ULTRAVIOLET LIGHT INDUCED RECOVERY TN ESCHERICHIA CO LI
OF RADIATION DAMAGED BACTERIOPHAGE DEOXYRIBONUCLEIC ACID

By

JOHN RANDALL SILBER

A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF

THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA
1977

ACKNOWLEDGEMENTS

I am very grateful for the guidance and encouragement given to
me by Dr. P. M. Achey during my tenure as a graduate student. I
would also like to thank Dr. L. 0. Ingram and Dr. K. P. Boyce for
their many helpful comments and discussions. I am particularly
indebted to Dr. Boyce for taking me under his wing while Dr. Achey
was on sabbatical leave.

I must also acknowledge the technical assistance given to me
by Barbara Hopp , Suzanne Vander Griend, David Grier and Dorcas
Arnold. Their competent aid made it possible to explore many more
facets of no' research project than I could have done by myself.
The constant wit and good cheer expressed by these persons added
inmesurabJy to the pleasure of working in the 1 ibr>r".t' ry .

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ii

TABLE OF CONTENTS iii

LIST OF TABLES v

LIST OF FIGURES vi

ABSTRACT vii

INTRODUCTION ... 1

UV Damage in DNA 1

Repair of Dimers 1

Photoreactivation 1

Excision Repair ?

Replication on Damaged Templates 3

Post-Replication Repair h

Inducible Recovery of UV Damage k

SOS Hypothesis k

SOS Repair 6

Gamma Ray Action on DNA 7

Use of Scavengers in the Study of Indirect Action .... 8

Gamma Ray Induced Damage in DNA 9

Repair of Gamma Ray Damage 10

Strand Break Repair 10

Repair of Base Damage 11

Inducible Recovery of Gamma Ray DNA Damage 11

Object of this Study and Experimental Approach ]_?

MATERIALS AND METHODS 13

Bacterial Strains 13

Bacterial Growth Conditions 13

0X17^ DNA Preparation 13

0X17^ DNA UV Irradiation 15

0X17*4 DNA Gamma Irradiations 15

UV Irradiation and Induction of E. coli 17

Calcium Treatment of E. Coli 18

Transfection and Biological Assay of 0X17!i DNA 18

Evaluation of UV Inducible Recovery Ability 19

RESULTS 2k

UV Induced Recovery of UV Irradiated DNA 2h

UV Induced Recovery of Gamma Irradiated DMA ho

DISCUSSION 60

BIBLIOGRAPHY 72

BIOGRAPHICAL SKETCH 80

LIST OF TABLES

TABLE I

Bacterial Strains 1*4

TABLE IT

Bimolecular Rate Constants of the Primary Water

Radiolysis Radicals with the Radical Scavengers Used in

This Study l6

TABLE III

Maximum Recovery Efficiencies and Their UV Eliciting Doses . . ?9

TABLE IV

Efficiency cf UV Induced Repair of 0X17*4

DNA in Three Excision Repair Defective Mutants Ul

TABLE V

Efficiencies of UV induced Recovery of 0X17*4

DNA Gamma Irradiated Under Various Conditions 58

TABLE VI

UV Sensitivities of Single Stranded and Replicative

Form 0X17*4 DNA Molecules in Various Unirradiated Hosts . . . 6l

TABLE VII

Dimer Yields at D37 for Single Stranded and

Replicative form 0X17 h DNA in Excision Repair Deficient Hosts . 63

LIST OF FIGURES

FIGURE 1 20

FIGURE 2 22

FIGURE 3 25

FIGURE k 21

FIGURE 5 29

FIGURE 6 31

FIGURE 7 33

FIGURE 8 35

FIGURE 9 37

FIGURE 10 !+2

FIGURE 11 UU

FIGURE 12 k6

FIGURE 13 ItH

FIGURE ih 50

FIGURE 15 52

FIGURE 16 5I4

FIGURE 17 56

Abstract of Dissertation Presented to the Graduate Council

of the University of Florida in Partial Fulfillment of the

Requirements for the Degree of Doctor of Philosophy

ULTRAVIOLET LIGHT INDUCED RECOVERY IN ESCHERICHIA COLI
OF RADIATION DAMAGED BACTERIOPHAGE DEOXYRIBONUCLEIC ACID

By

John Randall Silber
December 1977

Chairman: P. M. Achey

Major Department: Microbiology and Cell Science

The ability of ultraviolet light (UV) 0X17^ single stranded and

replicative form DNA molecules to produce whole phage when transfected

into UV irradiated, calcium treated E. coli K12 hosts was investigated.

When IT/ irradiated single and replicative form 0X1TU DNA molecules were

transfected into UV irradiated wild type hosts, an enhanced survival of the

phage producing ability of the DNA was observed over that seen when the

transfection was performed with unirradiated wild type hosts. A similar

increase in survival was also found for both types of 0X17;t DNA molecules

when they were transfected into E. coli deficient in excision repair

( uvrA- , uvrB- or uvrC- ) or recombinational repair (recB- or recB- recC-).

No enhanced survival was found with recA- or lex/V- E . coli hosts . The

level of the increased phage producing ability for single stranded 0X17^

DNA was 1.3 to 1.7 times greater than that for the replicative form DNA

in all genetic backgrounds which showed enhanced survival.

These results suggest the existence of a UV inducible recovery

system which participates in the recovery of UV irradiated 0X17^ DNA.

This recovery system in dependent upon recA and I ox A regulated function.';
but is independent of excision and recombi national repair. The
common genetic requirements for the increased survival of both forms of
0X11 h DNA suggest a common mechanism of recovery for both. The greater
survival of the single stranded molecule indicates that it is more sus-
ceptible to inducible recovery than the replicative form molecule which
may be due to the physical differences between the two.

The ability of gamma irradiated 0X17'* single stranded and replicative
form DNA to produce whole phage when transfected into a UV irradiated,
calcium treated E. coli wild type host was also investigated. By adding
appropriate radical scavengers to aqueous solutions of the DNA, it was
possible to specify which of the water radiolysis radicals was interact-
ing with the DNA. The maximal enhancement of phage producing ability for
both types of DNA was observed under conditions which removed the hydroxyl
radical allowing the hydrogen radical and the solvated electron to pre-
dominate. Scavenging conditions which removed the hydrogen radical and
solvated electron as well as the hydroxyl radical resulted in an enhance-
ment of survival only half as large as the maximum. The same was found
to be true for scavenging conditions which removed the hydrogen radical
and solvated electron only.

These results demonstrate the existence of a UV inducible repair
system which mediates in the recovery' of 0X17*1 DNA from gamma ray damage.
The mechanism of recovery here could possibly be the same as that which
affected the UV inducible recovery of UV irradiated 0X17 '4 DNA. From
these results, it can also be concluded that the hydrogen radical and
the solvated electron produce a type of damage that is more susceptible
to inducible recovery than the hydroxyl radical. The hydroxyl radical

appears to produce two classes of damage in both the single stranded and
replicative form DNA molecules. The first is susceptible to inducible
recovery while the second is refractory to it. This second class of
damage may inhibit recovery from damage caused by the hydrogen radical
and the solvatcd electron.

INTRODUCTION

The biological functions of deoxyribonucleic acid (DIM) are very
sensitive to ultraviolet (UV) and ionizing radiation. These radiations
produce physico-chemical alterations in DNA which may result in death
or mutations. There are enzymatic repair systems which remove the
biological damage. A brief discussion of radiation damage and its
-repair in the bacterium Escherichia coli follows. This will provide
background information pertinent to this dissertation which is directed
toward characterizing one of these repair systems.

