Chapter I 



The Replication of DNA in Cell-Free 

 Systems^ 



MAURICE J. BESSMAN 



McCollum-Pratt Institute, 



The Johns Hopkins University, 



Baltimore, Maryland 



I. Introduction 1 



II. Synthesis of Deoxyribonucleotides 3 



A. Origin of the Deoxj'ribosides 3 



B. Synthesis of Thymidylic Acid 6 



C. Synthesis of 5-Hydroxymethyldeoxycytidyhc Acid (dHMP) . . 10 



III. Phosphoiylation of Deoxyribonucleotides 12 



A. Deoxynucleoside Monophosphate Kinases 12 



B. Deoxynucleoside Diphosphate Kinases 16 



IV. Synthesis of DNA by Escherichia coli Polymerase 17 



A. Measurement of DNA Synthesis 17 



B. Requirements of the Reaction 20 



C. The Limited Reaction 23 



D. Nature of the Primer 25 



E. Nature of the Product 27 



F. Primer-Product Relationships 29 



G. Synthesis of Deoxyadenylate-Deox>'thymidylate and Deoxyguanyl- 



ate-Deoxycytidylate Polymers 40 



H. Substitution of Analogs 46 



V. Synthesis of DNA by Calf Thymus Polymerase 50 



VI. Synthesis of DNA in Bacteriophage-Infected Escherichia coli ... 54 



A. Bacteriophage-Specific Polymerase 54 



B. Glucosylation of 5-Hydroxymethylcytosine (HMC) 56 



VII. Conclusions 60 



Acknowledgments 60 



References 60 



I. Introduction- 



Most organisms can synthesize all the constituents of the DNA macro- 

 molecule from small molecular weight precursors which are also the raw 



' Contribution No. 371 of the McCollum-Pratt Institute, The Johns Hopkins 

 University, Baltimore 18, Maryland. 



^ The following abbreviations have been used in this chapter : RNA, DNA : 

 ribonucleic acid, deoxjTibonucleic acid; RNase, DNase: ribonuclease, deoxjTibo- 



1 



