Mol Gen Genet (1981) 184:471-478 © Springer-Verlag 1981

Characterization of Postreplication Repair in Saccharomyces cerevisiae and Effects of rad6, radl8, rev3 and rad52 Mutations Louise Prakash Department of Radiation Biology and Biophysics, University of Rochester, School of Medicine and Dentistry, Rochester, New York 14642, USA

Summary. Postreplication repair of nuclear DNA was examined in an excision defective haploid strain of yeast lacking mitochondrial DNA (radl pO). The size of the DNA synthesized in cells exposed to various fluences of ultraviolet light (UV) corresponds approximately to the average interdimer distance in the parental DNA. Upon further incubation of cells following exposure to 2.5 J/m 2, the DNA increases in size; by 4 h, it corresponds to DNA from uniformly labeled cells. The alkaline sucrose sedimentation pattern of DNA pulse labeled at various times after UV irradiation, for up to 4 h, does not change substantially, indicating that dimers continue to block DNA replication. A significant amount of postreplication repair requires de novo protein synthesis, as determined by its inhibition by cycloheximide. The rad6 mutant does not carry out postreplication repair, the rad18 and rad52 mutants show great inhibition while the rev3 mutation does not affect postreplication repair. Both recombinational and nonrecombinationalrepair mechanisms may function in postreplication repair and most of postreplication repair is error free.

Introduction

UV irradiation inhibits DNA replication in both prokaryotic and eukaryotic organisms. Pyrimidine dimers induced by exposure to UV light block progression of the replication fork, yielding DNA fragments that are smaller than those observed in unirradiated cells. In Escherichia coli (Rupp and HowardFlanders 1968) and some eukaryotic organisms (Lehmann 1972; Buhl et al. 1972, 1974; Sarasin and Hanawalt 1980), the size of the DNA synthesized following UV irradiation corresponds to the average distance between pyrimidine dimers. Incubation of cells following UV irradiation results in the conversion of the small DNA fragments to high molcular weight DNA, similar in size to that observed in unirradiated cells. This process has been referred to as °'postreplication repair." Studies of postreplication repair in prokaryotic and eukaryotic organisms have revealed differences which may reflect the nature of repair in these two types of cellular systems. In E. coli, postreplication repair occurs mostly via recombinational mechanisms (Rupp et al. 1971), whereas in mammalian cells, it is probably nonrecombinational and requires de novo DNA synthesis (Lehmann 1972; Buhl et al. 1972). DNA synthesized at long times after UV irradiation still contains gaps in E. coli (Rupp and Howard-Flanders 1968), Chinese hamster B14 (Meyn and Humphrey 1971) and ICR2A frog cells (Rosenstein and Setlow 1980), but is high molecular weight in CHO (Meyn and

Humphrey 1971), mouse L5178Y (Lehmann and Kirk-Bell 1972), human normal and xeroderma pigmentosum (XP) cells (Buhl et al. 1973), and rat kangaroo kidney cells (Buhl et al. 1974), even though dimers have not been excised from the DNA. An inducible fraction of postreplication repair may exist in E. coli (Sedgwick 1975), whereas the inducibility of postreplication repair in mammalian cells is open to question (Painter 1978; Park and Cleaver 1979). However, recent results on replication of UV irradiated SV40 DNA suggest that an inducible function allows for trans-dimer synthesis on the leading strand and gap filling opposite lesions on the lagging strand of the replication fork (Sarasin and Hanawalt 1980). Studies on the genetic control of postreplication repair, which have been informative in E. coIi (see Hanawalt et al. 1979 for review), have been difficult in the eukaryotic systems studied so far. Since our objective is to understand the molecular mechanism(s) and genetic control of postreplication repair in an eukaryotic organism amenable to achieving both of these ends, we have begun studies in the yeast, Saccharomyces cerevisiae. Mutations in nine different loci, all belonging to the same epistasis group, are involved in excision of UV induced pyrimidine dimers (see Prakash and Prakash 1980 for review). Mutants of another epistasis group enhance the UV sensitivity of excision defective mutants (Cox and Game 1974) ; many of these may be defective in postreplication repair. In this study, we report the characterization of postreplication repair of nuclear DNA in excision defective haploid yeast. Our results suggest that there is both inducible and constitutive postreplication repair in yeast. The rad6 mutant is deficient in postreplication repair, both radl8 and rad52 mutants are defective but not completely deficient in postreplication repair, while normal levels of postreplication repair take place in the rev3 mutant.

Materials and Methods

Strains. The radl-2 mutation, formerly designated uvs9, was isolated by Snow (1967), the rad6-1 mutation was isolated by Cox and Parry (1968), and the rad18-2 and rad52-1 mutations, formerly designated uxsl and xsl, respectively, were isolated by Resnick (1969). The rev3-1 mutation was isolated by Lemontt (1971a). pO derivatives of all the strains listed in Table 1 were used throughout these studies since p0 strains lack mitochondrial DNA (Goldring et al. 1970). Media. Cells were grown in synthetic medium minus uracil, as described in Prakash et al. (1980) and supplemented with 5 pg/ml