UV Damage in DNA

UV radiation produces a variety of alterations in DNA molecules.
Such damage includes cyclobutane dipyrimid i ne dimers (pyrimidine dimers
or dimers), pyrimidine hydrates, pyrimidine adducts and DNA-protein cross-
links (hh, 77). Most of the lethality and mutagenesis caused by UV can
be attributed to the presence of dimers rather than other photoproducts
in DNA. The basis for this statement is the fact that photoreactivation,
which acts specifically on pyrimidine dimers, can remove 50 to 90$ of
the dimers from bacterial DNA which results in a corresponding decrease
in the biological effect of UV (hh) . Thus, in the following discussion
of UV repair systems, only the repair of dimers will be considered.

Repair of Dimers

Fhotoreactivation
Photoreactivation results in the in situ splitting of pyrimidine
dimers (68). The mechanism of this repair employs an enzyme which

binds specifically to pyrimidinc dimers. When exposed to light (300-
U00 nra) , the enzyme-dimer complex is activated and the dimer is split
without breaking the phosphodiester backbone of the DNA molecule. In
concept, photoreactivation should not be error prone which is confirmed
by the observation that it is at least 90$ error free (92).

Excision Repair

A second repair pathway can remove diners in the absence of light.
In E. coli excision repair breaks the phosphodiester backbone in the
tide and then closes the resulting gap by de novo DNA synthesis (10, 75).

The incision step is absent in uvrA and uvrB mutants (kO) . These
mutants lack E. coli eorrendonuclease TIsan endonuclease which
specifically makes a nick on the 5' side of pyrimidine dimers (ll, 31).
The nick with 5' phosphoryl and 3' hydroxy! termini can be sealed by
polynucleotide ligase. Premature closure of this nick, which aborts
excision repair is observed in uvrC mutants (13, *l7). Also, uvrC ligts
mutants contain more nicks in their DNA after tJV irradiation than uvrC
single mutants (7h) . These results suggest that the uvrC gene product pro-
tects the incision from being resealed by ligase.

In wild type E. coliT excision of the dimer is carried out by DNA
polymerase I (19, 30, I48). Beginning at the '}' phosphoryl end of the
nick, DNA polymerase I, acting as a 5' to 3' exonuclease, degrades a
short segment (approximately 30 nucleotides) of the DNA strand con-
taining the dimer as oligonucleotides. The gap resulting from this
excision is filled concurrently by DNA polymerase I using the comple-
mentary strand as a template. In the absence of DNA polymerase I, DNA
polymerase II or III can perform the exci sion-resynthesis step (55, 82).

The final step of excision repair is the rejoining of the phosphodiester
backbone between the preexisting, undamaged DNA strand and the newly
synthesized DNA by ligase.

An alternative type of excision repair termed "long patch" (as
opposed to "short patch" described above) has been found in p_olA mutants
(31). The excision step of long patch repair removes at least 100 times
as many nucleotides as the short repair excision step. Long patch
repair is growth medium dependent, and is inhibited by chloramphenicol.
It requires the uvr A , recA, recB, lexA ant] p_olC gene products (95). The
common requirement for uyrA+ suggest that long patch repair and short
patch repair share the same incision step but differ in their excision-
resynthesis steps.

Replication on Damaged Templates
Bacterial DNA synthesized immediately after UV irradiation is
discontinuous (67, ?6). The post-irradiation molecular weight of the
newly synthesized DNA approximates the value calculated, for the inter-
dimer distance after a given UV dose. It has been proponed that dimers
block the progress of bacterial DNA replication (67). They do not in-
hibit subsequent reinitiation beyond the dimer at a new initiation site.
Thus, it is possible that gaps up to 1,000 nucleotides long may be left
in the newly synthesized DNA after irradiation (1»3). These gaps have
5' phosphoryl and 3' hydroxy! termini. They can be closed by DNA
polymerase I and ligase but only after the dimer has been removed by
photoreactivation. This suggests that dimers can block the polymerizing
activity of DNA polymerase I (12).

Post-replication Repair
The daughter strand gaps caused by replication of IIV damaged DNA
do not persist indefinitely. Instead, the gaps are filled and sealed
so that the molecular weight of the newly synthesized DNA becomes the
same as that of DNA synthesized on unirradiated template. This process
is called post-replication repair (67).

Post-replication repair occurs in E. coli by a variety of pathways
(65, 72, 97). All post-replication repair pathways are recA dependent
which suggests a recombinational mechanism (U?, 76). Roth the recB,
recC and recF recombination pathways have been shown to contribute
to post-replication repair (65, 97). recF mutants show a greater in-
hibition of post-replication repair than recB recC mutants, even though
both have the same sensitivity to UV (65). This indicates that the recB
recC product, exonuclease V, contributes to recovery from UV pre-
dominantly by some action unrelated to post-replication repair.

There are at least two post-replication repair pathways which are
controlled by gene products that are not involved in recombination. One
is dependent upon lexA while the other requires uvrD (29, 72, 96, 97).
Apparently these and the recombination dependent pathways are arranged
into a recA dependent, multi-branched post-replication repair pathway
in wild type E. coli (72, 97).

Inducible Recovery of UV Damage

SOS Hypothesis

In E. coli there are a number of UV inducible phenomena whicli
contribute to the recovery of UV damage. These include:

1. W-reaetivation of UV irradiated bacteriophage (21, 22, 36, )»9 , 90)

2. Error prone repair mutagenesis of bacteria and their phage
(6, 22, 71, 90, 93).

3. UV inducible cell division delay and fi lamentation (6l).

!;. Induction of a pathway of post-replication repair (.13, 71, 73,

97).

5. Inhibition of post-irradiation DMA degradation (53).

6. Cessation of respiration following irradiation (79).

The induction of all of these phenomena is recA and lexA dependent
and is sensitive to chloramphenicol indicating that de novo protein
synthesis is required for their expression ( 9'1 ) • A variety of agents
other than UV such as ionizing radiation, thymine starvation, naladixic
acid and non-permissive temperatures in dna b mutants will lead to
induction (9'0- Finally, all of these phenomena occur in excision
repair and post-replication repair deficient (other than recA or lexA)
cells (9^). This indicates that inducible recovery from UV damage
proceeds by mechanisms unique from the constitutive repair systems.

The GOG hypothesis of Radman is an attempt to find a common basis
for the participation of these phenomena in UV repair (60, 6l ) . The
hypothesis states that treatments which abruptly interrupt DNA replica-
tion triggers the coordinate expression and de novo synthesis of a set
of gene products which function by various mechanisms in the repair of
UV damage. The derepression of these gene products is jointly regulated
by the recA and lexA genes. Radman also proposes that the molecular
mechanism of some of the inducible repair phenomena involves an error
prone DNA polymerase. The repair functions involving such a hypothetical
polymerase are referred to as SOS repair.

SOS Repair
Biological evidence provided by experiments with single stranded
phage such as 0X17^ supports the error prone DMA polymerase mechanism
of SOS repair. The heart of the biological evidence lies in the fact
that during replication these phage must first convert their single
stranded genome into a double stranded repLicative form (50). UV
induced dimers inhibit this conversion by blocking DMA replication at
the point of the radiation lesion and prevent the reinitiation of
replication beyond it (5'*, 62, 63). This renders the DM molecule
biologically inactive. Increased survival with a concomitant increase
in mutagenesis (W-reactivation) has been observed when UV irradiated
single stranded bacteriophage infect wild type E. coll host which have
also been UV irradiated so that they express SOS repair (6, 21, 58, 83)
This can only occur if the damaged single stranded genome is converted
into its double stranded form.

There is also biochemical evidence supporting the error prone
DNA polymerase mechanism of SOS repair. When UV irradiated 0X1 7^4 DNA
or oligodeoxyadenylic acid primed polydeoxythymidylic acid have been
used as templates for DNA synthesis in a crude extract of UV irradiated
E. cold, both are converted to full length, intact, double stranded
forms (62). A high level of misincorporation of nucleotides also has
been observed in the experiments utilizing the polydeoxythymidylic acid
as a template (62).

