Investigating the Role of the Little Finger Domain of Y-family DNA Polymerases in Low Fidelity Synthesis and Translesion Replication

François Boudsocq,Robert J Kokoska,Brian S Plosky,Alexandra Vaisman,Hong Ling,Thomas A Kunkel,Wei Yang,Roger Woodgate

doi:10.1074/jbc.m405249200

Abstract

Dpo4 and Dbh are Y-family polymerases that originate from two closely related strains of Sulfolobaceae. Quite surprisingly, however, the two polymerases exhibit different enzymatic properties in vitro. For example, Dpo4 can replicate past a variety of DNA lesions, yet Dbh does so with a much lower efficiency. When replicating undamaged DNA, Dpo4 is prone to make base pair substitutions, whereas Dbh predominantly makes single-base deletions. Overall, the two proteins are 54% identical, but the greatest divergence is found in their respective little finger (LF) domains, which are only 41% identical. To investigate the role of the LF domain in the fidelity and lesion-bypassing abilities of Y-family polymerases, we have generated chimeras of Dpo4 and Dbh in which their LF domains have been interchanged. Interestingly, by replacing the LF domain of Dbh with that of Dpo4, the enzymatic properties of the chimeric enzyme are more Dpo4-like in that the enzyme is more processive, can bypass an abasic site and a thymine-thymine cyclobutane pyrimidine dimer, and predominantly makes base pair substitutions when replicating undamaged DNA. The converse is true for the Dpo4-LF-Dbh chimera, which is more Dbh-like in its processivity and ability to bypass DNA adducts and generate single-base deletion errors. Our studies indicate that the unique but variable LF domain of Y-family polymerases plays a major role in determining the enzymatic and biological properties of each individual Y-family member.

Highlights

Dpo4 and Dbh are Y-family polymerases that originate from two closely related strains of Sulfolobaceae
The thumb and finger domains are smaller than those found in high fidelity polymerases and in the ternary complex of Dpo4 with DNA and an incoming nucleotide; the primer-template is held between the thumb and little finger (LF) domains and buttresses against the finger domain [9]
To investigate the role that the LF domain plays in the enzymatic properties of Y-family polymerases, we constructed chimeric proteins in which the respective LF domains and the flexible linker that tethers the LF domain to the thumb domain were interchanged (Fig. 1B)

Summary

The abbreviations used are

DNA polymerase; LF, little finger; CPD, cyclobutane pyrimidine dimer. PolY Little Finger Domain Chimeras been determined and the ϳ2.5-kb dbh-containing sequence reported by Kulaeva et al [12] matches perfectly with the genomic sequence from S. acidocaldarius. Dbh originates from S. acidocaldarius and not S. solfataricus P1 as was originally thought. Structural studies of the two polymerases reveal that in addition to sharing high sequence homology, the fingers, palm, and thumb domains of the proteins are virtually superimposable. This suggests that the different enzymatic properties of the two enzymes might lie more in their sequence-divergent and structurally mobile LF domains. To investigate the role that the LF domain may play in determining the enzymatic properties of Y-family polymerases in general, we have constructed Dbh-Dpo chimeras in which the LF domains and the preceding linker have been interchanged (see Fig. 1B). By replacing Dbh LF with that of Dpo, the enzyme becomes more “Dpo4-like,” indicating that the LF domain is clearly a major factor in determining the physical and enzymatic properties of each polymerase. We discuss our observations in light of the crystal structure of Dbh and of the various Dpo4-DNA complexes that have been reported to date

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION