doctor of philo~
TRANSCRIPT
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RECOMBINATIONAL ANO PACKAGING SIGNALS
IN
HERPES SIMPLEX VIRUS
(
,
DEOXYRIBONU~LEIC ACID
by
SUSANNAH' LOUISE VAIj,MUZA, BSc., MSc.
A Thesis~ .
Sub.itted to the School of Graduate ~tudies
in Partial Fulfil.ent of the Require.ent~
for the Degree
Doctor of PhilO~
June, 19B5 ©
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\.
\')
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RECOMBINATION AND PACKAGING OF HERPES SIMPLEX VIRUS DNA
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DOCTOR OF PHILOSOPHY (1985)
(Medical Sciences)
McMASTER UNIVERSITY
Hamilton, Ontario
TITLE: Recombinational and Packaging Signals in Herpes Simplex Virus
~eoxyribonucleic Acid
AUTHOR: Susannah Louise Var~uza B.Sc. IMcMaster University)
M.Sc. IMcMaster Universityl
SUPERVISOR Dr. J.R. Smiley
NUMBER OF PAGES: x iv, 267
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ABSTRACT
H~rpes Si.pl~x Virus DNA displiYs i number of unusual(
features which
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Ihave been the subj~ct of intense scrutiny in a number of laboratories.
The genole is cOlposed of two segments, each of which IS flanked by
inverted repeats. These seglents invert freely with respect to each
other generating equilolir qUintities of faur different isolers. This
phenolenon, called s~g.ent inversion, WiS reput~d to be the result //
site specific recomhinition lechanisl operating on the terlinir-;;peat,
the 'i' cequence, which is part of the inverted repeits flanking each
seglent. The 'a' sequence/was also ilplicated is the cleavage~packaging
signil utilized by the .vlrus to process viral DNA concatelers. The
underlying lechanisl of this pr~cess was believed to be a double strand
break it i sp~ciflc site ~~i!!~~a ho 'a' sequences. The- lodels of. HSV
laturitlon w~r~ deflcl~nt, however, in explaining several phenolena,
nal~ly the tendency of the 'i' sequence to accululate tandem iterations
of Itself, the isy••etric distribution of these tandem iterations to one
end of the geno.e, but not to the other, and the ibility of defective
geno.es, 'which do not have tinde.ly iterited 'a' sequences, at least
"..J
initiilly, to be efficiently packaged. have shown that the "a"
sequence actuilly contains two signals for cleavage/packaging, not one,
that the cleivage occurs it specific distances ~rol these signals, not
In specific sequences, and that the cleavage mechanism results in a
duplicition of the cleavage signal and flinking DNA~ Furthermore, I have
deter.ined that the 'a' sequence is aei i target for site specific
recombination, and thit therp !~ better evidence to support the idea
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that legment inYer~ion il acco.plilhed by a nu.ber of related, but
independent mechanis.l, including generalized reco.bination.
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Sacchetti for lending me her excellent
Acknowledgements
This work is the distillation at many hours at stimulating conversation,
.shared ideas, disappointing ~ailures, and soaring triumphs. For much at
this owe a debt of gratitude to my sURervisor, Jim Smiley. His
critical appraisal has been ·invaluable. Thanks are also due to Silvia------,
editorial~ skills. To my
~ colleagues whose unqualified moral support helped me aver the rough
spats - Marnie Halpern and Eddy Littler in particular - I offer thanks
with gratitude and aftection. Finally, wards are inadequate to express
my debt to my husband, Petr Varmuza, whose boundless patience and sense
at humour have been the staying torces in ~y life •
.
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This Thesis is Dedicated
to the Memory of
my stepfather
Samuel Burke
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TABLE OF CONTENTS
INTRODUCTION
Recombination
Recombination During Meiolis and Mitosis in Eucaryotes. .Recombination in Procaryotes
Site Specific Recombination
Bacteriophage Recombination Systems
Special Recombination Mechanisms
Herpes Simple, Virus
General.,
Structure of HSV DNA
MATERIALS AND METHODS
RESULTS
Gene Conversion and/or Recombination ,in the "c" Inverted
Repeats
Sequence Identity of "a" Is Not Obligatory
Sequence Analyssis of the KOS Joint
Analysis of the Packaging Signal in Smal F
Recombinational Signals in Sma I F
Sequence Analysis of JK Termini
Sequence and Restriction Analysis of TK Stepladder
Cis-Acting Site Specific Recombination Signals Do Not
Map Within "a"
(v i i )
,
,
2
B
10
12
16
19
20
52
73
88
122
136
153
163
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DISCUSSION.'