0026-8925/81/0184/0471 / $01.60

472 Table 1. Genotypes of strains used

Strain

Genotype

LP841-13B

MATa ade] lys2-1 ural radl-2 pO

LP2627-1 B

MATa adel lys2-1 ural radl-2 rad6-1 pO

LP2614-11C

MATc~ arg4-17 his5-2 lys2-1 metl ural radl-2 rev3-1 pO

LP2596-5A

MAT:~ ural radl-2 radl8-2 pO

LP2630-12A

MATa adel lys2-1 ural radl-2 rad52-1 pO

10 ~ x

UV Irradiation of Cells. Asynchronously growing cells were irradiated in logarithmic phase at room temperature with constant stirring at a density of 0.5-1.0 x 1 0 7 per ml in growth medium in 150 m m x 20 mm Petri dishes. The UV source and its dosimerry were as described in Lawrence et al. (1974). Irradiation was carried out at a fluence rate of 0.1 J/mZ/s. After irradiation, all operations were performed in yellow light to avoid photoreactivation. The effective dose was determined from the survival of yeast irradiated on the surface of plates compared to the survival of yeast irradiated in suspension. Labeling and Treatment of Cells after UV Irradiation. Cells collected by filtration were resuspended in fresh medium at a density of 1 - 2 x 108 per ml. Pulse labeling was achieved as follows: 100 gCi [3H]-6'-uracil (Moravek Biochemicals, City of Industry, CA) was added to 1 ml cells and the suspension was incubated with vigorous shaking for 15 rain at 30 ° C. The pulse was terminated by adding 4 ml synthetic medium supplemented with 1.67 mg/ml uracil instead of uridine (high uracil medium), as described by Rivin and Fangman (1980). Cells were centrifuged, resuspended in 5 ml high uracil medium and incubated for varying times at 30 ° C. The incubation in high uracil medium free of radioactive uracil constituted the "chase". Preparation of Spheroplasts and Alkaline Sucrose Sedimentation. Cells collected by filtration were washed and resuspended in 1 ml water and transferred to microfuge tubes. To 1 ml cell suspension were added 0.1 ml 0.5 M EDTA, pH 9 + 25 gl 2-mercaptoethanol followed by incubation for 5 rain at room temperature. Cells pelleted by centrifugation were suspended in 1 ml 1.2 M sorbitol-0.1 M EDTA, pH 7.5 (SE). Zymolyase 5000 (Kirin Brewery Co., Ltd., Takasaki, Gumma Pref., Japan) made in SE was added to a final concentration of 1 mg/ml, and the suspension was incubated at 3 7 ° C for 5 rain, which resulted in conversion of cells to spheroplasts. A 0.3 ml aliquot of the spheroplast suspension was layered directly onto a 0.2 ml lysing layer, consisting of 0.79 M sorbitol-0.066 M EDTA-2.5 percent sarkosyl-0.3 N NaOH-0.7 M NaC1 on top of a 15-30 percent (w/v) linear alkaline sucrose gradient made in 0.3 N N a O H 0.7 M NaC1-40 m M EDTA-1 percent sarkosyl (pH 12.5). Centrifugation was carried out at 4 ° C in an SW41 rotor (Beckman) in a Model L5-65 ultracentrifuge for the times and speeds indicated in the figure legends. Fractions of 0.3 ml each (15 s) were collected from the bottom of the tube as described previously (Prakash 1975). An equal volume of 2 N N a O H was added to each fraction and alkali hydrolysis of R N A carried out by incubation of fractions for 90 rain at 60 ° C. Fractions were then cooled to 4 ° C and 30 gl of 1 mg/ml salmon sperm D N A was

0

6

0

• A

0

0 uridine. These conditions were found to be optimal for pulse labeling of D N A in these strains.

0

8

O

o

i

i

5

TIME (hrs) Fig. 1. DNA synthesis in radl pO cells (strain LP841-13B) exposed to various UV fluences. Exponentially growing cells exposed to 0 (©), 1.3 (e), 2.5 (zx), and 3.8 J/m x (D) of UV light were incubated at 30° C with 40 gCi/ml [3H]-6'-uracil. Alkali stable, acid-precipitable counts were determined as described in Materials and Methods in samples withdrawn at 0.5 h intervals after addition of label. At the time indicated by the arrow, a portion of the sample exposed to 2.5 J/m 2 was incubated in high uracil medium and samples withdrawn as indicated (A). Total counts incorporated at the end of 3 h were 97,483; 63,353; 52,131 and 31,496 for samples exposed to 0, 1.3, 2.5, and 3.8 J/m 2, respectively

added to each tube as carrier, followed by 0.3 ml cold 50% trichloroacetic acid (TCA). Fractions were kept on ice for 20 rain; acid precipitable counts were determined by filtering each fraction over a G F / C filter, rinsing each tube 5X with 3 ml cold 5 percent TCA followed by one wash with cold ethanol. Filters were dried under a Fisher Infrared Rediator lamp, placed in 10 ml Nalgene filmware bags (Sybron Corp.) in plastic scintillation vials, and 1.5 ml Liquifluor PPO, POPOP (New England Nuclear) toluene scintillation cocktail was added. After sealing the bags, radioactivity was determined in a Beckman LS-250 liquid scintillation counter. Recovery of counts was usually > 90 percent.

