Radiation Damage and Repair in Cell and Cell Components

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RADIATION DAMAGE AND REPAIR IN CELLS AND CELL COMPONENTS UDIATIOM INDUCED REPAIR Zoology Department, Duke University XPFF.OVE3 FOR RELEASFY OR

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RADIATION DAMAGE AND REPAIR I N CELLS AND CELL COMPONENTS RADIATION INDUCED REPAIR PROGRESS REPORT FOR U.S. DEPARTMENT OF ENERGY Zoology Department, Duke University

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Personnel; major thrusts; connnentary; publications* I Present status of knowledge regarding induced repair. 22 111 A separation of UV cross sections for repair induction IV Induced Radioresistance: an aspect of induced repair. and premutational damage in E.coli revertant mutagenesis. 38 VI W mutagenesis (His revertants) relative to p-recA production. 113 VIII The induction of prophage as measured by endolysin activity. 126

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RADIATION DAMAGE AND REPAIR IN CELLS AND CELL COMPONENTS Progress report: fourth n e w contract year. The group involved in the project includes Dr. D.J.Fluke, C.E.Bronner, a recent graduate at Duke University, S.Nolte, a junior.at the University of Colorado and Cynthia White, a sophomore at Bryn Mawr. The elucidation of the phenomenon of induced reBeZ, using the techniques available to us, remains our primary objective. In Appendix I we give an update of what has become an annual review of the situation. A great deal of what was shadowy has become clear, some of it because of our work. There remain to be determined almost all the detailed explanations of the process outlined in Appendix I and also clearing up some surprising difficulties in the scheme. These are later phenomena which have been ascribed to the induction of the recA gene product (p-red) are really manifestations of one process. As of our last repetr we concluded that this is true for septum Inhibition, Weiglereactivation, induced inhibition of post radiation DNA degradation, and One of our aims has been to see whether, in E.coli, the various with the additional concept of a premutational lesion, for UV mutagenesis. We felt that induced radioresistance and clearly related phenomena were not in exact relationship. During the year, by devising means of quaatitation of induced radioresistance, we have been able to show that the fraction of resistant cells and the fraction of p-recA induced reasonably match. We further give the interpretation of induced radioresistance as due to three effects of induction: (a) rendering molecular synapsis repair (the use of a homologous genome in repair) more probable by the combination between g-recA and single stranded DNA (b) the inhibition of post radiation DNA degradation and (c) the inhibition of septum formation, thus increasing the number of genomes

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available for "breakage-reunion gap filling" which we term molecular synapsis repair. The paper reporting this is given as Appendix IV: it has been submitted to Molecular and General Genetics and preliminary correspondence indicates that with some additional clarification it is acceptable. We feel that A prophage induction may perhaps be brought into line with p-recb induction with the consideration of the additional secondary aspects of (a) activation of p-red to make it enzymatically active and (b) the need to have the concentration of activated p-recA high enough to keep up with the rate of production of A-repressors. We continue to believe that more than one method of activation of p-red exists and that the concepts of diffusible and stationary activation introduced in our 1978-1979 report are valid and useful. Some of this is discussed in Appendix VIII. On the other hand we have the following less amenable findings. Revertants seem to be in more than one class and two of these can not easily be explained by the idea that p-re4 contains an "error-prone repair" enzyme that makes errors at mutagenic lesions. These two classes are the small revertan*ts we discussed at Williamsburg and the high dose revertants found'by ourselves and Doudney. We find that while there is W-reactivation and W-mutagenesis associated with X-ray damage of the character broadly called "direct action", the development of the two phenomena do not go together. This calls for more work. We further-flnd it hard to understand the lack of WeigleWe do& understand the lack of induction of p-red in the reactivation in uVP-. strains where the other phenomena are clearly apparent. uvr-recB-recF- strain which should have daughter strand gaps, supposedly a means of induction. The close proximlty of recP and dnaA on the genome may suggest the explanation. chad is a mutation in the initiation of During last year we began to use light we felt to be mostly photoreactivating in studies and we found a surprising variability in its action, some of which we reported in last years repzort. In order to be sure that our work is not confused by added action i n the 365nm region, we have set up two additional light facilities. One, BLB light, has been measured as primarily a broad band around 365- with a little 313nm light readily filtered out by window glass, and the other is a filtered "patio" light that pperates from 420nm and longer. We propose to use these

