The beta subunit sliding DNA clamp is responsible for unassisted mutagenic translesion replication by DNA polymerase III holoenzyme.
The replication of damaged nucleotides that have escaped DNA repair leads to the formation of mutations caused by misincorporation opposite the lesion. In Escherichia coli, this process is under tight regulation of the SOS stress response and is carried out by DNA polymerase III in a process that involves also the RecA, UmuD' and UmuC proteins. We have shown that DNA polymerase III holoenzyme is able to replicate, unassisted, through a synthetic abasic site in a gapped duplex plasmid. Here, we show that DNA polymerase III*, a subassembly of DNA polymerase III holoenzyme lacking the beta subunit, is blocked very effectively by the synthetic abasic site in the same DNA substrate. Addition of the beta subunit caused a dramatic increase of at least 28-fold in the ability of the polymerase to perform translesion replication, reaching 52% bypass in 5 min. When the ssDNA region in the gapped plasmid was extended from 22 nucleotides to 350 nucleotides, translesion replication still depended on the beta subunit, but it was reduced by 80%. DNA sequence analysis of translesion replication products revealed mostly -1 frameshifts. This mutation type is changed to base substitution by the addition of UmuD', UmuC, and RecA, as demonstrated in a reconstituted SOS translesion replication reaction. These results indicate that the beta subunit sliding DNA clamp is the major determinant in the ability of DNA polymerase III holoenzyme to perform unassisted translesion replication and that this unassisted bypass produces primarily frameshifts.
Tomer
G
G
Department of Biological Chemistry, Faculty of Biochemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
Reuven
N B
NB
Livneh
Z
Z
D004249:DNA Damage; D004258:DNA Polymerase III / Q000737:chemistry* / Q000378:metabolism*; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D016368:Frameshift Mutation; D007700:Kinetics; D046911:Macromolecular Substances; D016296:Mutagenesis; D010957:Plasmids; D015183:Restriction Mapping
D004277:DNA, Single-Stranded; D046911:Macromolecular Substances; D004258:DNA Polymerase III