Determination of Molecular Weights. D N A from phages T4c and T7, prepared as described by Kutter and Wiberg (1968), were used as molecular weight markers to calibrate the 15-30% linear alkaline sucrose gradients. The details of the calibration of gradients and determination of number average molecular weights (Mn) are given in Miller, Prakash and Prakash (manuscript in preparation). The methods used were similar to those of Hill and Fangman (1973), but our experimentally derived value of k, obtained from the equation of Burgi and Hershey (1963), was found to be 0.369. The values of 57x 1 0 6 and 13.2 × 106 daltons, for T4c and T7 D N A , respectively, half the values published for the native D N A s (Clark et al. 1980), were used in calculation of Mn from our alkaline sucrose gradients. Results

Incorporation of [ 3H]-6"-Uracil after UV Irradiation of radl po Cells The incorporation of 3H label into alkali stable, acid precipitable material was measured in the excision defective radl-2 strain exposed to various UV fluences. The rate of D N A synthesis decreased with increasing UV fluenee (Fig. 1). When high uracil medium was added to UV irradiated cells, no further incorporation of label into D N A was observed (Fig. 1). These conditions

473 I0

Table 2. Size of nuclear DNA synthesized in UV irradiated haploid radl pO cells Fluence (J/m z)

/t ':

0 1.3 2.5 3.8 5.0

: I-

:4

\

,i i F -t

O

~ 1 i

l'a

,< re"

".

I

'%

:

0 ~)0 bottom

/.J

75

r,,a i t

I,

-"

,, '

!

~

"...,...'

f

,:

50

25

0 top

RELATIVE DISTANCE SEDIMENTED

Fig. 2. Sedimentation in alkaline sucrose gradients of nuclear DNA from unirradiated and UV irradiated cells pulse-labeled for 15 min. Total counts layered for each gradient are given in parentheses. DNA from unirradiated cells of strain LP841-13B (radl pO) given a 15 min pulse and 0 min (. . . . . ; 53,459) or 30 min chase ( . . . . . ; 58,666) in high uracil medium; DNA from unirradiated cells labeled for several generations ( ; 52,549); DNA from cells exposed to 2.5 J/m 2 UV light, pulse labeled for 15 rain, then given a 0 rain chase ( . . . . . ; 25,620) in high uracil medium. No difference in sedimentation pattern was observed in cells given a 30 rain chase following exposure to 2.5 Jim 2 UV light prior to spheroplast formation. Centrifugation was at 4° C at 19,000 rpm for I6 h 37 min in the SW41 rotor

. ,,,,,

6 D:

>-

o~

\

o

<,~

:'

?'

< ne

;,,

t'i

z

x 2

\ iI ,{M L

ill

I

•

.,;:,

i

i

'1 t!

i"ffl

i

i

!

',~ .~'t ss ". :

\,:,.,t t, !it: k,

,

' i

75

' :i

,. ¢ . ]

..

!

', it

i..'!i

;i

,

•" "~'-"': IOO

"}i

tq } .." / l ,:i i ,J .!, :. :i

I

,

I:

: ~

"k,~'; i

i

I

50

,

,

i

,

Mn ( x 10

6)

Expected b

Observed

26.2 I5.1 10.3 8.0

136 29.I 17.7 12.3 7.8

i

o

IO

Number of dimers induced per single stranded DNA molecule ~

,

,

,

,

,

25

bottom top RELATIVE DISTANCE SEDIMENTED

Fig. 3. Sedimentation in alkaline sucrose gradients of nuclear DNA synthesized after exposure of cells to various UV fluences. Logarithmically growing cells of LP841-13B (radl po) were irradiated with various UV fluences, pulse labeled for 15 min, then chased for 30 min prior to conversion to spheroplasts. Centrifugation was at 4° C at 24,000 rpm for 17 h 22 min in the SW41 rotor. Total counts layered for each gradient are given in parentheses. DNA from ceils exposed to 1.3 ( ; 39,929), 2.5 (. . . . . ; 26,252), 3.8 ( . . . . . , 19,416) and 5 J/m 2 (. . . . . . . ; 14,585), respectively

0 4.2 8.0 12.2 16.0

After exposure to the fluences indicated above, cells were pulse labeled for 15 min and chased for 30 rain prior to spheroplast formation and sedimentation in alkaline sucrose gradients as described in Fig. 3 The observed Mn of 136 × 10~ daltons for single stranded DNA from the unirradiated tad1 po strain, which was smaller than the expected Mn of 267 x 106 daltons for single stranded DNA in an average yeast chromosome (Lauer et al. 1977), was used to calculate the number of dimers expected per single stranded DNA molecule as a function of fluence. The fluence rate used was 3.2 dimers per J/m 2 per 136 x 106 daltons of DNA, from the data of Unrau et al. (1973) b Calculated by dividing 136 x 106, the observed Mn of single stranded DNA from unirradiated cells, by n + 1, where n=the number of dimers induced at a given UV fluence

also prevented further incorporation of label into D N A of unirradiated cells (Rivin and Fangman 1980, and our results).

