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Cell Bi~Io~y Reviews (1991) 2~(2): Cell Bia~c~,.'V Reviews © Uni,,'e~hy of&e BazcI~ Co.try A review of DNA metabolism in Escherichia cell E. Balbinder and C. Waldren Dwarm..em of aioclwniaey, a~o;l~s~cs and G,~ics, University of Cok~rado Health Scie~ea C~, Den~'er, CO 80262. USA L Introduction The ongoing revolution in molecular biology is bringing increasing evidence that mutations am central to a humor of pathological ~ including cancer, bkth defects and in- herited ~. Thls ~g and the fact that a number of'mvin~nmental ageats, physical and chcmica/, a~ cap~le of cansing mutations, eaug~ and other l~holc~ie~ tun converted the study of mucuioas and tbe processes Icadin~ to them into a field of medical in~'es~. Tbe resulting scscarch has improved our undoes of DNA and the intricacies of its lism. We now view DNA as a dynamic entity and within this frantework have anak~d a ~ und~standing of how muta- tions may occur. It has also become clear that tl~ study of mutagen~s, is cannot be scpar~l from thin of DNA ~plic- afiun, repair and recombination. Although historically have davolop~i as separate r~carch discipfincs, we know today that they overlap significantly ami share rnsny enz~mat. ic functions so th~ a uni~d coaccpmal framework ~nbracing these different aspects of DNA metabolism is needed. We know that, in prokaryot~, enzymes pmicip~iag in these pro- cesses are organiz~ in batteries of opemns which respond mo~ or less coonfieatcly to a variety of axe, real sinuses such as radiation, gcnotoxic cizmicals, he~ ssatvatioa and othem An operon is a set of linked g~nes mmscribed as a anit from a single regulated promoter. Some of thcs~ opemns arc intc- gr-a~d into complex regul~ory netwodm and, to a large extent, mutagenesis is an inducible raspomc. One objective of this revi~'w is to show how these n~works overlap and connect DNA mc, abolism with overall cellular m~taboltsm. Our current undentanding of DNA n~e.~0olism is based in large part on studies coaducted with dividing Es~ker/ch/a coli cells, following the demonstration by Luri~ and'Dclbruck in 1943 (389) that mutations occur in repficating ceils in the absence of a selective environment. Rccentlyo attention has been focused on mutations caking place in non-dividing cells under stress. According to Cairns et ai (66) and or, has, such mutations can occur adaptively uader starvation conditions in respons~ to a selective agaric These ideas may usher a new ©ra in the study of DNA metabolism of particular importance to the the etiology of human geaetic diseases and cancer. This will be discussed in the last chapter of this monograph (VII Chapter). This ~cview has been wrinen iximmily for the use of grad- uam stud-'ms, but should be useful to reseasch~s who are not specialists in the various areas of DNA metabolism and, hopefully, to some specialists as well We will present the major ideas deveinped with the E. coil syswra as ~ guide to studies in eukaryotes, including yeast and mmiunalian cells. The subject is large and intricate and cannot b~ covered in great detail in a relatively short monograph. I~any ev.~llent reviews have been published in recent years a~d will be men- tioned where indicated. However. nee a single review amxnpting to imegr~e all the information about DNA metabolism into one large p;.ctur~ exists m da~c. This ica'oductory review hOl~'~ to fill this g~p. II. Maintaining the integrity of genetic information during DNA replication The accurate transmission of genetic "information from mother to daughter cell is a fundamental requirement of all forms of life. Most heritable mutations (alterations of the g~.nedc message) would be deleterious but a few could be beneficial for the evolution of the species. Thus, not all muta- tions need to be avoided. In this chapter we will review the strat~gies which E. coli has developed to deal with this dual challenge, i. e. maintenance of the integrity of its genome while allowing a eeaaln, small n~asme 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. co//chromosome. This is an exlremely complex process which re.quires the pre- cise interaction of a large number of proteins organized into a greplication machine~. The essential proteins at~ listed in tables ! and 2. It is beyond the seope of this monograph to discuss DNA replication in detail. This has been done in sev- eral excellent reviews, some of themquite recent (301, 406, 407, $33). We need here, however, to provide an overview of DNA repfication to allow the seader to understand how replic- ation fidelity is accomplished in the process. £. coil has a circular chromosome consisting of about 4 X 10' base pairs. It replicates bidisectionally (62), without being linearized, from a fixed origin of replication (or/C) at 83. 5 minutes on the E. coli genetic map. to a terminus (¢erC) situ- ated almost diametrically opposite from or/C in the region from 30-32 minutes in the map (2S, 407, 480). To accomplish this replication some major problems must be solved. The first has to do with the reciprocal polarity of the two strands of the double helix. Each strand of DNA has a chemical polarity defined by the asymmetry of the sugar-phosphate backbone: the two complemental, strands of a double helix have opposite polarities, one going in a 5' -~ -3" and its com- plement in a 3" --~ -5' direction, All DHA polymemses extend newly synthesized DNA chains only in a 5" --> -3" direction. however, so a problem arises'in having the same enzyme moving in only one direction along a replication fork and having simultaneously to synthesize the two DNA strands that are oriented in opposite directions. This problem has been solved, as illustrated in fig. ! by a semi discontinuous replication of the chromosome: one strand is made cominu- ously in a 5" ~ -3" direction (leading strand) and the other (lagging strand); which goes in its entirety in a 3' --> -5" direetinn, is synthesized discontinuously in short (100-|000 base pairs) 5' ~ -3" fragments nam~ after their discoverer Okazaki (see fig. !). This presents some peculiar difficulties a~d opportunities for mumgenesis which will be discussed l~er. The second ffrob!em in replic~icn is to unwind the dou- bl~ helix, i. e. separate the two strands in .-drawee of the 40000088