Analysis of unassisted translesion replication by the DNA polymerase III holoenzyme

DNA damage-induced mutations are formed when damaged nucleotides present in single-stranded DNA are replicated. We have developed a new method for the preparation of gapped plasmids containing site-specific damaged nucleotide s, as model DNA substrates for translesion replication. Using these substra tes, we show that the DNA polymerase III holoenzyme from Escherichia coli c an bypass a synthetic abasic site analogue with high efficiency (30% bypass in 16 min), unassisted by other proteins. The theta and tau subunits of th e polymerase were not essential for bypass. No bypass was observed when the enzyme was assayed on a synthetic 60-mer oligonucleotide carrying the same lesion, and bypass on a linear gapped plasmid was 3-4-fold slower than on a circular gapped plasmid. There was no difference in the bypass when stand ing-start and running-start replication were compared. A comparison of tran slesion replication by DNA polymerase I, DNA polymerase II, the DNA polymer ase LII core, and the DNA polymerase III holoenzyme clearly showed that the DNA polymerase III holoenzyme was by far the most effective in performing translesion replication. This was not only due to the high processivity of the pol III holoenzyme, because increasing the processivity of pol II by ad ding the gamma complex and beta subunit, did not increase bypass. These res ults support the model that SOS regulation was imposed on a fundamentally c onstitutive translesion replication reaction to achieve tight control of mu tagenesis.