Abstract
Dpo4 and Dbh are from two closely related Sulfolobus species and are well studied archaeal homologues of pol IV, an error prone Y-family polymerase from Escherichia coli. Despite sharing 54% amino acid identity, these polymerases display distinct mutagenic and translesion specificities. Structurally, Dpo4 and Dbh adopt different conformations because of the difference in relative orientation of their N-terminal catalytic and C-terminal DNA binding domains. Using chimeric constructs of these two polymerases, we have previously demonstrated that the interdomain linker is a major determinant of polymerase conformation, base-substitution fidelity, and abasic-site translesion synthesis. Here we find that the interdomain linker also affects the single-base deletion frequency and the mispair extension efficiency of these polymerases. Exchanging just three amino acids in the linkers of Dbh and Dpo4 is sufficient to change the fidelity by up to 30-fold, predominantly by altering the rate of correct (but not incorrect) nucleotide incorporation. Additionally, from a 2.4 Å resolution crystal structure, we have found that the three linker amino acids from Dpo4 are sufficient to allow Dbh to adopt the standard conformation of Dpo4. Thus, a small region of the interdomain linker, located more than 11 Å away from the catalytic residues, determines the fidelity of these Y-family polymerases, by controlling the alignment of substrates at the active site.
Highlights
Dpo4 and Dbh translesion polymerases show distinct mutagenic signatures and lesion bypass abilities
Using chimeric constructs of these two polymerases, we have previously demonstrated that the interdomain linker is a major determinant of polymerase conformation, base-substitution fidelity, and abasic-site translesion synthesis
Nucleotide incorporation rates were determined for both parental enzymes and for six chimeras constructed from all possible combinations of the polymerase domain, 15-amino acid linker and little finger” or polymerase-associated domain (LF/PAD) (Fig. 2, A–H, and Table 2)
Summary
Dpo and Dbh translesion polymerases show distinct mutagenic signatures and lesion bypass abilities. A large number of studies have focused on understanding the correlation between structure and function in the Y-family TLS polymerases [9] These enzymes share a core structure consisting of an N-terminal catalytic domain, with palm, fingers, and thumb subdomains (as are found in other families of DNA polymerases), and a C-terminal domain (unique to the Y-family) that is known as the “little finger” or polymerase-associated domain (LF/PAD) [10, 11]. Like Dpo, the LF/PAD is in contact with the fingers domain, docking into the major groove of the DNA duplex and positioning the primer-template junction at the active site for efficient catalysis This chimera was found to bypass an abasic site and display single nucleotide incorporation fidelity similar to Dpo. Just three residues in the interdomain linker control the enzyme conformation and influence fidelity by affecting the rate of nucleotide incorporation, without being in the vicinity of the active site
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