Alkaline Sucrose Sedimentation fo Pulse Labeled DNA from Unirradiated and UV Irradiated radl pO Cells In unirradiated cells, D N A synthesized during a 15 rain pulse sedimented slower in alkaline sucrose gradients than D N A from uniformly labeled cells (Fig. 2). A 30 rain chase in high uracii medium was sufficient to convert the initially slowly sedimenting D N A to the size of D N A from uniformly labeled cells. In unirradiated RAD+ po cells, it also takes about 30 rain for pulse labeled D N A to reach the size of D N A from uniformly labeled cells (Johnston and Williamson 1978). However, D N A synthesized during a 15 rain pulse in radl po cells exposed to 2.5 J/m 2 UV light sedimented slower in alkaline sucrose gradients than pulse labeled D N A from unirradiated cells (Fig. 2), and a 30 rain chase had no effect on the sedimentation rate of pulse labeled D N A from irradiated cells. No single strand breaks were observed in parental D N A following U V irradiation (results not shown). As the UV fluence increased, the D N A synthesized during a 15 rain pulse following U V irradiation decreased in size (Fig. 3). F r o m this fiuence response, an estimate of the correlation between the size of the D N A synthesized after U V irradiation and the number of pyrimidine dimers induced in parental D N A was made. The results in Table 2 indicate a good agreement between the size of the newly synthesized D N A and the average interdimer distance in parental DNA. In recent studies of diCaprio and Cox (1981), the size of D N A synthesized after U V irradiation in a radl p+ strain was about 6 x larger than the interdimer distance. However, in their studies, cells had been grown in yeast extract-peptone-dextrose (YPD) medium prior to U V irradiation, labeled after UV irradiation in Y P D medium for an unusually long period of 1 h, corresponding to twice the length of S phase, and then chased in unlabeled YPD medium. Such long labeling periods in their experimental conditions would make it difficult to determine

474

8/ / /

/ u o o

!

..;

!.' ,'kA:

i ,..t

:;

"l: : l >t-

I~

4

~ ~ "..

i/

i: :k~:'J s:

t

•t

F-

4 j

~.'! ~.1 | ::

~,t..i

O

•

O

ee, f~

q

)6'' bottom

'zg ....

go ....

i. ....

o top

RELATIVE DISTANCE SEDIMENTED

n-

°l )o bottom

75

50

25

o top

RELATIVE DISTANCE SEDIMENTED

Fig. 4. Sedimentation in alkaline sucrose gradients of nuclear DNA from cells incubated for different times after UV irrradiation with 2.5 J/m 2. Cells of strain LP841-13B (radl po) were pulse labeled for 15 min after exposure to UVandchased for 30 rain( . . . . . ); t h( ); 2 h (. . . . . ~ ; 3 h (. . . . . ) and 4 h ( ) in high uracil medium prior to conversion to spheroplasts. DNA from unirradiated cells given a 15 rain pulse followed by a 30 min chase gave a sedimentation pattern similar to the 4 h chase sample. Centrifugation was as in Fig. 2

Fig. 5. Sedimentation in alkaline sucrose gradients of nuclear DNA synthesized at different times after UV irradiation. Cells of strain LP841-13B (radl po) were exposed to 2.5 J/m 2, incubated in medium for various times, then pulse labeled for 15 rain and chased for 30 min prior to spheroplast formation. DNA from cells pulse labeled at 30 min ( . . . . . ); 3 h (. . . . . ) and 4 h ( . . . . -) after UV irradiation. DNA from unirradiated cells given a 15 rain pulse followed by a 30 rain chase ( ). Centrifugation was as in Fig. 2

the size of newly synthesized nuclear D N A due to the possibility of postreplication repair occurring during the labeling period. Their chase conditions are not likely to result in effective dilution of the radioisotope, since log phase cells grown in YPD apparently contain large enough precursor pools so that it is not possible to terminate a pulse by diluting the pools even with excess unlabeled precursors (Johnston and Williamson 1978). Another difficulty in interpreting the diCaprio and Cox results stems from the use of strains containing mitochondrial D N A . Since the position of the newly synthesized nuclear D N A after UV irradiation overlaps that of the mitochondrial D N A in alkaline sucrose gradients, it is not possible to make calculations of the size of the newly synthesized nuclear DNA. These considerations make interpretation of their results in various mutants difficult.

after UV irradiation no longer contains gaps. These observations suggest that the dimers have somehow been modified so that although still present, they can now be bypassed by the replication machinery. In yeast, we found that dimers continued to act as blocks for D N A synthesized at various times after UV irradiation, for at least up to 4 h. Pulse labeling of radl pO cells at 0.5, 3 or 4 h after exposure to 2.5 J/m 2 UV light did not substantially alter the sedimentation profile of the D N A , however, some fast sedimenting material was detected in D N A from cells pulsed at 3 and 4 h after UV irradiation (Fig. 5). This faster sedimenting component may arise from dimer-free templates since postreplication repair is complete by 4 h after UV irradiation.

Effect of Cycloheximide on Postreplieation Repair. Although Postreplication Repair in radl pO. We next determined the time required to convert the low molecular weight D N A synthesized following UV irradiation to high molecular weight. Ceils were UV irradiated at 2.5 J/m 2, pulse labeled for 15 rain and incubated in high uracil medium for various times. As the time of incubation in high uracil medium increased, the D N A sedimented faster, until by 4 h after the pulse, sedimentation was like that of D N A from uniformly labeled unirradiated cells or unirradiated cells given a 15 rain pulse and 30 rain chase (Fig. 4).

