Mismatch repair-independent increase in spontaneous mutagenesis in yeast lacking non-essential subunits of DNA polymerase ε

Abstract

Yeast DNA polymerase ε (Pol ε) is a highly accurate and processive enzyme that participates in nuclear DNA replication of the leading strand template. In addition to a large subunit (Pol2) harboring the polymerase and proofreading exonuclease active sites, Pol ε also has one essential subunit (Dpb2) and two smaller, non-essential subunits (Dpb3 and Dpb4) whose functions are not fully understood. To probe the functions of Dpb3 and Dpb4, here we investigate the consequences of their absence on the biochemical properties of Pol ε in vitro and on genome stability in vivo. The fidelity of DNA synthesis in vitro by purified Pol2/Dpb2, i.e. lacking Dpb3 and Dpb4, is comparable to the four-subunit Pol ε holoenzyme. Nonetheless, deletion of DPB3 and DPB4 elevates spontaneous frameshift and base substitution rates in vivo, to the same extent as the loss of Pol ε proofreading activity in a pol2-4 strain. In contrast to pol2-4, however, the dpb3Δdpb4Δ does not lead to a synergistic increase of mutation rates with defects in DNA mismatch repair. The increased mutation rate in dpb3Δdpb4Δ strains is partly dependent on REV3, as well as the proofreading capacity of Pol δ. Finally, biochemical studies demonstrate that the absence of Dpb3 and Dpb4 destabilizes the interaction between Pol ε and the template DNA during processive DNA synthesis and during processive 3' to 5'exonucleolytic degradation of DNA. Collectively, these data suggest a model wherein Dpb3 and Dpb4 do not directly influence replication fidelity per se, but rather contribute to normal replication fork progression. In their absence, a defective replisome may more frequently leave gaps on the leading strand that are eventually filled by Pol ζ or Pol δ, in a post-replication process that generates errors not corrected by the DNA mismatch repair system.

Figure 1. Processivity of the polymerase activity of Pol ε holoenzyme and Pol2/Dpb2 complex.

(A) A 50 nt long, ³²P-5′end-labeled, oligonucleotide was annealed to pBluescript II SK (+) ssDNA and used as a DNA substrate in the polymerization assay. (B) Shown is the image of extension products generated by four-subunit Pol ε and a two-subunit Pol2/Dpb2 complex, separated on a 8% denaturing polyacrylamide gel (for details see Materials and Methods). A DNA sequencing ladder with the identical template was used as a molecular weight marker on the right hand. Reaction times are indicated under each lane. (C) The termination probability at each position on the template was calculated for the four-subunit Pol ε holoenzyme and Pol2/Dpb2 complex (for details see Materials and Methods).

Figure 2. Processivity of the exonuclease activity of Pol ε holoenzyme and Pol2/Dpb2 complex.

(A) A 57 nt long, ³²P-5′end-labeled, oligonucleotide was annealed to a 75 nt oligonucleotide, creating a primer-template to be used as a DNA substrate in the exonuclease assay. (B) Shown is the image of degradation products generated by four-subunit Pol ε and two-subunit Pol2/Dpb2 complex, separated on a 12% denaturing polyacrylamide gel (for details see Materials and Methods). Reaction times are indicated above each lane. (C) The termination probability at each position on the template was calculated for the four-subunit Pol ε holoenzyme and Pol2/Dpb2 complex (for details see Materials and Methods).