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to try to elucidate the variability of the phenomena that were photoreact iv able, notably the ready photo react iv ability of septum inhibition and of revert ants formed i n lean medium, but not of the yield of p-recA, nor of revert ants formed stage.at high UV exposures. This is work in the planning The study of induced r e p a i r is rewarding simply In terms of 4 the intellectual effort to understand how E.coZi can use the recA and ZexA genes t o achieve repair and indeed other processes in response t o damage t o DNA. It is for this reason that we engage in the study. However, it has been widely thought that the process would find analogous behavior in many kinds of cells and, presumably, i n human cells. Atthe Gainesville Conference in 1975 it was freely but informally held that some of the mechanisms of cancer production would be seen as closely related t o induced repair phenomena. The importance t o evolution of a cell survival dependent on a stress was also discussed. these wider interests and consider the validity of motivation for research that they suggest. It is appropriate t o look a t Our work, and that of others, places the main characteristics of induced repair in three zones. First,the repression of two genes by a repressor capable of in activation by the gene product of one of them: in activation that requires the modification of that gene product t o become enzymatic. DNA damage is one vay of achieving t h a t modification. Second, the relation of the gene product tothe DNA itself : binding to single stranded regions and thus inviting the pairing of homologous regions of different duplexes, a process that can produce recombination and also the use of the second duplex t o give the information for repair of a damaged original duplex. This process would otherwise Third, the enzymatic action of the modified gene product is pro teo ly tic in some way and can act t o modify o r cleave other repressor s, notably prophage repressors. How would these appear in a cell such asa human cell? Take the second first : has a cell with 1,000 times the DNA ina nucleus which still makes fora crowded condition, and in which there is considerable combination between DNA and protein, anything like the advantage ;to be gained from holding a single stranded region open

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and making recombination more probable? It would seem that such a mechanism is already there in abundance and is also constitutive and not induced. Consider now the activation process. If this is, i n some way, due t o DNA damage then only the d if fusible activation (requiring small effect or molecules) i s of the slightest use. Whereas, i n an E.ooZi cell it takes 300 seconds for one enzyme molecule to find one particular fixed substrate,ina nucleus which is about 100 times greater in volume it would certainly take ten times longer. So fixed activation is not likely to have the necessary speed. However, to obtain small effect or molecules from the DNA requires a certain amount of DNA degradation after some stress and there is very little evidence for this. So this form of activation is unlikely to be found. W e are left with the Idea of a repressor capable of being lifted by a modified gene product and with the idea that the resulting enzyme that was now transcribed could operate 011 other pro zte i as. Thus t h e expectation is that: 1. "Stress" modification to lift repression can be found but that t h e "stress" is probably not DNA damage. 2. Such lifted repression would give a product that could carry into more than me action. Our most notable activity in this area this year was the delivery of six contributed papers atthe Biophysical Society in Denver, the Radiation Research Society i n Minneapolis and the American Society for Photobiology i n Williamsburg. Copies of the abstracts are included. The two papers we delivered at the Gatlinburg Symposium have now been published. Reprints of these are included. Two papers are actively in process of publication. The paper on Induced Radioresistance has been submitted and is belxq revised f o r conformity t o some suggestions for clarification. The paper on W mutagenesis is in the final stages of preparation. It is included i n this report.A paper on the observations i n strain JC7623 has been written in draft form and is also included in the report. The book for Marcel Dekker is actively moving. It is being revised for final form at the moment.

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In Progress & Nucleic Acid Research Comparative induction studies. aspect of induced repair. tion by ultraviolet light at -79OC. Photochemistry Photobiology, 32, (Submitted for Molecular and general Genetics). Ultraviolet cross sections for tion to induced repair. ultraviolet light and by X-rays i n E. coli AB2494 U3610-89 in different media. Program Abstracts, 9th Annual Meeting, American Society for Photobiology, pp 69-70 (1981, abstract). Induced repair phenomena in Induction of prophage by American Society for Photobiology, p 70 (1981, abstract) ? E. C. Pollard. Concluding remarks. I n Progress i n Nucleic -Acid Research Molecular Biology (W. E. Cohn, ed. 1, Vol. 26, 3 2 5 3 2 6 (1981). Program & Abstracts, 9 t h Annual Meeting,