From this Radman deduces the error prone DNA polymerase mechanism
for SOS repair. To account for these observations, the UV induced
polymerase must be able to polymerize DNA on a damaged template. Mis-
incorporation is presumably caused by the damaged nucleotides in the
template either being non-instructive or coding for a non-complementary

nucleotide (60, hi, 62). Alternative explanations surest that
misincorporation may be an inherent property of the induced polymerase
itself. The polymerase may be faulty in selecting complementary
nucleotides or it may be defective in a proof reading exonuclease
function similar to that of DNA polymerase I which can excise a mis-
matched nucleotide from a newly synthesized DNA strand (50, 52).
These alternative explanations are supported by the observation of
a mutator effect of SOS repair on unirradiated DNA (9k).

Treatments which elicit the inducible repair phenomena to date
have not been shown to result in the appearance of a new polymerase
with the properties necessary to accomodate the SOS repair mechanism
(9!0. If it is assumed that SOS repair includes W-reactivation of
double stranded bacteriophage and error prone repair mutagenesis of
bacteria, some speculations can be made as to the involvement of DNA
polymerases I and III i„ SOS repair. Polymerase I can be eliminated as
a participant since £olA mutants support W-reactivation of lambda phage
and UV induced bacterial mutagenesis (15, 03). It has been shown,
however, that dnaEts mutants do not show UV induced mutagenesis at non-
permissive temperatures. This implicates DNA polymerase III as having
a role in SOS repair (13). One speculation is that one of the inducible
recovery phenomena is the synthesis of a modifying factor which causes
DNA polymerase III to become an error prone DNA polymerase (9)4).

Gamma Ray Action on DNA

Gamma rays interact with aqueous solutions of DNA molecules in two
manners. In the first, gamma rays cause ionizations directly in the DNA
molecule resulting in physico-chemical alteration. This manner of

interaction is called direct action (?h). 'Hie second mode of interaction
is called indirect action (2k). The gamma rays in this case interact
with the water molecules of the solution causing them to become excited
or ionized. By a series of secondary reactions, the excited or ionized
water molecules break down to form a set of free radicals, the water
radiolysis radicals. The primary water radiolysis radicals are the
hydrogen radical, the solvated electron and the hydroxy! radical (26).
All of these radicals are very reactive with DM causing either electron
additions or abstractions which can alter the molecule (9, 18).

Use of Scavengers in the Study of Indirect Action

It is possible to remove one or more of the water radiolysis radicals
from a gamma irradiated aqueous solution of DNA molecules by the use of
radical scavengers. The best scavengers are chemical species which
have high bimolecular rate constants for their reaction with a given
radical and form reaction products which are innocuous to DNA. Thus,
certain radiolysis radicals may be prevented from reacting with the DNA
so that the effects of the remaining ones may be more clearly delineated.

By using the appropriate radical scavengers, it has been possible
to determine the contribution of the water radiolysis radicals to the
biological inactivation of DNA. Work with the single stranded DNA
molecule of 0X17^ has indicated that at least 95$ of the gamma ray
inactivation of its biological activity is due to hydroxyl radicals (8).
Other experiments with the same molecule hove suggested that the solvated
electron does not contribute at all to inactivation (9). A larpe increase
in the survival of the biological activity of 0X17!4 replicative form DNA
is seen when it is gamma irradiated under conditions which effectively

scavenge the hydroxy! radical while a much smaller increase in pro-
tection is seen when the hydrogen radical and the solvated electron
are scavenged (2). All of these observations support the contention
that the hydroxy! radical is the major radical species contributing
to the biological inactivation of DM in aqueous solution.

Gamma Ray Induced Damage in DMA

Gamma ray damage may be put into two broad groups; strand breaks
(single and double) and base damage. A strand break is a disruption
of the phosphodiester backbone of the DNA molecule. In vitro studies
with aqueous solutions of bacteriophage DMA molecules have shown that
the hydroxy! radical is almost exclusively responsible for producing
strand breaks (2). Strand breaks are detected in the DNA gamma
irradiated E. coli, but the complex chemical environment surrounding
cellular DNA makes it impossible to attribute the production of strand
breaks in this instance to any one of the water radiolysis radicals.

In DNA all four of the purine and pyrimidine bases can be altered
by gamma irradiation (l8, 38, 69). Unfortunately, the doses required
to produce these alterations are far greater than those needed to produce
a physiological effect (9). There are, however, two types of base
damage in irradiated DNA molecules which can be detected in the phy-
siological dose range. The first are alterations of thymine which pro-
duce a class of damage products of the 5, 6 dihydroxy l-dihydrothymine
type (17, 18). The other type of base damage is that which produces
apurinic and apyrimidinic sites in DNA (iR, 51). The hydroxyl radical
has been implicated in the formation of the 5, 6 hydroxyl-dihydrothymine
type of damage (6*4, 66).

10

Some indication of the relative contribution to lethality of the
two types of gamma ray damage has been determined from experiments with
bacteriophage DNA (9, 18). Almost 90% of lethality in gamma irradiated
0X17^ replicative form DNA and PM2 DNA has been attributed to base
damage (87). On the other hand, 50 to 95$ of the biological inactivation
of $XIjk single strand DNA is due to strand breaks (7, 9). Thus,
double stranded DNA is more susceptible to biological inactivation by
base damage than single stranded DNA even though the efficiency of
inducing base damage in single and double stranded DNA is very similar
(18). The chemical nature of the lethal base damage is not known and
the role of base damage in the in vivo inactivation of living cells has
yet to be determined (l8).

He pair of Gamma Ray Damage

Strand Break Repair
The repair of single strand breaks in E. coli appears to proceed
by several mechanisms dependent upon the chemical nature of the break
and post-irradiation conditions. It has been suggested that the repair
of single strand breaks produced under anoxic conditions may be mediated
by polynucleotide ligase (8)4, 85, 86). Under aerobic conditions most
strand breaks produced in polA+ E. coli are repaired in less than one
minute after irradiation while polA mutants show little repair under the
same conditions (8k). This type of DNA polymerase I dependent strand
break repair is absent if the irradiation is done under anoxic con-
ditions. Breaks generated under these conditions are closed very slowly
and only during incubation at 37° C in the presence of nutrients.
This slow type of strand break repair is missing in recombination de-
ficient E. coli (1*6, 81*).

11

In E. co 1 L no repair of double strand breaks nan boon found, and
the killing efficiency of such damage is assumed to be 100% ('45).
Repair of Base Damage

Removal of the 5, 6 dihydroxyl-dihydrothymine type of base damage
has been carried out in vitro using crude extracts of _E. coli (l?).
The involvement of the 5' to 3' exonuclease activity of DNA polymerase
T in the removal of the damage and of polynucleotide 1 igase in the last
sealing step has been demonstrated (33, 3'* ) - The uvtA yvrB endonuclease ,
correndonuclease II, has been shown not to be involved in the repair
of this damage ( l6 , 35). An endonuclease has been discovered in E. coli
which specifically acts on X-irradiated DNA (78). Whether or not this
endonuclease plays a role in the repair of 5, 6 dihydroxyl-dihydro-
thymine type of base damage has not been estab] ished.

An endonuclease which hydrolyzes the phosphodiester bond near
apurinic sites has been found in E. coli (88, 89). This hydrolysis
represents the first step in the repair of such sites. The in vitro
incubation of DNA with this endonuclease along with DM polymerase I and
polynucleotide ligase results in the removal of apurinic sites (88).

