Isolation and characterization of the RAD2 gene of Saccharomyces cerevisiae

Gene, 30 (1984) 121-128

121

Elsevier GENE

1066

Isolation and characterization of the RAD2 gene of Sacclrammyces cerevisiae (DNA repair; Escherichiu coli; yeast; incision; excision; pyrimidine dimers; deletion mutants; UV sensitivity)

David R. Higgins,

Louise Prakash *, Paul Reynolds * and Satya Prakash

Department ofBiology, Universityof Rochester, Rochester, NY 14627, Tel. (716) 275-3847, and *Department ofRadiation Biology and Biophysics, University of Rochester School of Medicine, Rochester, NY 14642 (U.S.A.), Tel. (716) 275-2656 (Received

March

(Revision

received

(Accepted

28th, 1984) May 7th, 1984)

May 14th, 1984)

SUMMARY

We have cloned the RAD2 gene of Saccharomyces cerevisiae and used it to determine the size and direction of its transcript and to make rud2 deletion mutants. The RAD2 gene encodes a 3.3-kb transcript and the direction of transcription is leftwards, from EcoRI towards BglII. Deletions of the RAD2 gene have no effect on viability of vegetative cells or spores, or on sporulation.

INTRODUCTION

Diverse mechanisms have evolved for incising DNA at pyrimidine dimers induced by UV. Phage T4 endonuclease V, a 16-kDal polypeptide, contains two activities, a pyrimidine dimer glycosylase and an AP DNA endonuclease which act on UV irradiated DNA in a sequential fashion. The glycosylase cleaves the N-glycosyl bond between the 5’ pyrimidine of a dimer and the corresponding sugar, while the AP endonuclease subsequently hydrolyzes the phosphodiester bond on the 3’ side of the AP site (Nakabeppu and Sekiguchi, 198 1; Nakabeppu et al., 1982). Micrococcus luteus UV endonuclease, an 18 kDa.l polypeptide, also acts in a similar manner (Haseltine et al., 1980; Grafstrom et al., 1982). Abbreviations: idinic;

ApR, ampicillin-resistant;

A, deletion;

polyadenylated;

kb, kilobases

UV, ultraviolet

0378-I 119/84/$03.00

0

AP, apurinic/apyrim-

or kilobase

pair;

poly(A)+,

light.

1984 Elsevier

Science

Publishers

In Escherichia coli, dimer incision requires three proteins, the products of the uvrA, uvrB and uvrC genes, which form a complex necessary for incision at pyrimidine dimers. The products of these three genes, which are proteins of M, 114000 (Sancar et al., 1981a), 84000 (Sancar et al., 1981b), and 70000 (Sancar et al., 1981c), respectively, together possess UV endonuclease activity, but unlike the T4 or the h4. luteus enzymes, do not have gloycosylase activity. The uvrABC-coded endonuclease makes two cuts in UV irradiated DNA, one at the eighth phosphodiester bond on the 5’ side of the pyrimidine dimer and the other at the fourth or fifth phosphodiester bond on the 3’ side of the pyrimidine dimer (Sancar and Rupp, 1983; Yeung et al., 1983). In eukaryotic organisms, excision repair appears to be a much more complex process. At least seven complementation groups have been identified for xeroderma pigmentosum, the human equivalent of the E. coli excision defective mutants (Keijzer et al.,

122

1979). In the yeast S. cerevisiae, ten genes are required for excision repair of pyrimidine dimers and at least five of these have been shown to be required for the first step - that of incision (Wilcox and Prakash, 1981; Reynolds and Friedberg, 198 1). Elucidation of the mechanism of incision at pyrimidine dimers in DNA in eukaryotic organisms requires the characterization of the proteins involved in this reaction. We have been using S. cerevisiae as a model eukaryotic system in which to answer this question and have already cloned four of the genes involved in dimer incision, RADl (Higgins et al., 1983a), RAD2, the subject of this communication, RAD3 (Higgins et al., 1983b), and RADIO (unpublished results). It is hoped that the isolation of the RAD genes involved in dimer incision will facilitate the identification and characterization of the proteins they encode and enable us to elucidate the dimer incision mechanism utilized in this organism. In this paper, we describe the isolation of the RAD2 gene by complementation of rad2 mutants, show that the RAD2 gene encodes a 3.3-kb transcript, and that rud2 deletion mutants are viable.

