicol in-resistant transformants showed
that three different DNA sequences transformed cells to aphidicol in resistant.
All three produced slightly increased levels of pol I activity in crude
extracts. These DNA sequences are being subcloned to determine the ends of the
aphidicol in-resistance genes by deletion mapping. An internal fragment of the
aphidicolin-resistance gene will then be cloned into the yeast integration vec-
tor YIp5 for a gene disruption experiment to determine the requirement of the
intact gene for cell viability. The pol I gene is expected to be essential for
viabil ity .



225



PHS 6040 (Rev. 1/84)



GPO 914-918



Z01 ES 61040-01 LG



(^



Research Project:



Mature of Problem: Yeast DNA polymerase I (Pol I) is the replicative DMA poly-
merase^ Because DMA synthesis occurs only in the S-phase of the cell-division
cycle, Pol I activity must be controlled. In procaryotes, there are many pro-
tein factors that influence and regulate the activity of their replicative DMA
polymerases. In yeast, such control is also expected to involve many different
proteins. These proteins, the nature of their interactions with yeast Pol I and
how they regulate Pol I activity on native templates are the subjects of this
research project.

Objectives : The objective is to elucidate the elements affecting and regulating
yeast Pol I and to determine their mechanisms of actions.

Experimental Approach and Scientific Justification : Purified yeast Pol I is
being used to identify proteins that stimulate its synthetic activity on various
defined DMA templates such as activated calf thymus DNA or primed ssDMA.
Stimulation of synthetic activity was chosen because it is easily measured and
it is known that accessory proteins of procaryotic DNA polymerases have stimula-
tory effects on synthesis in vitro .

The extensively purified yeast Pol I will be used to detect and isolate proteins
that stimulate synthesis on defined templates. This approach has been useful in
identifying a yeast DNA primase required for Pol. I activity when unprimed ssDNA
is the template. In vivo , however, ssDNA resulting from replication fork move-
ment is likely to "b~e coated with single-stranded DNA binding proteins.
Therefore, the effect of yeast single-stranded DNA binding proteins on the reac-
tion catalyzed by DNA primase-Pol I on ssDNA will be explored. Templates
resembling a replication fork will be constructed and used to probe the require-
ments for simultaneous strand separation and strand elongation. Such a replica-
tion fork template may be obtained by annealing the (-) strand of bacteriophage
M13mpl8 RF DNA (linearized with a restriction enzyme) to circular (+) strand
M13mpl9 DNA. These two DNAs are complementary except for the 54-base multiple
cloning sites which are in opposite orientations. The annealed products will
therefore have a single-stranded gap on the (+) strand and a single-stranded
tail from the (-) strand.

Large amounts of purified Pol I enzyme are required to detect and characterize
the proteins that stimulate Pol I activity. The ever-present problem of pro-
teolysis in yeast and of low yields has made purifying Pol I very laborious. For
this and other reasons, we are attempting to clone the gene for yeast DNA Pol I
in order to overproduce it in and purify it from _E. col i . The method of cloning
utilizes the sensitivity of Pol I to inhibition by the drug aphidicolin. A
yeast mutant permeable to aphidicolin is unable to grow in the presence of the
drug. Acquisition of the gene for Pol I in a high copy number plasmid may have
a gene dosage effect that will allow the cells to grow in the presence of aphi-
dicolin. Thus, aphidicolin-resistant transformants will be isolated from a
yeast genomic library and the insert DNA conferring the aphidicolin resistance
will be characterized. At the same time, a yeast strain possessing a Pol I
enzyme mutated to aphidi col in-resi stance is required to confirm that any



226



Z01 ES 61040-01 LG

aphidicolin-resi stance genes code for Pol I. Transformation of the aphidi col in-
resistant mutant with a fragment of the Pol I gene in the integrating vector
YIp5 should result in the replacement of the aphidicol in-resistant Pol I gene
with a wild-type Pol I gene. The resulting transformant would then be
aphidicol in-sensitive and would contain an aphidicol in-sensitive Pol I activity.

Recent Accomplishments and Significance to Biomedical Research : Yeast Pol I has
been extensively purified, used tor raising antibodies and antibody-linked
Sepharose has been made to purify a large amount of Pol I enzyme in a short time
and with minimum effort. At the same time, several proteins which stimulate the
purified Pol I have been purified. These include three different single-
stranded DNA binding proteins (14,000, 20,000 and 38,000 daltons), RNase H
(68,000 daltons) and DNA-dependent ATPase (63,000 daltons). The nature of these
stimulation reactions has been investigated. This study strongly suggests that
RNase H and DNA-dependent ATPase are subunits or accessory proteins of Pol I.

Transformation of the aphidicolin-sensitive permeable mutant with a yeast geno-
mic DNA library in a high copy number shuttle vector resulted in several dif-
ferent transformants resistant to aphidicol in. Four plasmids, two of which
share common DNA sequences, contain the aphidicol in-resistant DNA sequences.
Deletion mapping to determine the ends of the aphidicol in-resistance genes will
be followed by gene disruption experiments to determine if these sequences are
required for cell viability. Any aphidicol in-resistance gene that is not essen-
tial is very unlikely to be the gene for DNA Pol I.