Dimers Continue to Inhibit DNA Synthesis at Various Times after UV Irradiation. We determined whether dimers in template D N A continue to inhibit D N A synthesis at various times after exposure to UV irradiation, since in some eukaryotic cells such as C H O (Meyn and Humphrey 1971), mouse L5178Y (Lehmann and Kirk-Bell 1972), human normal and XP (Buhl et al. 1973) and rat kangaroo cells (Rosenstein and Setlow 1980), even though dimers remain in the D N A , the D N A synthesized at long times

there seems to be an inducible component of postreplication repair in E. coli (Sedgwick 1975), results obtained in eukaryotes are not clear. Some of the difficulty in the interpretation of experiments using the protein synthesis inhibitor cycloheximide (CH) in mammalian cells to assess the inducibility of postreplication repair stems from the fact that CH inhibits both initiation of D N A synthesis as well as D N A chain elongation (Cummins and Rusch 1966). In yeast, however, CH inhibits only initiation of D N A synthesis and D N A replication can continue in the absence of protein synthesis, once D N A synthesis has already initiated (Hereford and Hartwell 1973; Williamson 1973). Thus, unlike the situation in other eukaryotic organisms, essentially a complete round of D N A synthesis can take place in cells treated with 100 gg CH/ml, which produces an immediate cessation of protein synthesis in asynchronous cells. The D N A synthesized under these conditions is normal in size, has the density of nuclear D N A , and functions in cell division (Hereford and Hartwell 1973).

475

A. !

!%..

-

I

* i

i ~,

"l

, %

t:

It

!

ti?.,.,i"

/ s

,I .,:1

j

',\ fi

.

l

, ca

2

t

o 't

>I> I-0
/

:

.......J,,' ~;'~~'~k'."Z ..'" '

o

'"?./i-;':..';'..-.'..,."':""." 'i'. . . . . . . . . .

0

8

I

B.

i

i

=

1

i

i

,

,

,

,

i

,

,

,

,

,

,

,-

B.

,tl

re"

6

-

a. 6 ca

i

I: I:

o

?,~!1

t'¢.i

,i

"]

{ ,~ / \ /~"

I--.

\

",

"

!;

./.y/ 0

"/

"t

0

o

2 i~

'i!

/-Ni

I,-

/

-

i/

4

:

",Z-:

0::

¢ 2

,',

111 I :

[!

',".!.1t t ~,

: :

~J:

-

">~"?""

I00

75

50

25

bottom RELATIVE

0

top DISTANCE

8

C.

SEDIMENTED

Fig. 6A, B. Effect of cycloheximide on postreplication repair. A Cells of LP841-13B (radlp °) were UV irradiated at 2.5 J/m 2, pulsed for 15 min and chased for 30 min with (. . . . . ) or without ( - - ) 100 gg CH/ml followed by a 2.5 h chase without CH. D N A from UV irradiated cells pulsed for 15 rain and chased for 30 min ( . . . . . ). Centrifugation was as in Fig. 2. B As in Fig. 6A except that label was chased 3.5 h after removal of CH

• i"-

4

.1

.."

I' Fig. 7A-C. Postreplication repair in radl radn po mutants. Strains were exposed to 2.5 J/m 2, pulse labeled for 15 rain and chased for 30 rain ( . . . . . ) or 4 h ( . . . . . ). DNA from unirradiated cells given a 15 min pulse and 30 min chase ( - ). Centrifugation was as in Fig. 2. A LP2627-1B (radl rad6 p0). B LP2596-SA (radl rad18 po). As above except that one sample was exposed to 2.5 J/m 2, pulse labeled for 15 rain and chased for 30 min with 100 lag/ml CH followed by an additional 3.5 h chase without CH (. . . . . . ). C LP2614-11C (radl rev3 po)

In o r d e r to d e t e r m i n e the effect o f C H on postreplication repair in yeast, cells were U V irradiated at 2.5 J i m s, pulse labeled and chased for 30 rain in the presence o f 100 gg C H / m l , followed by an a d d i t i o n a l 2.5 h chase in the a b s e n c e o f CH. Cells treated with C H did n o t show the same extent o f p o s t r e p l i c a t i o n repair

1%l ~ i

I Ift t

II t tj

I00 75 50 25 0 bottom top RELATIVE DISTANCE SEOIMENTED

as was observed in u n t r e a t e d cells (Fig. 6A). Similar results were o b t a i n e d in C H - t r e a t e d cells chased for 3.5 h after r e m o v a l o f C H (Fig. 6B). N o effect o f C H was o b s e r v e d o n c o n v e r s i o n o f low molecular weight to high m o l e c u l a r weight D N A in unirradiated cells. In addition, C H did n o t affect the viability o f

476

i

/i ,,--\,

>N

complete postreplication repair to take place in the radl RAD6 pO mutant. Substantial inhibition of postreplication repair occurs in the radl rad18 pO mutant exposed to 1.3 (data not shown) and 2.5 J/m 2 UV irradiation (Fig. 7B), and the postreplication repair that does take place is not inhibited by CH, unlike results obtained in the radl pO single mutant. In the radl rev3 pO mutant, on the other hand, no inhibition of postreplication repair occurs and the process is complete by 4 h after UV irradiation (Fig. 7C), as is observed in the radl pO single mutant. Thus, although each of these three genes (rad6, radl8 and rev3) belong to the same epistasis group, their effect on postreplication repair is quite different.

..

/!!

~! i

'7...':

!,i

4

o

I,"

i

"~ii

"

:

I."