MATERIALS

AND METHODS

(a) Yeast and bacterial strains

The following yeast strains were used: 7799-5C, his4-17 ura3-52 RAD+ (obtained from G. Fink); DBY747, MATa h&3-dl leu2-3 leu2-112 trpl-289 ura3-52 RAD+ (obtained from D. Botstein); GP19-21C, MATa ade2-1 trpl-289 ura3-52

MATa

RAD’ ; LP2641-lA, MATa lys2-1 trpl-289 rad2-6; LP2641-4C, MATa ade2-1 lys2-1 trpl-289 ura3-52 rad2-6; LP2691-14C, MATa his3-Al leu2-3 leu2-112 4~2-1 trpl-289 ura3-52 rad2-6; LP2789-lB, MATa adel his3-Al his7 leu2-3 leu2-112 ura3-52 rad2-5; LP2804-5B, MATa his1 his3-Al leu2-3 leu2-112 trpl-289 ura3-52 rad2-2; LP2804-18A, MATa his1 leu2-3 leu2-112 ura3-52 RAD + ; LP2805-8B, MATa his3-Al trpl-289 ura3-52 rad2-3. E. coli strain HBlOl, obtained from H. Eberle,

was used to propagate all plasmids while JM 103 was used to propagate Ml3 derivative phages. Yeast strains containing plasmids were grown in synthetic media lacking uracil to maintain selection for the plasmid-bearing cells.

(b) Plasmids

Plasmids used were YEp24, which is pBR322 containing part of the yeast 2~ plasmid with its origin of replication and the yeast URA3 gene (Botstein et al., 1979; Carlson and Botstein, 1982) and YIPS, which is pBR322 containing the yeast URA3 gene with no yeast replication origins (Botstein et al., 1979; Scherer and Davis, 1979). YEp24 is maintained in high copy number in yeast because of the 2~ replication origin which confers the ability to replicate autonomously in yeast. In addition to these two plasmids, we constructed two other vectors to facilitate subcloning the RAD2 gene. The first, designated pTB199, was derived from YIPS. The 2.2-kb EcoRI fragment of the yeast 2~ circle DNA from YEp24 was subjected to BAL3 1 digestion to remove the EcoRI sites. The termini were filled in with the Klenow fragment of E. coli DNA polymerase I and the resulting fragment was ligated into YIp5 that had been linearized with Bali. The recombinant plasmid, designated pTB 199, contains a unique EcoRI site in the pBR322 portion of the plasmid and two Hind111 sites, one in the pBR322 portion 31 bp from the EcoRI site, and one in the 2~ yeast DNA insert. In order to remove the Hind111 site in the 2~ insert, plasmid pTB 199 was subjected to partial digestion with HindIII, the linear DNA purified by electroelution from an agarose gel, the termini filled in with the E. coli DNA polymerase I Klenow fragment and joined with T4 DNA ligase. Two types of plasmids were obtained, one in which the Hind111 site located in the pBR322 DNA had been destroyed and one in which the Hind111 site located in the 2~ insert had been destroyed. This latter plasmid, designated pTB220, was used for subcloning. Growth of E. coli containing plasmids was as described by Norgard et al. (1979) but with nucleosides omitted. The cleared lysate procedure of Clewell and Helinski (1969) was used to purify plasmid DNA from E. coli but with 0.1 y0 Triton X-100 instead of Brij 58. (c) Transformation

procedures and UV irradiation

Transformation of yeast was carried out by treating intact cells with lithium acetate to promote DNA uptake (Ito et al., 1983). The response of various yeast strains to UV irradiation was deter-

I23

mined from spot tests or survival curves following exposure to UV irradiation as described previously (Prakash and Prakash, 1977).

were prepared by cloning the 2.0-kb BgZII-EcoRI fragment of pLP1 (Fig. 1) into M13mp8 and M13mp9 phages (Messing and Vieira, 1982) and DNA labeled as described by Hu and Messing (1982). About lo6 cpm were used for each RNADNA hybridization. All autoradiographs were exposed on Kodak XL- 1 (X-Omat) film at -70’ C with DuPont Lightning Plus intensifying screens.