Inducible Recovery of Gamma Ray DNA Damage

There are several reports of SOS type repair of X or gamma ray
damaged DNA. X-irradiated 0R, a single stranded bacteriophage showed
W-reactivation when infected into U~V or X-irradiated E. coli hosts (58).
However, similar experiments with X-irradiated lambda phage resulted
in no W-reactivation (36). Increased survival of the colony forming
ability of gamma irradiated E. coli which had previously received a
smaller UV or gamma ray dose has been reported (59)- This indicates that
SOS repair plays a role in the repair of gamma ray damaged cellular DNA.

12

Object of thin Study and Experimental Approach

The objective of this dissertation is to characterize further
the UV induced recovery in E. coli of radiation damaged bacteriophage
DNA. The specific goals of this study are:

1. Comparison of the efficiency of UV induced recovery of UV
irradiated single and double stranded DNA.

2. Determination of the relationship of UV induced recovery of UV
irradiated single and double stranded DNA to excision repair
and recombination.

3. Determination of the role of the various gamma ray water
radiolysis radicals in producing damage in single and double
stranded DNA susceptible to UV inducible recovery.

k. Comparison of the UV induced recovery of UV irradiated versus

gamma irradiated DNA molecules.
5. Evaluation of whether or not the proposed mechanism of SOS re-
pair will explain the UV induced recovery of both single and
double stranded DNA.
The experimental approach to meeting these objectives employs a
purified 0X17^ DNA-calcium treated E. coli transfection system. Calcium
treated E. coli hosts which have or have not been induced by UV irradia-
tion are transfected with UV or gamma irradiated 0X17*1 single stranded
and replicative form DNA. The ability of the transfected, irradiated
DNA molecules to produce whole phage is then measured by an infective
center plaque assay. Inducible recovery is indicated by a greater
survival of phage producing ability when the irradiated DNA is trans-
fected into UV irradiated hosts as opposed to unirradiated hosts.

MATERIALS AND METHODS

Bacterial Strains

The genotypes for all strains and their references appear in Table
I. All strains are E. coli K12 and were derived from AB1157 with
the exception of HFl»7ll| which is a C strain E. coli. The nomenclature
is that proposed by Demerec etal (23). The relevant genetic markers
affecting radiation repair were checked by UV sensitivity.

Bacterial Growth Conditions

All cells were grown at 37° C with aeration in medium BX (8 gm KC1,
0.5 gm NaCl, l.l gm NHfcCl, 0.8 gm sodium pyruvate, 23 mg KR2POU , 20 mg
MgCl2, 0.11 gm CaCl2, 25 mg Na?S0]4 , 11. M gm Trizma-HCl , 3.32 gm Trizma
base, 10 mg uracil, 2 rug thymine, 15 gm Rneto casamino acids - vitamin
free, 20 mg tryptophan, 20 mg thiamine and 1( gm glucose per liter of
distilled water). Culture growth was followed using a Klett-Summerson
colorimeter with a blue filter.

0X1 7 i* DNA Preparation

Single stranded 0X171* DNA was prepare.! by two phenol extractions
at room temperature of bacteriophage 0X17*4 am3,alysis defective mutant
(lh). The double stranded, replicative form 0X17^ DMA was prepared by
using the method previously described by Achey et al. with the exception
that the column step was replaced by dialysis (l). Both DNA preparations
were extensively dialyzed against 0.001 M sodium phosphate buffer, pH 7.2
or 0.05 M Tris, pH 7.2 as a last step in their preparation.

1 3

Ik

TABLE I

BACTERIAL STRAIN:

Strain
AB115T

AB188H
AB1885
AB1886
AB1889
AB2U63
ABP.kjO
A B2 149)4
JC5519

Genotype

argE3 , his-'*, lent; p_roA?,
thr-1 , str-31, galK2 , lacYl,
xy!5 , ratl-l, ara-lU , tsx-33,
sup Eh k, thi-1

uvrC3'4

UvrB5

uvrA6

irvrA35

recA13

recB21

lexAl
recB2l recC22
thy uvrA sup_X

Reference

1*0
HO

ko
ko
91
91

91
14

15

0X17Jj DNA UV Irradiations

The UV source was three 15 watt General Electric L15T8 germicidal
bulbs. The intensity of the bulbs could be regulated by altering the
voltage to them with a rheostat. Two dose rates were used to irradiate
the DNA. A rate of 1.0 J/m2/s was used for delivering doses less than
100 J/m2. Higher doses were delivered at, a rate of 10 J/m?/s. Dose
rates were determined with an International Light Germicidal/Erythemal
Radiometer model IL570.

A 0.1 ml sample of the single and double stranded DNA in 0.05 M
Tris, pH 7.2 at a concentration of 5.0 Mg/ml were placed in a 7 cm
watch glass and exposed to a series of increasing UV doses while being
stirred. After each dose, 10 yl aliquots of the irradiated DNA were
diluted 100 fold into 0.05 M Tris, pH 7-2. The diluted DNA was kept
on ice in anticipation of the biological assay.

0X171' DNA Gamma Irradiations

Gamma irradiations were carried out in a custom built cobalt-60
gamma irradiator (32). Sample holders were positioned in the source
so that dose rates of it. 7 rads/s and l85 rads/s were available. The
dose rates were determined by ferrous- feri-ic sulfate dosimetry.

The DNA concentration at irradiation was 5-0 Ug/mL and the DNA
was in 0.001 M sodium phosphate buffer during irradiation. This buffer
was chosen since it does not appreciably affect the radiosensitivity
of the DNA in solution (l). The scavengers used in these experiments,
the radicals with which they react, the reaction bimolecular rate
constants and the reaction products formed are listed in Table II.
None of the scavenger-radical reaction products formed react with DNA
(9, 95). All of the samples (0.2 ml volume) were flushed immediately

16

TABLE IT

BIMOLECULAE RATE CONSTANTS OF THE PRIMARY WATER RADIOLYSIS
RADICALS WITH THE RADICAL SCAVENGERS USED IN THIS STUDY

Scavenger Bimolecular Rate Constant (M~-l sec-1)*

Hydroxy! Hydrogen Solvated

Radical Radical Electron

bee reterence

26.

7 x 10

No

°2 — 2.6 x 10 10 1.9 x 10-10

,9

17

prior to irradiation with either water saturated nitrogen or oxygen.
The samples were bubbled for at least 5 minutes at a flow rate of
60 cc/min to assure saturation with the gas. The concentration of
potassium iodide was 0.05 M whenever it was used as a scavenger. This
concentration ensures maximal scavenging of the hydroxy! radical (9).

Samples of the irradiated DNA solution were diluted 100 fold
immediately into 0.05 M Tris, pll 7.2. The diluted DNA was kept on
ice in anticipation of the biological assay.

UV Irradiation and Induction of E. coli

E. coli were grown in medium BX to a density of 1.7 x 108 cells/ml
(Klett=100). 25 ml aliquots of this culture were centrifuged at 10,000
rpm for 5 minutes to pellet the cells. The supernatant was discarded
and the pelleted bacteria were resuspended in 25 ml of phage buffer (7
gra Na2HP01;, 3 gm KH^POlt, 5 gra NaCl , 0.12 gin MgSOl, , 0.01 gm CaCl? arid 0.01
gm gelatin per liter of distilled water). The resuspended bacteria were
then poured into a 12 cm diameter, flat-bottomed dish and exposed to UV
while being vigorously stirred. For total doses greater than 10 J/m.2
a dose rate of 0.1 J/m2 was used. The irradiated culture was then
centrifuged as before, the supernatant discarded and the pellet re-
suspended in 2S ml of fresh medium BX. This culture was then incubated
in the dark for 50 minutes at 37° C with aeration to permit expression
of the inducible recovery system (59). The unirradiated control
samples were treated identically with the exception that they received
no UV irradiation.

lb

Calcium Treatment of E. eoli

The calcium treatment procedure is essential ly that of Taketo (80).
Immediately after the 50 minute post-irradiation incubation, 20 mi
camples of the cultures were centrifuged as before and the supernatants
discarded. The pellets were resuspended in 10 mi of an ice cold solution
of 0.05 M CaClo, 0.005 M MgCl2 and 2% polyethylene glycerol (57, 8l).
The resuspended bacteria were kept in an ice water bath for 30 minutes
before being centrifuged and the supernatant discarded. This final
pellet was resuspended in 1.0 ml of the 0.05 M CaCl2, 0.05 M MgClo and
2% polyethylene glycerol solution and then stored overnight at 0° 0
prior to being used to assay the biological activity of the 0X.17U DNA,

All steps subsequent, to the UV irradiation were performed in
yellow lights to prevent photoreactivation .