",

Effect of tad52, a Recombination Deficient Mutant, on Postreplication repair

unirradiated or UV irradiated cells, and no D N A degradation after treatment of UV irradiated cells with CH was observed.

Since most of postreplication repair in E. coli is recombinational, while in mammalian cells, it is probably nonrecombinational, we then determined what role recombination plays in postreplication repair in yeast. For this purpose, we examined the process in the rad52 mutant, which is defective in spontaneous and radiation induced homologous mitotic recombination (Resnick 1975; Prakash et al. 1980; Malone and Esposito 1980), in meiotic recombination between homologous chromosomes (Prakash et al. 1980; Game et al. 1980), and in UV induced sister chromatid recombination in haploids (Prakash and Taillon-Miller 1981). If recombinational repair plays an important role in postreplication repair in yeast, we might expect the rad52 mutant to be blocked in postreplication repair. However, even though rad52 strains are recombination deficient, postreplication repair is only partially inhibited in the radl rad52 pO strain, and the postreplication repair that does take place is not inhibited by CH (Fig. 8). No significant degradation of newly-synthesized D N A was detected in cells of ali the double mutant strains given a 4 h incubation following UV irradiation.

Effect of Genes in the rad6 Epistasis Group on Postreplication Repair

Discussion

In order to understand the genetic control of postreplication repair in yeast, we have determined the effect of various mutations, conferring sensitivity to UV and affecting error prone repair. Like recA mutants of E. coli, rad6 mutants of yeast are very sensitive to the lethal effects of UV, ionizing radiation and alkylating agents (Cox and Parry 1968 ; Lawrence et al. 1974; Game and Mortimer 1974; Prakash 1974). In addition, like the recA + gene of E. coli, the RAD6 gene is required for radiation mutagenesis (Lawrence et al. 1974; Lawrence and Christensen 1976). However, unlike recA, rad6 mutants are proficient in spontaneous and UV induced homologous recombination in diploids (Hunnable and Cox 1971; Montelone et al. 1981) as well as sister chromatid mitotic recombination in haploids (Montelone et al. 1981). We have examined the effect on postreplication repair of the rad6-1 mutation and of two other mutations of the rad6 epistasis group: radl8-2 and rev3-1. The radl8 mutants are very UV sensitive and, like rad6 mutants (Prakash 1977), are proficient in excision of pyrimidine dimers (Reynolds and Friedberg 1981). The rev3 mutants are only moderately UV sensitive (Lemontt 1971a) but show no UV induced mutagenesis at a wide variety of loci (Lemontt 1973 ; Lawrence and Christensen 1979). These mutants are also proficient in genetic recombination (Lemontt 1971c). Postreplication repair is blocked in the radl rad6 pO mutant given a 4 h incubation period in growth medium following UV irradiation (Fig. 7A), even though this is a sufficient time for

The size of the nuclear D N A synthesized after UV irradiation in the excision defective tad1 mutant of yeast corresponds approximately to the average distance between dimers in parental D N A and upon subsequent incubation of cells in growth medium, the D N A assumes the molecular weight observed in uniformly labeled unirradiated cells or in pulse-chased D N A in unirradiated cells. This process of postreplication repair is complete within 4 h after exposure to 2.5 J/m 2 of UV light. The alkaline sucrose sedimentation profiles of D N A from cells pulse labeled at different times after UV irradiation indicate that dimers remain as blocks for D N A synthesis for at least 4 h after UV irradiation. The use of mutants showing different capacities for genetic recombination and UV induced mutagenesis might allow us to assess the role of these processes in postreplication repair• The rad6 mutant is recombination proficient but deficient in UV induced mutagenesis and carries out little or no postreplication repair. This might suggest that genetic recombination does not play a significant role in postreplication repair. The results obtained in the rad52 mutant are consistent with recombinational repair contributing significantly to postreplication repair, although not as much as in ~F. coll. The apparent contradiction with the rad6 and rad52 mutants as to the role of recombination in postreplication repair could be reconciled by assuming that in the tad6 mutants, the observed UV induced genetic recombination in haploids occurs at times other than S phase, e.g., during

O

i

IO0 bottom

t

i

~

75

i

,

,

,

,

50

,

,

,

,

i

i

i

i

25

0 top

RELATIVE DISTANCE SEDIMENTED

Fig. 8. Postreplication repair in the radl rad52p ° mutant. Strain LP2630-12A (radl rad52p °) was exposed to 2.5 J/m z, pulse labeled for 15 rain and chased for 60 min ( . . . . . ) or 4.5 h (. . . . . ). One sample was exposed to 2.5 J/m 2, pulse labeled for 15 min and chased for 30 min with 100 gg/ml CH followed by an additional 4 h chase without CH (- . . . . . . ). DNA from unirradiated cells given a 15 min pulse and a 60 rain chase ( ). Centrifugation was as in Fig. 2. It was necessary to give the radl rad52 pO strain a 60 rain chase rather than a 30 rain chase, as was done with all the other strains, since strand joining, even in unirradiated cells, was apparently slowed down in this mutant