(d) Purification of nucleic acid, RNA blotting, hybridization and autoradiography A modification of the Hirt (1967) procedure was used for purification of plasmid DNA from yeast. The method of Ish-Horowitz and Burke (198 1) for DNA extraction from plasmid or cosmid cultures was used for screening large numbers of E. coli containing plasmids. DNA restriction fragments were electrophoresed as described (Higgins et al., 1983b). Total RNA was prepared from exponentially growing yeast cells as described (Reed et al., 1982). Poly(A) + RNA was obtained by binding total RNA to oligodeoxythymidylate cellulose (type 3, Collaborative Research) as described (Higgins et al., 1983a). RNA was fractionated by electrophoresis in formaldehyde agarose gels, transferred to Gene Screen (New England Nuclear), and hybridized with 32P-labeled DNA, using Method II as described by the manufacturer, which does not utilize dextran sulfate. Single-strand DNA hybridization probes

r

I

kb 0

1

RESULTS

(a) Isolation of the RAD2 gene We isolated the RAD2 gene of S. cerevisiae by complementation of a rad2-2 mutant for UV resistance using a yeast genomic bank constructed in the vector YEp24 by Carlson and Botstein (1982). This DNA was used to transform the uracil requiring (Ura-), UV sensitive (Rad-) strain LP2804-5B (ura3-52 rad2-2) to uracil prototrophy (Ura’ ). Approx. 5000 transformants were screened for UV resistance by spot testing and one UV-resistant (Rad’) colony was obtained. Plasmid DNA from

1

I

1

1

I

I

I

I

1

2

3

4

5

6

7

8

9

IO Vector

Complementatlon of rad2

pLP1

YEp24

+

pLP5 pLP6

YEp24 Ylp5

+ +

pLP6

pTBl99

pLP7

YEp24

1

pLP9

Ylp5

1

PLPlO

pTB220

Plasmid H*V

A

Bg K Bg

SI

___

I

l-l

I

K’

1

w

I

5’

3’

Fig. 1. Restriction DNA segment and asterisks

map of the yeast DNA insert in plasmid

in pLPI,

pLP1 and complementation

given by the open bar, is in the BamHI

denote the Sau3A-BumHI

junctions

between

the insert and the plasmid.

A,AvuI;B,BamHI;Bg,BglII;E,EcoRI;H,HindIII;K,KpnI;

S,SalI;

to yield pLP7. AvuI, BglII and EcoRV sites were mapped is not meant to indicate

their absence

and XbuI. The open bars below the map of plasmid The 2.0-kb BglII-EcoRI

fragment

generate

probes for hybridization

single-stranded

The wavy line with the arrowhead

used to construct represents

pLPl plasmid

subclones.

Insert of the yeast

sites are not present

DNA segments

pLP9 was also inserted

of pLPI

of transcription

portion

of pLP1

in pLP1: PstI, PvuII, SucII, SphI vectors

and M13mp9

the size and direction

of the RAD2 mRNA.

are as follows:

theKpnIsitedestroyed

from the rightward

cloned in various

into M13mp8

YEp24 DNA

enzymes

K+denotes

pLP8 and their omission

The following

represent

Symbols for restriction

to poly(A) + RNA from yeast to determine the direction

of pLPI

SI,SucI;V,EcoRV;X,XhoI.

only in plasmid

from that DNA segment.

pattern

site of the tet gene of YEp24. The thin line represents

phages

as indicated. and used to

of the RAD2 transcript.

this yeast strain was prepared by a modification of the Hirt (1967) procedure and used to transform E. coli HBlOl to ApR. Plasmid DNA isolated from the E. coli ApR transformants converted the ura3-52 rad2-2 strain LP2804-5B to Ura+ and Rad+ . The complementing plasmid, designated pLP 1, was shown to carry a 9.3-kb insert of yeast DNA, whose restriction map is given in Fig. 1. Plasmid pLP 1 was also found to restore wild-type levels of UV resistance to rad2-3, rad2-5 and rad2-6 mutants (Fig. 2). (b) Genetic mapping of the DNA segment complementing the rudd mutants

10 1

o*ooo’o 1

20I

30I

UV FLUENCE Fig. 2. Survival

after UV irradiation

without

BAD2 insert-containing

various

grown

in minimal

nutrients selective

medium.