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U3610-89 In different media. Program and Abstracts, 9th Annual Meeting, American Society for Photobiology, pp 69-70 (1981, abstract). provide varyin(: numbers of genome equivnlents in DNA content ;ier cell. and also tlic lurthcr effcct n f I>rrJcimin.-intlv3 h ‘ j - iim “h1.ic.k I iClit” on this and glucose + casamlno acids) ;iiid Iti b r ; i i i i !tv;irt in1 tisiriri, vitli. divtslon times and prcdlctcd genome rquivalcnie slioun i t i ttir tahls.. l’vrnsinc and 15 minutes of NUV exporure. Change of grouch rate of E . c ~ l i b v ‘.-ariacicm \>f the grouch medium can medium time (min) per cell ratio per surviving C N Revertants are more numeroue Fn the lean-grovn cells, emphasizing complete genomes, slow formation of initiators and l o v genome numbers per cell. The reduction of revertants in such cella by 365-em exposure is understandable by enhanced opportunity for PER during long intervals between initiators of DNA replication. The high incidence of revertants at low genoae numbers suggests some distinction between error prone and

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Ultraviolet cross sections for i i & liar order resp anse dt la qes c exposures. 3ur results at five iV u a vel mg rh s fra 23L.jto 313 nm can be represented reasonably by the following relation : respectively. snow wavelength dependence concordant vich DXA ib s or pci on and differ In magnitude by about 300.000. Quantum e if ici enc is s can be 10'- fork : in relation to one E. coli qrnonc 3nd for kl in relation to 10 co 15 base pairs.IC is suggested th ac the i at t cr number relates to the specificS LCC S for suppressor mutation. and chat t ne vnoli genome may !x the carte t for error-prone repair induction.ISe k, cross o ect ion is roughly DSA-i.ndicative and simllrr t o K] i n magnitude. Lts exponent. z , is cio s ebc to :, averaging over the five uirvelengthr. ICcan also be remarked thatd pusstblr? cross ses rion for CV-induced decline of induced error-prone repair (i:)is nov indicated fa be nil. tion to induced repair.

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phenomena arwcialed with induccd repair in wmpariwn with striiin .AR?470.rer.b . The phenomena thc rffccrivcncss of Wciglc reactivation of U V damaged A phage. 4 ) the presence 0 1 induced radiorcWe find that JC7b23 is iiiucli iiiurc rc%i*iani 11) both S-royr o t d CIV than is :\H?470.l h c pnduction wcun very rudily. l a d i n g to lung Libmenis that rctain colony-Tornling ability. and the frsction \cptuiiiinhibited a g r m with thc yield of prccA. Wcigle reactivation is rc;idily tibscrvcd. There 1s no iriducrrl radiorcsislance. nor i.i.prd. Thc yield of histidine revertanis i s found to be quite low. Wc can suggest th;lt septum inhibition rct:rins multiple duplcxcz of DNA. prt1ittittig r o p i r by nlolecular synaprs. (Work Lambda induction. associated with the induaion of the recA gene. is held in repression by the l e r A gunc prtduci (p-lcxt\). I)\/\ daiiiagc ctinverts the basal level of the rrcrl gcne product ( p r c c h ) to an ;iciivc limi capable 01 inactirdting ihc rccA rcprcswr. thereby allowing morc prccA synthesis. Activald tlic nuiirbcr t i l IainbJa repres.wrs f ~ l l sk l u w the minimum ncsxssary to prevent prophage induction. In the L.t.4 inutant p-lcxr\ cannttt k inactivated by the activatcd p r c c A and therefore the rcrA gcnc 1s' not induced. Though no p-rccA induciim has k e n detected in the lt.v.4. lysogen. for ultraviolet(UV) rc\pm\c. wiii1:ir 1 4 1 ih;it t i l w i l d I>F1)Migcns. A t ;tboui live percent of the wild-typc nwximum inductilln rdi ; : t it iii iii,tciitatcs ciitiugh h rcprewirs IO alluw phage expression in a small fraction of the cells. sr;l? iriduc:ion t d A W : I ~ tithsrv.iblc in broth iiicdium but was onlj marginally r e n in nlinimal niediunl.

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revert ants in 5. coli WU3610-89. Program and Abstracts,9th Annual Meetinq, American Society for Photobiology, p 70 (1981, abstract) .u- - Ocr studies of leucine revert ants in g. coli LT3610-89. a uvr tyr --Leu Bir strain. have encountered some cos pli cations of CV mutagenic expression in this system. The most notable is a class of small revert ant colonies.best distinguished from noma1 revert ant colonies sicer four 239 incub.+tion 3t 37'C. Feu such colonies are seen on phtrs from non-C': controls. 0-10 or so per ml plated. In contrast. as xanv JS :30 nore :hat are di luc able. (3ackground revert ant dilution benavior is mot ner of the complications …