Tobacco Documents Online

Cell Biology Revtew~ @ University of the B~sque Country A review of DNA metabolism in Escher chia coil E. Balhinder and C. Waldren D~m+rIment ff Bior)u~.qry, aiophysic~ an~ Gen~irz, UnP~rsiry of C~l~r~do He~d~h $~ience~ C~n~r, D~nvo, CO 80262. USA L Introdu~on The ongoing revclu~on inmolecular biolo~-/is bringing increasing evidence that mutations are central to a number of padm~@cal [necemes including canc~, bLnh defects and in- of'envin~nmenla/agents, physical and chemical, are capable of causing reunions" cancer and other lXUholo@es has converted the study of reunions sad the ~ leadlng to them into ~ field of medical inter¢~ The resulting resemch Ires improved o~r undenlanding of DNA and tho intflc~es of its meutho- lism. We now view DNA as a dynamic e~tity and wlddn this frmnewosk have ntla~ed a belier ~S of how mum* fious may occur. It has also become clear that the study of mutagen .mis cannot be separate~l from that of DNA replic- ation, xepakand ~combination. Altbeugh historically thee have developed as separa~ research disciplines, we know today that they overlap siguificanfly and share msay .es~zymat- ic functions so th~ a unified conceptual framework embracing tbese different aspects of DNA metabolism is needccL We cesses arc org~nlzcd in bat~ties of operonn which respond more or less coordinately to a variety of cxtenml slre~es such as mdintion, genotoxic chemicals, heat, starvation and others. An operon is a set of linked genes transcnq~ed as a unit from a singl~ regulated promoter. Some of these opemns are inte- grated into complex ret~muo~ netwod~ and. to a lm~e extent, mu .tagenesis is an inducible t~'ponse. One objective of this ~evidw is to show how the~e netwodcs overlap and connect DNA metabolism with overall cellidar metabolism. Our cuaent undez~anding of DNA n~embolism is based in large part on stt~es cmducted with dividing Escherichia coli cells, following the demonsu'afion by Luria and'Dellm~ in 1943 (389) that mutations occur in replic~ing cells in the absence of a selective environment. Recently, atvmtion has been focused on mutationsmking place in non-dividing cells under stores. According to Cairns et a/(66) and others, such mutations can oc~tr adaptively under starvation conditions in response to a selective agent. These ideas may usher a new em in the study of DNA metabolism of particular importance in the the etiology of human genedo diseases and cancer. This will be discussed in the last chapter of this monograph (VII Chapter). This review has heen vaiuen pdmadly for the use of grad- uate students, but should be useful to ~sce~hes who are not specialists in the various a~eas of DNA metabolism hopefully, to some specialists as well. We will present the major ideas developed with the E. coil system as a guide to studies in eukatTotus, including y~ and madm~alian cells. TI~ subject is larse sad inuicate and cmunot he covered in gre~ detail in a re ol~ively short mmuogr~.ph. Mm~y excellent + reviews l~ve been pobli~hed in recer~ yem~ and will be men- tioned where indicazed. However, not a single review ~empting to imegmte all fl'.~ infcnn~on about DNA metabolism Lure on~ la~e picture exL~ts to daY~. T/ds L-'~.rocluctory ~eview hop~s to fill this H. Ma/ntaining the integrity of geaeffc informa6on during DNA replication The accurate transmission of genetic "information from mother to d~u. ghter cell is a fundamental requirement of all forms of life. Most heritable mutations (alterations of the g.enetic message) would be deleterious but a few could be I~meficial for the evointina of the species. Thus, not all muta- tions need to be avoided. In this chapter we will review the strategies which E. colt has develo .i~1 to deal with this dual challenge, i. e. maintenance of the integrity of its genome while allowing a certain, small measure of diversity to ensure the survival of the species in a changing environment. A. DNA Replication .Maintaining the integrity of the genetic information is an integral part of the replication of the E. coli chromosome. This is an exucraely complex process which rc~t_ uh'es the pre- cise interaction of a ~ nmnber of Inoteins organized into a ~replication machine~. The essentinl proteins e~e listed in tables I and 2. It is beyond the scope of this monograph to discuss DNA replication in detail~~ This has been done in sev- eral excellent reviews, some of themquke recent (301, 406, 407~ 533). We need here~ however, to provide an overview of DNA replication to allow the reader to undersland how replic- ation fidelity .is accomplished in the process. £. coli has a ciscular chrom~me consisting of about 4 X .10" base pai~. It replicates bidirectinmslly (62), without being lineadzed, from a fixed origin of replication (or~C) st 83. 5 minutes on the £. coli genetic map, to a terminus (terC) situ- ated almost diamet~:ally opposite from or~C in the region from 30-32 minutes in the map (2~, 407, 480). To accomplish this repltcadon some major problems must be solved. The first has to do with the ~ecipmcal polarity of .the two strands of the double helix. Each strand of DNA has a chemical polexity defined .by the asymmetry of the sugar-phosphate backbone: the two cmnplemenmry strands of a double helix have opposite, polarities, one going in a 5' --~ -3" and its corn: plement in a 3' -.~ -$" dkection. All DNA polymerases extend newly synthesized DNA chains ~tly in a 5" --~ -3' dir~tion. however, so a problem arises'in having the same enzyme moving in only one direction along a rcp!ic~_tion fn~ m~d h~tving simultaneously to synthesize the two DNA strands that are oriented in opposite directions. This problem has been solved, as illustrated in fig. I by a semi discontinuous • replication of the chromosome: one strand'is made continu- ously in a 5" --~ -3' dit~'cdon (leading strand) and the other (lagging strand), which goes in its entirety in a 3" direction, is synthesized discontinuously in short If00-1000 b~se palm) 5° .-~ -3" fragn~nts named after their dL~overer Okazaki (see fig. l). This presents some peculiar difficulties and opportunities for mutsgenesis v,'h~ch will be discussed l~er. The s~ond prob]em in replicaxion is to unwin~l t]~ dou- ble helix, i. e. ~ep~e ~ two str',m~ in .~dvance of 40000071