(Prakash

ura3-52

0,

leu2-112 /ys2-1 trpl-289 v,

LP2789-1B

of yeast strains plasmids.

was

with and

strains

as

RAD+; trpl-289

0,

were grown in

described

previously his1

MATa his1

LP2804-5B,

ura3-52 rad2-2;

0,

LP2804x ,

A, LP2804-5B[YEp24];

LP2691-14C,

MAT

his3-Al

IeuZ-3

ura3-52 rad2-6; H, LP2691-14C[pLPl];

MAT adel his3-Al his7 IeuZ-3 leu2-112 ura3-52

rad2-5; V, LP2805-8B, MATa his3-Al rrpl-289 ura3-52 rad2-3; 4, DBY747[pLP9];

this strain contains

the deletion oftheRAD2

gene. The survival ofthe rad2-3 strain LP2805-8B strain

LP2789-1B

containing

and the rad2-5

the rad2 complementing

pLP1 was similar to that of the RAD+ strain

insert

Cells were

with the appropriate

containing

A, LP2804-5B(pLP5];

LP2804-5B[pLPlO];

(c) Location of the RAD2 gene in the cloned DNA

1977). 0, Strain LP2804-18A,MATa

IeuZ-3 leu2-112

SB[pLPl];

40I

(J/m*)

supplemented

Irradiation

and Prakash,

leu2-3 leu2-112 his3-Al

medium

except that plasmid

The 6.4-kb HindIII-XhoI fragment of pLP1 was ligated into Hind111 + SalI-restricted YIPS, a plasmid lacking sequences required for autonomous replication in yeast, and therefore capable of transformation only by homologous integration into the yeast genome (Scherer and Davis, 1979). The plasmid generated, designated pLP8 (Fig. 1) was used to transform the ura3-52 rud2-2 strain LP2804-5B to Ura + . All Ura + transformants were radiation resistant, indicating that RAD2 function is present in the 6.4-kb HindIII-XhoI fragment. Three Ura+ Rad’ transformants were mated to the ura3-52 Rad’ strain 7799-5C and diploids sporulated. Genetic analysis of 16, 17 and 40 tetrads from these three crosses yielded only Rad’ spores, indicating that integration of the plasmid pLP8 had occurred at the rad2 site and that we had indeed cloned the RAD2 gene.

plasmid

LP2804-18A.

In order to localize the RAD2 gene within the cloned DNA insert, we tested the ability of various DNA fragments to confer UV resistance on the rad2-2 strain LP2804-5B. As expected, plasmid pLP5, constructed by removing the 3.6-kbXhoI-Sal1 fragment of pLP1, was found to retain the ability to complement rad2 (Figs. 1 and 2). Thus, the RAD2 gene, whether present in multicopy as in plasmid pLP5, or in single copy as in plasmid pLP8, confers wild-type levels of UV resistance. The 4.8-kb EcoRIBamHI fragment of pLP1, cloned in the 2~ vector pTB 199 to generate plasmid pLP6, did not complement rad2 (Fig. 1). Further localization was achieved

125

resulting blunt ended linear DNA was religated to generate plasmid pLP7, lacking the KpnI site originally present in pLP1. Since pLP7 did not contain Rad2 function (Fig.l), the RAD2 gene must extend to the left of the KpnI site. The plasmid pLPl0 was constructed in order to determine how far to the right of the KpnI site the RAD2 gene extends. The 4.5-kb HindIII-EcoRI fragment of pLP5 was inserted into Hind111 + EcoRI-restricted pTB220 to generate the plasmid pLP10, which was found to lack Rad2 function (Figs. 1 and 2). All these results taken together suggest that the RAD2 gene extends from the left of the KpnI site to the right of the EcoRI site at 4.5 kb, and that the 2.0-kb BglII-EcoRI fragment between 2.5 kb and 4.5 kb is a fragment internal to the RAD2 gene. As expected, plasmid pLP9, containing the 2.0-kb BglII-EcoRI fragment cloned in YIPS, transformed a RAD2 strain to Rad- (Figs. 1 and 2 and see section e below).