"
~
Conclusions Drawn From This Work
Sequence Iden-tity of the Inverted "c" Repeats is not
Obligatory
•Heterologous "a" Se~uences Are Tolerated
No Site Specifying a Cis Acting Signal for SIte Specific
Recombination Maps Within the "a" Sequence
192
193
195
197
Both T~rmirii of Defec!ive Genomes Contain "a" Sequences 199
The Cleavage Signal Sequence Resides Within a Subfragment
of. ·"a" Which .Does Not Con·tain the Cleavage Site\
Cleavage Occurs at a Specific Distance from the Cleavage
Signal
200
201
There are Probably Two Cleavage Signals
The "." Sequence Amplification is a Direct Consequence
of the CJeavage Reaction
Intermolecular Ligation May Occur in HSV DNA
Models For Cleavage/Packaging of HSV DNA
l 202
203
204
Old Data 206
Model 207
Model II 210
Asymmetric Distribution of Tandem "a" Sequ~nces 211
3'OH Protruding Ends on Viral DNA 216
"a" S~quence Stereotypic Amplification and Reduction 218
Multiple Cleavage Signals in HSV and Other Herpesviruses 222/
Relationship Between Cleavage M~chanisms and Segment Inversion 226
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Ta~le of Contents ecntd.
APPENDIX - PLASMID MAPS
REFERENCES'
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•
~
• LIST OF ABBREVIATIONS
~erpes Simple, VlrUS
'Herpes Simple, Virus Type 1
Herpes Simplex Virus Typ e Strain KOS
Herpes Simplex Virus Type Strain KOS.
Herpes Simplex Virus Type Strain F
Herpes Simplex Virus Type ·1 Strain 17
Herpes Simplex Virus Type I Strain USA-B•
Herpes Simplex Virus Type
Herpes Simplex Virus Type 2
Human Cytomegal ovi rus
Epstein-Barr Virus
Varicella Z~ster Virus
Simian Virus 40
Thymidine Kinase'I,
Thymidine Kinase Deficient
Adenosine triphosphate
Deoxyguanosine monophosphate
Deoxyadenosine monophosphate
Deoxythymidine monophosphate,
De_oJlycytoslne monophosphate
base pair
kilobase pair (bp x 103)
kilobase pair
kilodalton
(x)
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Strain Justin
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Abbreviatioos contd.
MOl
pfu
32p
DNA
Multiplicity of Infection
plaque forming unit
radioisotopic phosphorus
Deoxyribonucle~c Acid
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. .
(x i )
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List of Figures and Tables contd.
Fig. 24: KOS joint sequence
Fig. 25: Map of Test Fragments
Fig. 26: Blot of. Defective DNA
Fig. 27: pcaDI Map
Fig. 2B: Blot of caDI Defectives
Fig. 29: Marker Rescue Screening Gel
Fig. 30: PvuII, EcoRI Digests of Insertion Mutant Viruses
Fig. 31: Pvu[I, Sma I Digests of KOSF10, TK blot
Fig. 32: Two Models of. "a" Sequence Amplification
Fig. 33: Smal Digest KOSF10, Smal F blot
Fig. 34: MaR of KOSF10 insert
Fig. 35: pTKLl3 Sequence Gels
'.Fig. 36: Sequences pTKLl3, pRIS3, pSBFI4
Fig. 37: pRIS3 Sequence Gels
Fig. 3B: Colony Hybridization
Fig. 3~: fstI, PstI/SmaI, and BamHI Digests Cloned Second Steps
Fig. 40: Maps of pRIaa6, pRlaa3, and pRIaa35
Table 2: Rest~iction Analysis of Second Step Clones
Fig. 41: Sequence Gels pRlaa6 BamHI Fragment, 5' and 3' TK
Fig. 42: Sequence Gels pRIaa6 SstI I Fra,9ment
Fig. 43: Sequence Gels pRIaa6 and pRlaa3 Sstll Fragments
Fig. 44: Sequences of pRlaa6 and pRlaa3
Fig. 45: Sequencing Strategy and Results-Table 3: Restriction Analysis of KOSFIO and KOSA23
(xiii)
121
125
129
132
134
, 139
14 1
144
147
149
151
159
162
166
16B
171
172
175
177
179
181
184
187
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list 0/ Figures and Tables contd.
Fig. 46: Deletions Produce Segments Without Origins
Fig. 47: Cleavage/Packaging Models I and I I
Fig. 48: Models of Asymmetric Distribution 0/ II a II
---,-Fig. 49: Clonal Analysis of KOSF10
Fig. 50: Terminal Sequences From Various Herpesviruses
((x i v)
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209
214
220
224
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INTROOUCT~~/
When Gregor Mendel's brilliant insights emerged from obscurity
at the beginning of this century, bi?logy moved firmly Into the
scientifIc realm. The mechanIsm of heredity could be seen as a process
that obeyed Newtonian-like laws, and could therefore be studIed with
scientific approaches based on experimentation of cause and effect.
The r.pid expansion of knowledge in the field of molecular
biology has produced a set of fairly flexible rules by which most
organisms seem to conduct their inner affairs. say flexible because
for every "rule" there are usually exceptions of one form or another
(Nature is something of an opportunist). Indeed, even Mendel, whose
~"~Manded too perfect by some (Whitehouse, 1977),
apparently fai ed to make observation of one basic' feature of heredIty
•which may eventually hold the key to an understandIng of larger
experIments
mysterIes such as evolution. That feature is the ordered linkage of
genes within a chromosome. It was discovered by early geneticists
attempting to repeat Mendel's experiments wIth other organisms and
expanded lIbraries of 'characters" or genes. Even after linkage was
established as a general feature of heredity, however, exceptions ~gain
were found toethe new' "rule'. These were discovered to be the result of
a fairly ubi~uitous process, known as recombination, in which alleles
of different genes appeared to move from one chromosome to another.