477 G2 phase and in diploids, in G1 (Fabre 1978) and G2 phases, whereas the tad52 mutants are defective in U V induced recombination in all phases. The role of the R A D 6 gene may be regulatory whereas the R A D 5 2 gene may participate directly in recombinational postreplication repair. Results with the rev3 mutant, which is entirely deficient in UV induced mutagenesis, but proficient in postreplication repair suggest that error prone repair could at best account for only a small c o m p o n e n t of postreplication repair and, therefore, most of postreplication repair in S. cerevisiae is error free. A substantial a m o u n t of postreplication repair in radl is inhibited by CH, suggesting that a portion of postreplication repair is inducible, or that a c o m p o n e n t which affects the efficiency of postreplication repair is inducible. In b o t h radl radl8 and radl rad52 mutants, C H does not affect the postreplication repair that takes place indicating that the R A D I 8 and R A D 5 2 genes are probably involved in inducible postreplication repair. In conclusion, our results suggest that b o t h recombinational and n o n r e c o m b i n a t i o n a l repair mechanisms play a role in postreplication repair and most of postreplication repair is error free. Acknowledgements. I thank Patricia Taillon-Miller for technical assistance, This work is based on work performed partially under NIH grant GM1926I and under Contract Number DE-AC02-76EV03490 with the U.S. Department of Energy at the University of Rochester Department of Radiation Biology and Biophysics and has been assigned Report No. UR-3490-2016. References Boram WR, Roman H (1976) Recombination in Saccharomyces cerevisiae : a DNA repair mutation associated with elevated mitotic gene conversion. Proc Natl Acad Sci USA 73:2828-2832 Buhl SN, Setlow RB, Regan JD (1972) Steps in DNA chain elongation and joining after ultra-violet irradiation of human cells. Int J Radiat Biol 22:417 424 Buhl SN, Setlow RB, Regan JD (1973) Recovery of the ability to synthesize DNA in segments of normal size at long times after ultraviolet irradiation of human cells. Biophys J 13:1265-1275 Buhl SN, Setlow RB, Regau JD (1974) DNA repair in Potorus tridactylus. Biophys J 14:791 803 Burgi E, Hershey AD (1963) Sedimentation rate as a measure of molecular weights of DNA. Biophys J 3:309-321 Clark RW, Wever GH, Wiberg JS (1980) High-molecular-weight DNA and the sedimentation coefficient: a new perspective based on DNA from T7 bacteriophage and two novel forms of T4 bacteriophage. J Virol 33:438-448 Cox, BS, Game J (1974) Repair systems in Saccharomyces. Mutat Res 26 : 257 264 Cox BS, Parry JM (1968) The isolation, genetics and survival characteristics of ultraviolet-light sensitive mutants in yeast. Mutat Res 6:3755 Cummins JE, Rusch HP (1966) Limited DNA synthesis in the absence of protein synthesis in Physarum polycephalum. J Cell Biol 31:577583 diCaprio L, Cox BS (1981) DNA synthesis in UV-irradiated yeast. Mutat Res 82:69-85 Fabre E (1978) Induced intragenic recombination in yeast can occur during the G1 mitotic phase. Nature 272:795-798 Game JC, Mortimer RK (1974) A genetic study of X-ray sensitive mutants in yeast. Mutat Res 24:281-292 Game JC, Zamb TJ, Braun RJ, Resnick M, Roth RM (1980) The role of radiation (rad) genes in meiotic recombination in yeast. Genetics 94:51~68 Goldring ES, Grossman LI, Krupnick D, Cryer DR, Marmur J (1970) The petite mutation in yeast. Loss of mitochondrial deoxyribonucleic acid during induction of petites with ethidium bromide. J Mol Biol 52:323 335