Transfection and Biological Assay of 0X17h DNA

The transfection procedure is also basically that of Taketo (80).
100 Ul of the diluted, irradiated DNA and POO Ml of the calcium
treated bacteria were mixed together and incubated in an ice water
bath for 30 minutes. The transfection mixture then was incubated at
37° C for three minutes before being returned to the ice water bath for
an additional 5 minutes before plating.

The infective centers produced by the above procedure were
detected by a plaque assay. This consisted of plating appropriately
diluted samples of the transfection mixture with 10 ml of top apar
(2.5 gm NaCl, 2.5 gm KC1 , 10 gm Bacto-tryptone , 10 gm Bacto-agar and
6 ml of 1 N NaOH per liter of distilled H20) seeded with the indicator
strain E. coli HF'li7.U. The plates were incubated at 37° c for lj to 6
hours .

19

All of the above steps were; done in yellow lights to prevent
photoreactivation .

Evaluation of UV Inducible Recovery Ability

The ability of UV inducible recovery to promote the survival
of the phage producing ability of irradiated 0YAjh DNA was determined
by comparing the dose survival curves of the DNA in a UV induced host
and an unirradiated host. As shown in Figures .1 and 2 using data from
this study, UV induced recovery was indicated by the dose survival
curves for the 0X171* DNA in the UV irradiated host having a shallower
final slope than that for the unirradiated host.

Rather than comparing sets of dose survival curves, the level of
UV induced recovery is best measured, by the percent efficiency of
repair which is determined by the expression :

% E = (log S2 - log Si) /(log ^2 - log °>n) x 100
(5, 25). As shown in Figures 1 and 2, So is the survival of the un-
irradiated 0X17*4 DNA molecule (fraction survival equals 1.0), S] is the
survival of the 0X17U DNA irradiated with dose D when transfected into
a UV induced host and S2 is the survival of the 0X17)4 DNA at dose D
when transfected into an unirradiated host. So, S^ and Sp have been
determined from dose-survival curves which are the average of at least
three independent experiments. The efficiency of recovery expresses
the change in the final slope of the survival curve of the irradiated
DNA from steeper to shallower in the UV induced host as the fraction of
potentially lethal damage which is removed by UV induced reapir.

Figure 1. Dose survival curve of UV irradiated 0X17*4 single stranded

DNA transfected into E. coli AB1157. Triangles, unirradiated
E. coli AB1157 as host; circles, UV irradiated (70 J/m?)
E. coll AB1157 as host. The parameters necessary for
determining the percent efficiency of repair are shown.

21

20 D

D0SE(J/m2)

40

Figure 2. Dose survival curve of UV irradiated 0X171* replicative form
DNA transfected in E. col.i AB115T. Triangles, unirradiated
E. coli AB115T as host; circles, IJV irradiated (70 J/m2)
ii- c°li as host. The parameters necessary for determining
the percent efficiency of repair are shown.

23

200 D 400

DOSE (J/m2)

000

RESULTS

UV Induced Hocovery of UV Irradiated DNA

Figures 3 through 9 show dose induction kinetics plots of the
efficiency of UV induced recovery of UV irradiated single stranded and
implicative form 0X1?U DNA in wild type, uvrA, uvrC , recB, recB recC,
recA and lexA hosts. Such plots illustrate the change in the amplitude
of the efficiency of UV induced recovery as the UV inducing dose to
the host is varied.

The wild type, uvrA, uvrC, recB and recB recC host all show UV
induced recovery of both the single stranded and implicative form 0X17*4
DNA while the recA and lex A hosts show no UV induced recovery. In
all hosts showing UV induced recovery, the amount of recovery for
both types of DNA molecules changes in a parallel fashion as the UV
inducing dose is increased. In all cases, however, the efficiency of
recovery is always 1.3 to .1.7 times higher for the single stranded DMA
molecule than for the implicative form.

Table III lists the maximal efficiencies of UV induced recovery
and the UV inducing doses that elicit them for the hosts which show
UV induced recovery. The wild type strain AB1157 shows the greatest
efficiency of recovery. At best, a single stranded 0X171* DNA molecule
can recover from an additional 60% of the potentially lethal UV
damage not repaired by other repair mechanisms while the replicative
form DNA molecule can recover only from h^% of such damage. The other
hosts show only 5k% to 13% of the wild type efficiency of recovery of

2k

Figure 3. Dose induction kinetics plot for E. coli AB1157. Circle
single stranded 0X17'* DNA, triangles, replicative form
0X17!i DNA.

DOSE (J/rrf)

Figure U . Dose induction kinetics plot for E. coli AB1B86 uvrA. Circles
single stranded 0X171* DNA; triangles, replicative form 0X17lt
DNA.

28

6 9

DOSE (J/m2)

2

Figure 5. Dose induction kinetics plot for E. coli ABI88I4 uvrC.

Circles, single stranded 0X17^ DM; triangles, replicative
form 0X17)( DNA.

30

8 12

DOSE (J/m2)

Figure 6. Done induction kinetics plot for E. coli AB2ll70 recB.

Circles, single stranded 0X17,( DNA; triangles, replicatii
form 0X1 7^ DNA.

32

DOSE (J/m )

Figure 7- Pose induction kinetics plot for E. coli JC5519 recR recC.
Circles, single stranded 0X17^' DNA; triangles, replicative
form 0X17)< DNA.

3h

DOSE (J/rrf )

Figure 8. Dose induction kinetics plot for E. coli AB2I463 recA.

Circles, single stranded 0X17Ji DNA; triangles, replicative
form 0X17k DNA.

36

20

rr

>

o
o

LJ

cr

& l0

>-

o

LJ
O

u_

LJ
f-

LJ

o
cr

LJ

3 6 9

DOSE (J/m2)

12

Figure 9- Dose induction kinetics plot for E. coli AB^Q.'t lexA.

Circles, single stranded 0X.17'* DNA; triangles, replicate
form 0X1 7 ;i DNA.

38

LlI
>

O

o

Ld

20

0 20

DOSE (J/m2)

30

39

TABLE I IT

MAXIMUM RECOVERY EFFICIENCIES AND THEIR UV EI.ECITING DOSES

AB115T

70

AB1186

uvrA

5

0

abii81*

uvrC-

8

0

AB2l*70

recB"

15

0

JC5519

recB-

recC"

20

0

AB2U6?,

recA~

-

AB2'*9l*

lexA"

__

Host Dose j/m2 Efficiency Relative

oT Recovery Efficiencies***

SS* RF** SS RF

60.0 1+5-0 1.0 1.0

Ul.O 27.2 0.68 0.6o

1*5-2 27.2 0.75 0.60

39-9 26.6 0.67 0.59

32.2 20. U 0.5't 0.1*5

0 0 0 0

0 0 0 0

"Single stranded 0X11 h DMA

**Replicative form 0X17Jt DNA

***Relative to the wild type strain

/

Uo

the single strand DNA molecule and hr)% to 60% of the efficiency of
recovery of the replicative form molecule.