kb

3.3

1

2

Fig. 3. Transcript

encoded

(2 fig per lane) from yeast hyde-1.5% hybridized cloned

agarose

by the RADZ gene. Poly(A)+RNA cells was fractionated

gels, transferred

to the 2.0-kb BglII-EcoRI

into M 13mp9. Lane

on formalde-

to gene screen paper, fragment

and

ofthe RAD2 gene

1, poly(A) + RNA from the RAD +

strain DBY747 carrying

the plasmid pLP1; lane 2, poly(A) + RNA

from DBY747

the plasmid

carrying

YEp24.

(d) The size of the RAD2 transcript and its orien-

by removing the KpnI site of pLP1 to generate the plasmid pLP7. pLP1 was linearized with KpnI and the 3’ extensions removed by utilizing the 3’ + 5’ exonuclease activity of T4 DNA polymerase. The

tation

To identify both the transcript encoded by the RAD2 gene as well as determine transcription di-

URA

l--2.0

kb+ X

V CHROMOSOMAL RADP LOCUS

A

Bg KBgSI ,. .. .. .

** - * -a HOMOLOGOUS INTEGRATION AT RADP LOCUS

Fig. 4. Generation

of a duplicated

deletion

the RAD2 gene. The 2.0-kb BglII-EcoRI pLP9. As indicated

diagrammatically,

at the RAD2 locus flanking segment,

RAD2

of the RAD2 gene by homologous

fragment integration

the YIp5 sequence.

region DNA; hatched

I

segment,

integration

ofRAD2 gene was ligated into BumHI by homologous Symbols

recombination

for restriction

enzymes,

at the RAD2 locus of an internal

+ EcoRI-restricted

YIp5 to generate

at the RAD2 locus will generate

a duplicated

as given in Fig. 1; thin line, pBR322

yeast URA3 gene; heavy line, yeast chromosomal

DNA.

segment

of

the plasmid deletion

sequences;

open

126

rection, we cloned the 2.0-kb BglII-EcoRI fragment internal to the BAD2 gene, into BamHI-EcoRIrestricted M13mp8 and M13mp9 phages described by Messing and Vieira (1982). The recombinant phages containing the inserts were used as templates for DNA synthesis to generate radioactively labeled single-stranded probes (Hu and Messing, 1982). These probes were then used in hybridizations to poly(A)’ RNA obtained from the Rad’ strain DBY747 containing either the BAD2 insert present on a multicopy plasmid, pLP1, or the plasmid YEp24 without any additional insert. The 2.0-kb BglII-EcoRI fragment hybridized to poly(A) + RNA from DBY747 containing the plasmid pLP1 (Fig. 3, lane 1) and revealed a transcript of 3.3 kb, but under these conditions no hybridization was detected when poly(A)‘RNA is prepared from DBY747 lacking the RAD2 gene on a multicopy plasmid (Fig. 3, lane 2). In addition, these hybridization results were obtained only when the 2.0-kb BglII-EcoRI fragment is cloned in M13mp9 and not with the same fragment cloned in M13mp8 (results not shown). Since the restriction sites of M13mp9 are arranged in the order 5’ - BamHI - EcoRI - 3’ and those of M 13mp8 are 5’ -EcoRI - BamHI - 3’, these results indicate that the 3.3-kb mRNA encoded by the RAD2 gene is transcribed from EcoRI towards BglII, as indicated in Fig. 1. (e) Effect of rud2 deletions

