Biochemistry Of Dna Replication

The synthesis of a new DNA molecule is catalyzed by DNA polymerase III. Its substrates are the four deoxyribonucleoside triphosphates, dATP, dCTP, dGTP, and dTTP. DNA poly-merase III catalyzes the formation of a phosphodiester link (Fig. 4.3 on page 69) between the 3'-hydroxyl group of one sugar residue and the 5'-phosphate group of a second sugar residue (Fig. 5.2a). The base sequence of a newly synthesized DNA strand is dictated by the base sequence of its parental strand. If the sequence of the template strand is 3' CATCGA 5', then that of the daughter strand is 5' GTAGCT 3'.

DNA polymerase III can only add a nucleotide to a free 3'-hydroxyl group and therefore synthesizes DNA in the 5' to 3' direction. The template strand is read in the 3' to 5' direction. However, the two strands of the double helix are antiparallel. They cannot be synthesized in the same direction because only one has a free 3'-hydroxyl group, the other has a free 5'-phosphate group. No DNA polymerase has been found that can synthesize DNA in the 3' to 5' direction, that is, by attaching a nucleotide to a 5' phosphate, so the synthesis of the two daughter strands must differ. One strand, the leading strand, is synthesized continuously while the other, the lagging strand, is synthesized discontinuously. DNA polymerase III can synthesize both daughter strands, but must make the lagging strand as a series of short 5' to 3' sections. The fragments of DNA, called Okasaki fragments after Reiji Okasaki who discovered them in 1968, are then joined together by DNA polymerase and ligase (Fig. 5.1).

DNA Synthesis Requires an RNA Primer

DNA polymerase III cannot itself initiate the synthesis of DNA. An enzyme called primase is needed to catalyze the formation of a short stretch of RNA complementary in sequence to the DNA template strand (Fig. 5.1). This RNA chain, the primer, is needed to prime (or start) the synthesis of the new DNA strand. DNA polymerase III catalyzes the formation of a phosphodiester link between the 3'-hydroxyl group of the RNA primer and the 5'-phosphate group of the appropriate deoxyribonucleotide. Several RNA primers are made along the length of the lagging strand template. Each is extended in the 5' to 3' direction by DNA

DNA polymerase adds a C to the growing chain

DNA polymerase makes a mistake and adds an A to the growing chain

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