There is an order of magnitude difference in the range of the UV
inducing doses which give maximum efficiency of recovery. The doses
for the different hosts are inversely related to their relative
sensitivities to UV. The wild type strain ABU 57 is the least
sensitive to UV and shows the greatest maximal UV inducing dose while
strains AB1886 uvrA and AB188U uvrC are the most sensitive to UV
and show the smallest maximal UV inducing dose. Strains A,°'i70 recB
and JC5519 recB recC have intermediate UV sensitives and reflect this
in their maximal UV inducing doses.

Table IV lists the efficiencies of UV induced recovery for single
stranded and replicative form 0X17'* DNA in three hosts all lacking
a functional correndonuclease II and are all defective in the incision
step of excision repair. Two of these hosts, AB1R86 and ABI889, are mutant
at the uvrA gene but at different loci while the third host, ABI885,
is mutant at the uvrB gene. Little difference is seen in the
efficiencies of UV induced recovery in these three hosts at the two
UV inducing doses used.

UV Induced Recovery of Gamma Irradiated DM

Figures 10 through 17 show the plots of percent survival of phage
producing ability versus gamma ray dose for single stranded and rep-
licative form 0X17^ DNA when UV induced or uninduced E. coli AH1157
were the host. The UV inducing dose is 70 J/m2. In all instances there
is greater survival of phage producing ability with the UV induced host
than the uninduced host. Table V summarizes the efficiencies of UV in-
duced repair observed under the various radiation conditions employed.

Ul

TABLE IV

EFFICIENCY OF UV INDUCED REPAIR OF 0XJ Y'i DNA
IN THREE EXCISION REPAIR DEFECTIVE MUTANTS

Sing

le Stranded 0X1 7J*

Dose (J/m2)
To Host

188s uvrB5

1886 uvrA6

I889 uvrA35

2.5
7.5

35.9
36.2

38.2
33.6

35.8
kk.3

RepI]

Lcative

Form 0X1 7'j

Dose (J/m2)
To Host

1885 uvrP5

1886 uvrA6

1889 uvrA35

2.5
7-5

30.0
22.7

23. 9
21.2

22. U

20.8

Figure 10. Survival of the phage producing ability of gamma irradiated
0X17^ single stranded DNA bubbled with nitrogen. Circles,

unirradiated E. coli AB1157 as host; squares, UV irradiated
(70 J/m2) E. coli AB1157 as host.

»«3

00 200 300 400
DOSE (rods)

Figure 11. Survival of the phage producing ability of gamma irradiated
0X17*4 replicative form DNA bubble with nitrogen. Circles,
unirradiated E. coli AB1157 as host; squares, UV irradiated
(70 J/m2) E. coli as host.

;<5

2.0 4.0 6.0 8.0

DOSE (kilorads)

Figure 12. Survival of the phage producing ability of gamma irradiated
0X17*4 single stranded DMA bubbled with oxygen. Circles,
unirradiated E. coli AB1157 an host; squares, LTV irradiated
(70 J/m2) E. coli AB1157 as host.

hi

200 400 600
DOSE (rods)

800

Figure 13. Survival of the phage producing ability of gamma irradiated
0Xl'jh replicative form UNA bubbled with oxygen. Circles,
unirradiated E. coli AB115T as host; squares, LP/ irradiated
(70 J/m2) E. coli AE1157 as host.

Uq

4.0

8.0 12.0

DOSE (kilorads)

16.0

mm

Figure Ik. Survival of the phage producing ability of ga™,a irradiated
0X171* single stranded DNA in the presence of 0.05 M potassii
iodide and bubbled with nitrogen. Circles, unirradiated E. coli_
AB1157 as host; squares, UV irradiated (70 J/m?) E. coli AB1157
as host.

5.1

2.0 3.0

DOSE (kilorads)

4.0

Figure 15- Survival of phage producing ability of gamma irradiated 0X171*

replicative form DNA in the presence of 0.05 M potassium iodide
and bubbled with nitrogen. Circles, unirradiated E. coli AB115T
as host; squares, UV irradiated (70 J/m?) E. coli AJ31157 as
host .

5"!

100 200

DOSE (kilorads)

300

Figure l6. Survival of phage producing ability of gamma irradiated 0X17 1j
single stranded 0X171! DNA in the presence of 0.05 M potassium
iodide and bubbled with oxygen. Circles, unirradiated E. coll
AB1157 as host; squares, ITV irradiated (70 J/m2) E. coli AB1157
as host.

55

5 30 45 60

DOSE (kilorads)

Figure 17. Survival of phage producing ability of gamma irradiated 0X17*4

replicative form DM in the presence of 0.05 M potassium iodide
and bubbled with oxygen. Circles, unirradiated E. coli AB1157
as host; squares, UV irradiated (70 J/m2) E. coli AB1157 as
host.

57

200 400 600 800

DOSE (kilorads)

58

TABLE V

EFFICIENCIES OF UV INDUCED RECOVERY OF 0X17*4 DNA
GAMMA IRRADIATED UNDER VARIOUS CONDITIONS

*Single stranded 0X17** DNA
**Replicative form 0X17U DNA

Radical Radical s Efficiency

Scavenger Reacting with DNA of Recovery

RF4H

?5.3 28.6

Np Solvated electron

Hydrogen radical
Hydroxy I radical

02 Hydroxyl radical 20.8 1 9 . 9

I- Solvat.pd Electron 1*8.7 ^6.5

Hydrogen radical

I- and Op None (direct action) 22. h 20.0

59

Several observations are apparent from Table V. First, for a
given radiation condition the efficiency of I IV induced recovery is
the same for both types of 0X17U DNA molecules. Secondly, radiation
conditions which effectively scavenge the hydroxy! radical produce the
largest efficiency of recovery. Thirdly, the efficiencies of recovery
that result from no radical attack on the DNA (direct action) or
hydroxy! radical attack alone are the same. Conditions which do
not scavenge any of the radicals result in efficiencies of recovery
which are only slightly higher than those of direct action or hydroxyl
radical attack.

DISCUSSION

The purpose of this dissertation was to elucidate some of the
properties of UV induced recovery in E. cohi of LTV and gamma irradiated
single and double stranded DNA molecules. Five objectives were set
forth in the Introduction as a framework for doing this. What follows
is a discussion of how this study meets these objectives.

Figures 3 through 7 and Table IV show that in every strain in which
UV induced recovery of UV damaged DNA occurred, the efficiency of
recovery of the single stranded 0X17^ DNA molecule is more than that
of the double stranded replicative form molecule. There are several
possible explanations that could account for this difference. One
could be that when both types of molecules are given UV doses which
will reduce their phage producing capacities to the same level, the
replicative form molecule will contain more damage than the single
stranded molecule. This greater amount of damage presumably could
inhibit the UV induced recovery mechanism from restoring the molecule
to a biologically active state. The yield of pyrimidine dimers in
both single stranded and replicative form 0X171* DNA is known (20, 37).
The number of dimers present after both types of DNA molecules have
been given a UV dose which results in equal phage producing ability in
an induced host can be computed. The host chosen for this calculation
must possess only the UV induced recovery system as its only method
of repairing UV damaged 0X17|4 DNA. The data in Table VI must be
consulted to choose such a host.