This ability of genetic material to ~ove around and stIll maintaIn -
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order has fasCInated genetICIsts for decades. In:- the process of~.
unravelling the mysterIes of genetIc re.comb.i~ation,,,;jnvestigators have
discovered some amazing tricks devise~Jy natu~e to preserve and expand
genetic inffrmation.
B~SQm~lQ~~i~Q j •
B~sQm~iQ~~iQQ ~~~iQg ~~iQ!i! ~Q~ ~i~Q!i! lQ ~~S~~YQ~~!'
Mendel '5 experf;ents measured the random assortment of unl inked
genes during sexual reproductio~. Later work showed that the fre~uency
of co-segregation of two lInked markers reflected (inversely) the
distance between them; close markers frequently segregated together,
while distant markers segregated separately. This property allowed
geneticists to order, or map, different genes along a chromosome. Thus,
recombination became a tool as well as a phenomenon:
Sexually reproducing org~nisms recombine their chromosomes
during meiosis. This allows even greater mi.ing of genetic material
than that afforded by rando. assort~ent of chromosomes, presumably to
ensure variation within the population as a hedge against genetic
catastrophe. Recombination is believed to occur at an early stage of
meiosis, after DNA replication. The chromosomes form a structure called
a "synaptonemal complex" characterized by the synapsis of homologous
chromosomes along a po'lymeric protein backb9ne. Shortly after it .is
formed, chiasmata can be observed at various sites along the paIred
chromosomes. Chiasmata have been positively correlated WIth chromosome
cross-overs and with recombination frequency. For example, male
Drosophila and the anthers of Frltillaria Japonlca do not exhIbit
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chlasmata during melOSlS and do not recombIne theIr genes ICatcheslde,
1977; Henderson, 1970).
Nature has klndby supplied geneticists with wonderful tools to
study recombination in the form of fungi and yeasts. These organisms
retain the property of independent growth of both haploid and diploId
forms, although in some fungi the diploid stage is s~rt and highly
specialized. Moreover, in some fungi, the meiotic products (the spores)
_are conveniently ordered in the ascus according to the planes of
division of the original precursor cell. Many fungi undergo a
post-meiotic mitosis, yielding 8 spores from-a. single diploid cell. The
eight spores are thus analogous to the eight strands of DNA within the
interacting chromosomes at the diplotene stage of meiosis during which
chiasmata form, and, by deductive re~soning, .durlng which recombInation
takes place. ThlS allows the Investigator to follow the fate of all the
participating chromatids during meiosis (reviewed in Catcheside, 1977;
St ah 1 , 1979). This is an important concept in the study "of
recombination because it ensures an "unbiased" sampling of the
population. Analysis of ascospores, referred to in the trade as tetrad
analysis, has provided biology with some basic ground rules of the
behaviour ·of recombining chromosomes. First, distant markers almost
always recombine reciprocally, ie. there is no net loss of genetic
material during recombination, only rearrangement. Second, this rule
does not apply to very close markers. Tetrad analysis of ordered asci
from Neurospora crassa and Sordaria fimicola occasionally yields
ascospores with asymmetrically distributed genes. These are referred to
as 116:2 11" "5:3", and lla.berrant 4:4" tetrads, and were originally
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dIscovered durIng tetrad analySIS of spores produced by a cross of two
different straIns of Neurospora which dIffered at a locus defining
spore colour. Most ascospores contained 4 coloured (wild type - wt):4
colourless (mutant - m) spores, indicating that no rearrangement of the
spore colour gene with respect to the centromere occurred. Some con-
tal ned 2 wt:2 m:2 wt:2m spores, indicating that the spore colour gene
had been exchanged between the two internal chromatids. The beauty of
ordered asci is that the products reflect exchanges between individual
chromatids within the division plane. Thus, a 2:4:2 arrangement
reflects recombination between non-adjacent chromatids withIn the
division plane. Rare ascopores with' 6:2 (or 2:6) and 5:3 (or 3.5)
arrangements were also detected. These asci were interpreted as the
result of nonreciprocal exchange of genetIc informatIon within the
spore colour locus. The best mechanistic explanation of the phenomenon
so far is the repair of mismatched heterodupleles formed during r~eom-
bination, or, gene conversion.
The observation
patterns exhibited by
of gene
~fferent
conversion
organisms,
durIng meiosIs, and the
allowed investigators to
inf~r mechanisms of recombination which encompassed this unusual, trait.
All of these models begin with the assumptIon that gene conversion is a
consequence of the mechanism employed by the cell to recombine its
chromosomes. This interpretation is based on the observation that
approximately 50X of the time conversion is assoc~ated with recombin
ation of £~~Q~iQg ~~~~~~§.
Models of recombination are themselves subject to evolution, as
data from different organisms becomes avaIlable. The first widely known
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