A rud2 deletion (A) mutation was generated by the method of Shortle et al. (1982). The plasmid pLP9, which contains the 2.0-kb BglII-EcoRI fragment internal to the RAD2 gene, can transform uru3 auxotrophs to Ura + only by homologous integration in the yeast. genome, since pLP9 does not contain any yeast DNA sequences necessary for autonomous replication in yeast. Although homologous integration could occur at either the uru3 or rud2 loci, the introduction of a double-strand cut in the 2.0-kb BglII-EcoRI fragment, results in directed integration at the BAD2 locus (Orr-Weaver et al., 1983). Therefore, the Rad + uru3-52 strain DBY747 was transformed to Ura + with pLP9 DNA that had been linearized by limit digestion with SacI, causing preferential integration at the RAD2 site and generating a duplicated rud2A (Fig. 4). All of the Ura+ transformants obtained were found to be UV sensi-

tive. Ten of the UV-sensitive transformants were tested for allelism with rud2 by crossing each to the haploid rud2-6 strains LP2641-1A and LP2641-4C. All the diploids were UV sensitive, indicating that the Ura+ transformants contained deletions of the RAD2 gene. To inquire about the viability of spores containing rud2A, five of the Ura+ rud2A transformants were mated to the uru3-52 Rad’ strains 7799-5C or GP19-21C. Diploids were sporulated and 13-24 tetrads analyzed from each cross. In all five crosses, both Ura+ Rad and Ura- Rad + spores were obtained, demonstrating that rud2A-bearing spores are viable. Since we observed only Ura + Rad _ and Ura- Rad’ spores, these results provide further confirmation that integration of the plasmid pLP9 had occurred at the RAD2 locus in the strain DBY747. The rud2Alrud2A diploids were proficient in sporulation and produced viable spores. However, the sensitivity to UV irradiation of a haploid carrying a rud2A was somewhat greater than the most sensitive rud2 point mutant tested (Fig. 2).

DISCUSSION

We have isolated a plasmid containing a 9.3-kb insert of yeast DNA which complements the rud2 mutants. A DNA fragment from this insert was cloned into the integrating vector YIp5 and the plasmid shown to transform yeast by homologous integration at the rud2 site in the yeast genome, thus confirming that we had cloned the RAD2 gene. Our subcloning results locate the RAD2 gene in the 6.0-kb DNA fragment from the Sau3A/BamHI fusion junction of the yeast insert DNA with the vector sequences on the left at about 0.4 kb to the X/z01 site on the right, at 6.4 kb (Fig. 1). Further subcloning indicates that the R4D2 gene is present from somewhere to the left of the KpnI site at 2.1 kb to the right of EcoRI at 4.5 kb. The R4D2 gene has also been cloned by Naumovski and Friedberg (1984) and their results show the RAD2 gene to be present in a 4.5-kb DNA fragment from near to the left of the KpnI site to near to the XhoI site. We have cloned an internal 2.0-kb BgfII-EcoRI fragment in phages M13mp8 and M13mp9 and used these recombinant phages to determine the RAD2

127

transcript size and the direction of transcription. The RAD2 gene codes for a 3.3-kb transcript and the direction of transcription is leftwards from EcoRI towards BglII (Fig. 1). The 3.3-kb RAD2 transcript could code for a polypeptide of about 115 kDal, similar in size to the uvrA protein of E. coli. The RADl and RAD3 genes encode transcripts of 3.1 kb and 2.5 kb, respectively (Higgins et al., 1983a,b), which could code for proteins of about 110 and 90 kDal. Deletions of the RAD2 gene created by integration of the internal 2.0-kb BglII-EcoRI fragment in the chromosomal RAD2 gene, have no effect on viability of vegetative haploid cells, sporulation or spore viability. However, rad2ds show somewhat greater UV sensitivity than the rud2 point mutant alleles examined (Fig. 2). The rudlds also have no effect on cell viability (Higgins et al., 1983a), whereas the rud3As are recessive lethals (Higgins et al., 1983b; Naumovski and Friedberg, 1983). The RADl and RAD2 genes, therefore, seem to be more specific to excision repair than the RAD3 gene, which in addition to its role in excision repair, is required for other vital cellular processes.

Clewell,

D.B. and Helinski,

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conversion

D.R.:

Supercoiled

circular

in Escherichia coli: purification

complex

to an open circular

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and induced

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R., Park, L. and Grossman,

pyrimidine

dimer-containing

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DNA. A 5’ dimer DNA glyco-

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mechanism

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deficient

xeroderma

pigmentosum.

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P.D.S.

and

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183-190.

We thank Dennis Dumais for help with initial screening of transformants. This investigation was supported by grants CA35035 to S.P., GM19261 to L.P. from NIH, and Contract Number DE-AC0276EV03490 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-3490-2392.

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