60

61

TABLE VI

UV SENSITIVITIES OF SINGLE STRANDED AND REFLJCATIVE
FORM 0X1TH DNA MOLECULES IN VARIOUS UNIRRADIATED HOSTS

Strain

AB2U63 recA"

n3T (J/m2)

Single Strand Replicative Fo

rm

AB115T wild type 8.5 75

ARllSU uvrC~ 8,5 -,£

AB188? uvrB" 9.1

15

AB1886 uvrA- 9,i 13

ABI889 uvrA" 8.5 ]6

■ 0 70

AB2h70 recB~ 9.0 70

JC5519 recB" recC~ 8.3 70

62

Table VT lists the D37 doses determi nor! in this study for the
inactivation of single stranded and replicative form 0XL7U DNA in
unirradiated hosts. The D37 dose is that dose which on the average
causes one lethal event for every DNA molecule in the irradiated
population. It is a measure of the LTV sensitivity of the 0X17h UNA
in a particular host and a measure of the repair capability offered
by that host. The higher the D37 dose, the greater the recovery from
UV damage. Table VI shows that the sensitivity of the single stranded
0X17*4 DNA is the same in all unirradiated hosts indicating that
excision repair and recombination do not participate in the recovery
of this molecule from UV damage. On the other hand, the replicative
form molecule is more sensitive in excision repair deficient hosts
than in the wild type or recombination deficient strains. This indicates
that excision repair plays a role in the recovery of the UV irradiated
replicative form molecule. The wild type and recombination deficient
hosts, however, have the same UV sensitivities indicating that re-
combination does not contribute to the recovery of the replicative form
molecule. Thus, the only recovery mechanism available in excision
repair deficient mutants for single stranded and replicative form 0X17^
DNA is the UV induced recovery mechanism.

Using UV irradiated excision repair deficient strains, Table VII
shows the number of dimers calculated to be present in single stranded
and replicative form molecules which have been exposed to a D37 dose
of UV. The number of dimers is approximately the same. Thus, the
difference in single stranded and replicative form 0X1 7U DM UV in-
duced recovery cannot be explained by different amounts of UV damage
in each type of molecule.

63

TABU'' VI T

DTMER YIELDS AT D37 FOR STNfJLE .STRANDED AND
REPLICATIVE FORM 0X1'P» DIM ' IN EXCISION REPAIR DEFICIENT HOSTS

Strain* Number of Miners**

Single Stranded Replicative Form

1835 uvrB- l.l

1886 uvrA- 1.0

1889 uvrA- 1 .0

1

2

1

0

1

1

*The hosts have been irradiated with 2.5 J/m2 of UV
**Number of dimers = D3Y dose x dimer yield

6)4

Another possibility could be that the action of IJV induced

recovery could produce configurations of the replicative form molecule
which are susceptible to nuclease attack. Degradation or a double
strand break would result in the inactivation of the DNA molecule.

A third possibility is to assume that the single stranded 0X17>»
DIM molecule is a better substrate for UV .induced recovery than the
replicative form. This explanation is particularly attractive if
it is assumed that a polymerase like that of SOP> repair mediates in
the UV induced recovery of 0X171* DNA. Such a polymerase might be
better suited for asymmetric rather than semi-conservative DNA
replication for a variety of reasons. Steric hindrance by one
strand of the replicative form molecule could inhibit the ability of
the polymerase to use the other strand as a template for DNA synthesis.
Also, the polymerase may lack the ability to interact properly with
other proteins which normally play a part in DNA replication. This
could result in a lowering of the activity of a replication associated
function, such as the ability to unwind duplex DNA, that is necessary
for the efficient use of double stranded DNA molecules as a template.
The net result of this or of any of the other possible explanations
described in the above paragraphs would he to lower the efficiency of
UV induced recovery for the replicative form molecule.

The results of this study show that the UV induced recovery of
UV irradiated single stranded and replicative form 0Xllh DNA does
not depend upon the uvrA , uvrB, uvrC , recB and recC gene products
but does depend upon the recA and lexA gene products. This indicates
that the UV induced recovery of 0X17^ DNA like the UV induced recovery
of other bacteriophage and bacterial systems proceeds by a mechanism

65

independent of excision repair and recombination mediated by the reel1.
recC pathway (22, 9b). Examination of Table 1TI, however, shows that
the levels of UV induced recovery in the excision repair and recombina-
tion deficient strains are anywhere from 75% to h^% of the efficiency
of recovery for the wild type host. This indicates that it is possible
to modulate the level of LTV induced recovery. The best explanation
of the disparity in the levels of efficiency of recovery is to
assume that the missing gene products play a role in producing the
inducing signal which ultimately turns on the UV induced recovery
mechanism. It has been suggested that the inducing signal in UV
irradiated E. coli may be the products resulting from the repair of
UV damage to the bacterial UNA (Qh). The uvrA, uvrB, uvrC, recB and
recC gene products all play a role in the metabolism of irv irradiated
bacterial UNA. It might be that all of the UNA degradation products
produced by the action of these gene products act as the inducing
signal in some cooperative manner. Removing one set of these products
could lessen the likelihood of a successful induction event. This
would result in fewer induced host cells in the irradiated population
which in turn would result in a lowering of the efficiency of UV
induced recovery with respect to the wild type host.

That the differences in the efficiencies of UV induced recovery
are not due merely to metabolic or physiological peculiarities among
the various uvr strains used in these experiments is demonstrated
in Table IV. All three strains share a common defect, the inability
to perform the incision step of excision repair. Two of the defects
map in separate loci in the uvrA gene while the other is in the uvrB
gene. In all three strains, however, the efficiencies of UV induced

66

recovery for both forms of 0X]7'i DMA at two different UV inducing
doses to the host are essentially the same. This indicates that
the level of efficiency of recovery is dependent upon a shared
step in the metabolism of UV irradiated bacterial DNA.

The wide range of UV doses required to elicit maximum efficiency
of recovery for 0X17'i UNA in the various hosts shown in Table III
Ls also observed in other systems (22, Q'i) . Wilkin explains this
variation by assuming that pyrimidine dimers must persist in bacteria]
DNA if induction is to occur (9>i). It is then assumed that repair
proficient cells can effectively remove dimers from their DNA before
the inducing signal can be generated. Thus, a. large UV dose is
required to insure that at least one dimer will escape being repaired
long enough so that it can participate in the induction process.
For cells less proficient in repairing dimers, a smaller dose is
required to ensure induction. Therefore, the most repair competent
strain AB1157 has the highest UV induction dose while the least
repair competent strain AB1886 uvrA" has the lowest UV induction dose.
The strains AB2l*70 recB- and JC5519 recB- recO are intermediate in
their UV repair capabilities and reflect this in their intermediate
UV inducing doses (39, 91).

Table V compares the efficiency of UV induced repair which re-
sulted from the transfection of E. coli AB1157 irradiated with 70 J/m2
of UV with gamma irradiated 0X1 7*) DNA. For all irradiation conditions,
UV induced recovery of both types of DNA molecules is observed. The
level of the efficiency of recovery, however, is dependent upon what
water radiolysis radicals are attacking the DNA. The greatest amount
of recovery is seen when the hydrogen radical and solvated electron

67

are the damaging species and the small est amount of recovery is seen
when the DNA is damaged either by the hydroxy! radical a] one or by
direct action. The results tabulated in Table V also suggest that
each radiation condition may produce two sets of damage. One set of
damage is susceptible to UV induced recovery while the second set
is not. The fraction of total damage susceptible to UV induced
recovery in the population of irradiated DNA molecules would be equal.
to the efficiency of recovery for a given radiation condition. If
this assumption is true, Table V suggests that the yields of both
types of these damages are equal in both types of 0X1 7't DNA.

The argument for susceptible and non-susceptible classes of
damage is further supported by the fact that when the action of the
hydroxyl radical is added to that of the hydrogen radical and solvated
electron, the efficiency of UV induced recovery is severely reduced.
This suggests that the hydroxyl radical ran produce damage which
masks the recovery from damage caused by the other two radical species.

