E. coli DNA Polymerase I

DNA Polymerase I is a single polypeptide chain of M r 103,000. In addition to its polymerase activity, the purified enzyme has two nuclease activities. The first is a 3' to 5' exonuclease that degrades single-strand DNA from the 3' end and the second is a 5' to 3' exonuclease that degrades base-paired DNA from the 5' terminus. DNA polymerase I can also cleave RNA from a duplex containing one strand each of RNA and DNA. The 3'-5' exonuclease activity serves a "proofreading" function to improve the accuracy with which a DNA template is copied. This activity removes an improperly base-paired nucleotide from the growing 3' end of a polydeoxynucleotide chain, giving the polymerase activity a second chance to insert the correct nucleotide specified by the template.

The 5'-3' exonucelase activity plays two known roles. The first of these is the excision of RNA primers in lagging strand replication (Figures 24.4 and 24.5) via a nick translation mechanism in which the 5'-3' exonuclease excises ribonucleotides just as the polymerase is replacing them with deoxyribonucleotides. The second known function of the 5'-3' exonuclease is to cleave nucleotides from DNA. This may be an important function for repair of DNA that has been damaged by radiation or chemicals. Again, the mechanism involves nick translation.

Limited proteolytic cleavage of DNA polymerase I yields a small N-terminal fragment (M r = 35,000) and a large C-terminal fragment (M r = 68,000). The large fragment, which contains the polymerase and 3'-5' exonuclease domains, is also called the Klenow fragment. The small fragment contains the 5' exonuclease domain. The molecule appears to bind DNA like a hand grabbing a bar.

See also: E. coli DNA Polymerases, Eukaryotic DNA Polymerases, Figure 24.8, Table 24.2, Base Excision Repair

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