Hanawalt PC, Copper PK, Ganesan AK, Smith CA (1979) DNA repair in bacteria and mammalian cells. Annu Rev Biochem 48:783 836 Hereford LM, Hartwell LH (1973) Role of protein synthesis in the replication of yeast DNA. Nature 244:i29-131 Hill WE, Fangman WL (1973) Scission of Escherichia coli deoxyribonucleic acid in alkali. Biochem 12:1772-1774 Hunnable EG, Cox BS (197I) The genetic control of dark recombination in yeast. Mutat Res 13 : 297-309 Johnston LH, Williamson DH (1978) An alkaline sucrose gradient analysis of the mechanism of nuclear DNA synthesis in the yeast Saccharomyces cerevisiae. Mol Gen Genet 164:217-225 Kutter EM, Wiberg JS (1968) Degradation of cytosine-containing bacterial and bacteriophage DNA after infection of Escherichia coli B with bacteriophage T4D wild-type and with mutants defective in genes 46, 47 and 56. J Mol Biol 38:395-411 Lauer GD, Roberts TM, Klotz LC (1977) Determination of the nuclear DNA content of Saccharomyces cerevisiae and implications for the organization of DNA in yeast chromosomes. J Mol Biol 114:50%526 Lawrence CW, Christensen RB (1976) UV mutagenesis in radiationsensitive strains of yeast. Genetics 82 : 207 232 Lawrence CW, Christensen RB (1979) Ultraviolet-induced reversion of cycl alleles in radiation-sensitive strains of yeast. III. rev3 mutant strains. Genetics 92 : 397-408 Lawrence CW, Stewart JW, Sherman F, Christensen R (1974) Specificity and frequency of ultraviolet-induced reversion of an iso-l-cytochrome c ochre mutant in radiation-sensitive strains of yeast. J Mol Biol 85:137 162 Lehmann AR (1972) Postreplication repair of DNA in ultravioletirradiated mammalian cells. J Mol Biol 66:319 337 Lehmann AR, Kirk-Bell S (1972) Postreplication repair of DNA in ultraviolet-irradiated mammalian cells. No gaps in DNA synthesized late after ultraviolet irradiation. Eur J Biochem 31:438-445 Lemontt JF (197Ia) Mutants of yeast defective in mutation induced by ultraviolet light. Genetics 68 : 21-33 Lemontt JF (1971b) Pathways of ultraviolet mutability in Saccharomyces cerevisiae. I. Some properties of double mutants involving uvs9 and rev. Mutat Res 13:31I 317 Lemontt JF (1971c) Pathways of ultraviolet mutability in Saccharomyces cerevisiae. II. The effect of rev genes on recombination. Mutat Res 13:319 326 Lemontt JF (1973) Genes controlling ultraviolet mutability in yeast. Genetics Supplement 73 : 153 159 Malone RE, Esposito RE (1980) The RAD52 gene is required for homothallic interconversion of mating types and spontaneous mitotic recombination in yeast. Proc Natl Acad Sci USA 77 : 503-507 McKee RH, Lawrence CW (1979) Genetic analysis of gamma-ray mutagenesis in yeast. I. Reversion in radiation-sensitive strains. Genetics 93:361 373 Meyn RE, Humphrey RM (197i) Deoxyribonucleic acid synthesis in ultraviolet-light irradiated Chinese hamster cells. Biophys J 11 : 295301 Montelone BA, Prakash S, Prakash L (1981) Recombination and mutagenesis in rad6 mutants of Saecharomyees cerevisiae: evidence for multiple functions of the RAD6 gene. Mol Gen Genet 184:410-415 Painter RB (1978) Does ultraviolet light enhance postreplication repair in mammalian cells. Nature 275:243-245 Park SD, Cleaver JE (1979) Postreplication repair: questions of its definition and possible alteration in xeroderma pigmentosum cell strains. Proc Natl Acad Sci USA 76:3927 3931 Prakash L (1974) Lack of chemically induced mutation in repairdeficient mutants of yeast. Genetics 78 : 1101-1118 Prakash L (1975) Repair of pyrimidine dimers in nuclear and mitochondrial DNA of yeast irradiated with low doses of ultraviolet light. J Mol Biol 98:781 795 Prakash L (1977) Repair of pyrimidine dimers in radiation-sensitive mutants rad3, rad4, rad6 and rad9 of Saccharomyces cerevisiae. Murat Res 45:13-20 Prakash L, Prakash S (1980) Genetic analysis of error-prone repair systems in Saceharomyees eerevisiae. In: Generoso WM, Shelby

478 MD and deSerres FJ (eds) DNA repair and mutagenesis in eukaryotes. Plenum Press, New York, p 141 Prakasb L, Taillon-Miller P (198I) Effects of the tad52 gene on sister chromatid recombination in Saccharomyces cerevisiae. Curr Genet 3 : 247-250 Prakash S, Prakash L, Burke W, Montelone BA (1980) Effects of the RAD52 gene on recombination in Saccharomyces cerevisiae. Genetics 94: 31-50 Resnick MA (1969) Genetic control of radiation sensitivity in Saccharomyces cerevisiae. Genetics 62 : 519-531 Resnick MA (1975) The repair of double strand breaks in chromosomal DNA of yeast. In: Hanawalt PC and Setlow RB (eds) Molecular mechanisms for repair of DNA. Plenum Press, New York, p 549 Reynolds RJ and Friedberg EC (1981) Molecular mechanisms of pyrimidine dimer excision in Saccharomyces cerevisiae: Incision of ultraviolet-irradiated deoxyribonucleic acid in vivo. J Bacteriol 146 : 692-704 Rivin CJ, Fangman WL (1980) Cell cycle phase expansion in nitrogenlimited cultures of Saccharomyces cerevisiae. J Cell Biol 85:96-107 Rupp WD, Howard-Flanders P (1968) Discontinuities in the DNA synthesized in an excision-defective strain of Escherichia coli following ultraviolet irradiation. J Mol Biol 31:291 304

Rupp WD, Wilde CE, Reno DL, Howard-Flanders P (1971) Exchanges between DNA strands in ultraviolet-irradiated Escherichia coli. J Mol Biol 61:25-44 Rosenstein BS, Setlow RB (1980) DNA repair after ultraviolet irradiation of ICR2A frog cells. Pyrimidine dimers are long acting blocks to nascent DNA synthesis. Biophys J 31 : 196-206 Sarasin AR, Hanawalt PC (1980) Replication of ultraviolet-irradiated Simian virus 40 in monkey kidney cells. J Mol Biol I38:299-319 Sedgwick SG (1975) Inducible error-prone repair in Escherichia coli. Proc Natl Acad Sci USA 72:2753 2757 Snow R (1967) Mutants of yeast sensitive to ultraviolet light. J Bacteriol 94: 571-575 Unrau P, Wheatcroft P, Cox B, Olive T (1973) The formation of pyrimidine dimers in the DNA of fungi and bacteria. Biochim Biophys Acta 312:626-632 Williamson DH (1973) Replication of the nuclear genome in yeast does not require concomitant protein synthesis. Biochem Biophys Res Comm 52:731 740 Communicated by B.A. Bridges Received August 10, 1981