The chemical nature of the susceptible and new-susceptible
classes of gamma ray induced damage in 0X17*4 DNA cannot be identified
by this study. Some inferences can be made about the nature of the
susceptible gamma ray damage in both types of the 0X17*4 DNA molecules,
however. It is most likely that this type of damage is base damage
rather than strand breaks. A single strand break converts the
covalently closed, single stranded 0X17!t DNA molecule info a linear
one. The linear molecule has no biological activity. Unless the
UV induced recovery mechanism can convert a single stranded, linear
molecule back to a covalently closed one, an activity which has
never been reported, some variety of base damage probably constitutes

63

the susceptible .lesion. The same may be true of the replicative
form molecule since single and double strand breaks only contribute
to 10% of gamma ray lethality (87). If base damage is indeed the
susceptible damage, then all radiation conditions produce this
damage to a. greater or lesser degree.

The levels of UV induced recovery of gamma irradiated 0X17*4 DNA
are the same for both single stranded and replicative form molecules.
This is in contrast with the efficiency of UV induced repair being
higher for irradiated single stranded molecules than for UV irradiated
replicative form molecules. This indicates that gamma irradiated 0X17^
DNA is an equally good substrate for UV induced recovery regardless
of whether the molecule is single or double stranded. Damage such
as UV induced pyrimidine dimers cause greater helix distortion in
duplex DNA molecules than the single strand breaks and base damage
caused by gamma rays (17). The lack of such distortion in a UV
irradiated single stranded molecule may account for its greater
recovery than that found for the UV irradiated replicative form.
It does not explain why UV irradiated single stranded DNA shows a
higher efficiency of recovery than its gamma irradiated counterpart.
Apparently other characteristics of the radiation induced damages
different from helix distortions account for these particular re-
sponses of the two forms of DNA molecules to the damage produced by
the two types of radiation.

In many respects the UV induced recovery observed in this study
is similar to the W-reactivation of single and double stranded
bacteriophage (21, 22, 36, 1)9, 58, 83, 90). Here irradiated, purified
DNA is used to transfect UV irradiated, calcium treated E. coli

69

rather than irradiated bacteriophage infecting irradiated, whole
bacteria. The phage and DNA show a greater recovery from the lethal
effects of radiation when they infect or transfect UV irradiated
hosts. Another similarity between the UV induced recovery of
irradiated (/iXlfh DNA and W-reactivation is their common requirement
for the recA and lexA gene products. Also, both processes are
mediated by a recovery mechanism which is independent of excision
repair and recombination since both occur in excision repair defective
and recombination defective (other than recA) mutants. Based on
these similarities, the UV induced recovery of irradiated 0X17^
single and double stranded DNA and W-reactivation are essentially
the same.

Biological and in_ vitro biochemical evidence indicate that W-
reactivation of single stranded DNA occurs by the conversion of the
UV damaged, single stranded molecule to a fu.l 1 length, covalently
close! replicative form (60, 6l, 9'-0 . Tins replicative form is
necessary if whole phage are to be produced (50). Radman's proposed
SOS repair mechanism which involves a hypothetical polymerase capable
of effecting such a conversion could ^ery nicely account for single
stranded bacteriophage W-reactivation. Such a mechanism could also
account for the UV induced recovery of UV irradiated 0X1714 single
stranded DNA.

The UV induced recovery of UV irradiated repl.icat.ive form 0X17'i
DNA can also be explained by the same mechanism. The sequence of 0X17U
DNA replication after the formation of the parental rep] icative form
leads to the production of daughter replicative form molecules which
in turn serve as templates for the production of progeny single

stranded DNA molecules (27, 23, 70 ). Ml of the DNA synthesis in
these steps is asymmetrical. A pyrimidine dimer in the complementary
strand of the replicative form could block any further DNA replication
since this strand is the primary template; for daughter replicative
form and progeny single stranded DNA synthesis. UV damage in the
complementary strand which blocked DNA replication would render
the replicative form biologically inactive. The hypothetical
SOS repair polymerase would be able to use the damaged viral strand
as a template for further DNA replication. Thus, this mechanism
could restore the biological activity of the UV damaged replicative
form molecule.

From the data in Table VI, the yield of dimers at D37 in the
single stranded and replicative form 0X17^ DNA molecules in an
unirradiated excision repair deficient host can be calculated. They
are 0.75 and 0.88 respectively. These numbers are approximately
equal to 1.0, the expected number if only one dimer v/ere required in
the single stranded DNA molecule or in the template strand of the
replicative form molecule to inactivate it. This represents evidence
supporting the contention that a dimer in the comp] ementary strand
of the replicative form molecule is a lethal event. In turn this
supports the speculation that the same mechanism which mediates in
the UV induced recovery of single stranded 0X1 7^ DNA could participate
in the recovery of the replicative form molecule.

The recovery of gamma ray damaged 0X1 fh DNA could be carried
out by the same mechanism described above for the UV irradiated
molecules if it is assumed that gamma ray damage can block the
asymmetric DNA replication necessary for a single stranded or

replicntive form 0X17Jt DNA molecule ultimately to produced whole
phage. Whether or not gamma ray damage can block bacteriophage DMA
replication is not known. Since UV irradiation of the host bacteria
results in the greater recovery of the biological activity of both
gamma and UV irradiated 0X.17'i DNA, it. in attractive tn assume that
the UV induced recovery of both types of damage is mediated by a
common system. However, the differences in the chemical structures,
the repair and the biological consequences of known UV and p.amma ray
DNA damages suggest that it is very possible that the UV induced
recovery of gamma irradiated 0X17^ DNA could occur by a completely
different mechanism than that which effects the recovery of UV
irradiated 0X17*J DNA. Whether or not UV induced recovery of gamma
irradiated 0X17*4 DNA can be carried out by a SOS repair type of
polymerase cannot be surmised until the effects of gamma ray damage
on bacteriophage DNA replication have been determined.

To summarize, this study demonstrates that both single and
double stranded 0X17'* DNA damaged by either UV or gamma, radiation is
susceptible to UV inducible recovery. The results presented suggest
that the amount of inducible recovery is dependent upon the type of
damage introduced into the DNA molecules and whether the molecule
is single or double stranded. Also, the data shown indicate that
the level of expression, of the inducible recovery system can be
nodulated by the action of gene products normally involved in DNA
repair. It can be surmised that, the inducible recovery of both types
of DNA whether UV or gamma irradiated is mediated by a common mechanism
possibly that proposed by SOS repair. The results of this study do
not. unambiguously support this conjecture which can only be verified
by further lines of experimentation.

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79

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BIOGRAPHICAL SKETCH

John Randall Silber war? born January 10, 19^9 in Philadelphia,
Pennsylvania. In 195U he moved to St. Petersburg, Florida, where
he graduated from Northeast High School in June of 19^7 • Attending
the University of Florida, he received a Bachelor of Science degree
in physics in December 197-1 • He returned to the University of
Florida in September of 1973 where he in currently a doctoral
candidate in the Department of Microbiology and Cell Science.

■Be

I certify that I have read this study and that in my opinion it
conforms to acceptable standards "f sohol ar.ly presentation and is fill
adequate, in scope and quality, as a thesis for the decree of Doctor
of Philosophy.

rl(Jj\/ ill lLLu.

Phi Hip M. Achey /~
Assistant Professor of Microbiology
and Cell Science

T certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and qualtiy, as a thesis for the degree of Doctor

of Philosophy.

-K . <■„■,< f)L.o

Richard P. Royoe ~^
Professor of Biochemistry and
Molecular Biology

T certify that 1 have road this study and that, in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a thesis for the degree of Doctor
of Philosophy.

Lonnie 0. rngram

Associate Professor of Microbiology

and Cell Science

This dissertation was submitted bo the Graduate Faculty of the
Department of Microbiology and Cell f-cience in the College of Arts
and Sciences and to the Graduate Council, and was accepted 'is partial
fulfillment of the requirements for the degree of Doctor of Philosophy,

December 1977

Dean, Graduate Hchoo]

UNIVERSITY OF FLORIDA

lillllVllllllII

3 1262 08553 2868