Plans for Future :

Enzymology of DNA Synthesis Involving DNA Pol I : (1) We will continue to iden-
tify protein factors that stimulate pol 1 synthetic activity. Previously
overlooked column fractions (such as ssDNA-cellulose flow-through fractions) may
contain stimulatory factors. More extensive purification of pol I fractions may
also reveal more proteins that interact with pol I. (2) Templates and reaction
conditions that approximate isolated steps during DNA synthesis will be deve-
loped. Primed and unprimed ssDNA will serve as templates for leading and lagging
strand DNA synthesis. The addition of ssDNA binding proteins and other pol I
accessory proteins may more faithfully mimic in vivo conditions. The enzymology
of DNA synthesis at a replication fork will be studied using a double-stranded
circular template with a single-stranded gap and a single-stranded tail. Pol I,
after filling in the gap, will encounter conditions similar to a replication
fork. The protein factors required for strand separation may then be determined
using this template.

Cloning and Genetics : (1) Once isolated, the mutant strain containing an aphi-
di col in-resistant DN~A pol I will be mapped to determine the locus for the pol I
gene. (2) After verifying that the pol I gene has been cloned, we will attempt
to have it expressed and over-produced in E_. col i . (3) The cloned pol I gene
will also be used for site-directed in vitro mutagenesis in order to isolate
temperature-sensitive pol I mutants. These jts mutants will be used to isolate
extragenic suppressors to detect and study proteins that interact with pol I in
vivo.



Publications
None



227



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE
NOTICE OF INTRAMURAL RESEARCH PROJECT



PROJECT NUMBER



Z01 ES 65021-13 LG



PERIOD COVERED

October 1, 1984 through September 30 ,



1985



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.)

Investigation of Germinal Mutation Induction in M1cj3



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation)

PI: F. M. Johnson Research Geneticist



Others:



L. C. Skow
M. L. Snell
Mar jo Smith
D. P. Lovell
S . E . Lewi s



Senior Staff Fellow
Bio. Lab. Technician
Postdoctoral Fellow
Statistician
Senior Geneticist



LG, NIEHS

LG, NIEHS
LG, NIEHS
LG, NIEHS
BIBRA
RTI



COOPERATING UNITS (if any)

Research Triangle Institute, Life Sciences Group, Research Triangle Park N C
British Industrial Biological Research Association, Carshalton Surrey
England '



LAB/BRANCH



Laboratory of Genetics



SECTION

Eukaryotic Gene Structure Section



INSTITUTE AND LOCATION

NIEHS, NIH, Research Triangle Pa r k , N orth Carol in a 27709



TOTAL MAN-YEARS:

5.0



PROFESSIONAL:

3.0



OTHER:



-2J3-



CHECK APPROPRIATE BOX(ES)

□ (a) Human subjects

□ (a1) Minors

□ (a2) Interviews



□ (b) Human tissues fp (c) Neither



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.)

The objective of this project is to detect natural and induced mutations in
mice for the purpose of providing understanding of the specific molecular
events involved in germinal mutation and the effects of these events on the
life, form and function of the mammalian organism. Results are relevant to
cases of human exposures to mutagens and the potential for increased risk
of genetic disease that may accompany mutagen exposure. The problem is
approached by detecting mutations at specific biochemical loci with
electrophoretic methods, by conducting characterization studies on the
mutant genes and gene products, and by examining the animals for expressed
physical abnormalities correlated with mutation rate increases and with
specific induced-mutant genotypes. The methods have led to successful
identification of more than 20 ethyl nitrosourea-induced mutants affecting
proteins such as malic enzyme, a hemoglobin and phosphoglucomutase, but
thereis little evidence to suggest that the induction of the detected
mutations has been accompanied by any increased incidence of adverse gene
expression. Perhaps the best example of a significant genetic disorder
(with homology in man) we have discovered by electrophoresis (a
P -thalassemia) was found to have originated spontaneously. Furthermore
our most recent analysis of skeletal variation shows a reduction in the'
frequency of naturally occurring variation as the statistically most
significant effect of mutagen treatment. The results raise questions as to
the extent hypotheses of human genetic risk based upon increased mutation
rates are indicative of elevated probabilities for significant genetic
damage.



228



PHS 6040 (Rev. 1/84)



GPO 91 4-918



Z01 ES 65021-13 LG

PROJECT DESCRIPTION

NATURE OF THE PROBLEM : Current perceptions of human genetic risk implicate
elevated mutation rates as a source of increased genetic disease in human
populations subjected to exposure to mutagenic agents in the environment.
Although many test methods provide for the efficient detection of mutations, the
mouse affords unique experimental opportunities to analyze efficiently the
effects of mutations in a mammal with many established homologies with man.
Therefore with mice, an effective means to detect mutations and to provide



Online LibraryNational Institute of Environmental Health ScienceAnnual report : National Institute of Environmental Health Sciences (Volume 1985) → online text (page 22 of 114)