Yeast Rev1 protein promotes complex formation of DNA polymerase zeta with Pol32 subunit of DNA polymerase delta

Abstract

Yeast DNA polymerase (Pol) delta, essential for DNA replication, is comprised of 3 subunits, Pol3, Pol31, and Pol32. Of these, the catalytic subunit Pol3 and the second subunit Pol31 are essential, whereas the Pol32 subunit is not essential for DNA replication. Although Pol32 is an integral component of Pol delta, it is also required for translesion synthesis (TLS) by Pol zeta. To begin to decipher the bases of Pol32 involvement in Pol zeta-mediated TLS, here we examine whether Pol32 physically interacts with Pol zeta or its associated proteins and provide evidence for the physical interaction of Pol32 with Rev1. Rev1 plays an indispensable structural role in Pol zeta-mediated TLS and it binds the Rev3 catalytic subunit of Pol zeta. Here, we show that although Pol32 does not directly bind Pol zeta, Pol32 can bind the Rev1-Pol zeta complex through its interaction with Rev1. We find that Pol32 binding has no stimulatory effect on DNA synthesis either by Rev1 in the Rev1-Pol32 complex or by Pol zeta in the Pol zeta-Rev1-Pol32 complex, irrespective of whether proliferating cell nuclear antigen has been loaded onto DNA or not. We discuss evidence for the biological significance of Rev1 binding to Pol32 for Pol zeta function in TLS and suggest a structural role for Rev1 in modulating the binding of Pol zeta with Pol32 in Pol delta stalled at a lesion site.

Fig. 1.

Complex formation of Pol32 with Rev1. (A) Physical interaction of Pol32 with Rev1. Pol32 or Rev1 was mixed with GST–Rev1 (lanes 1–4), or Rev1 was mixed with GST–Pol32 (lanes 5–8). Approximately 3 μg of each protein was used for this study. After incubation, samples were bound to glutathione-Sepharose beads, followed by washing and elution of the bound proteins by SDS-sample buffer. Aliquots of each sample before addition to the beads (L), the flow-through fraction (F), last washing fraction (W), and the eluted proteins (E) were analyzed on a SDS-12% polyacrylamide gel developed with Coomassie blue. A control experiment was also done for GST binding with Pol32 (lanes 9–12) and Rev1 (lanes 13–16). (B) Association of Pol32 with Rev1 in yeast cells. Pol32–FLAG and Rev1–HA proteins in crude cell extract (lanes 1 and 2); in the absence of tagged Pol32, Rev1-HA is not immunoprecipitated (lane 3); when Pol32-FLAG is pulled down by the anti-FLAG affinity gel, Rev1-HA is coimmunoprecipitated (lane 4).

Fig. 2.

Mapping of regions in Rev1 and Pol32 involved in physical interaction. (A) (i) Schematic representation of wild-type Rev1 and its truncated forms. Yeast Rev1 protein (985 aa) has a BRCT domain toward its amino terminus and has the 5 conserved motifs (I–V) characteristic of Y family Pols, and motif V is followed by the PAD. All 5 motifs, including the PAD, are indispensable for Rev1 DNA synthetic activity. The region at the carboxyl terminus of Rev1 is referred to as carboxyl-terminal domain (CTD). The residues that remain in the truncated protein are indicated in parentheses. (ii) Schematic of 350-residue Pol32 protein and its truncated forms. The interaction data shown in B and C are summarized. (B) The PAD of Rev1 is necessary and sufficient for interaction with Pol32. Pol32 was mixed with GST–Rev1–2 (lanes 1–4), GST–Rev1–3 (lanes 9–12), GST–Rev1–4 (lanes 17–20), or GST–Rev1–5 (lanes 25–28). In reciprocal experiments, GST–Pol32 was mixed with Rev1–2 (lanes 5–8), Rev1–3 (lanes 13–16), Rev1–4 (lanes 21–24), or Rev1–5 (lanes 29–32). (C) Pol32 region involved in interaction with Rev1. Rev1 was mixed with GST–Pol32–1 (lanes 1–4), GST–Pol32 -2 (lanes 5–8), and GST–Pol32–3 (lanes 9–12). For both B and C, ≈3 μg of each protein was used for the study. After incubation, samples were bound to glutathione-Sepharose beads, followed by washing and elution of the bound proteins by SDS-sample buffer. Aliquots of each sample before additions to the beads (L), the flow-through fraction (F), last washing fraction (W), and the eluted proteins (E) were analyzed on a SDS-12% polyacrylamide gel stained with Coomassie blue.

Fig. 3.

Formation of a Polζ–Rev1–Pol32 complex. (A) Polζ does not directly bind Pol32. GST–Polζ was mixed with Pol32 (lanes 1–4) or GST–Pol32 was mixed with Polζ (lanes 5–8) and examined for the binding of Polζ with Pol32. (B) The Rev1–Polζ complex binds Pol32. The Rev1–Pol32 complex was mixed with GST–Polζ (lanes 1–4) or Rev1 was first mixed with GST–Polζ for 2 h at 4 °C and the preformed GST–Polζ–Rev1 complex was then mixed with Pol32 for 1 h at 25 °C (lanes 5–8). Approximately 2 μg of each protein was used for this study. After incubation, samples were bound to glutathione-Sepharose beads, followed by washing and elution of the bound proteins by SDS-sample buffer. Aliquots of each sample before additions to the beads (L), the flow-through fraction (F), last washing fraction (W), and the eluted proteins (E) were analyzed on a SDS-12% polyacrylamide gel developed with Coomassie blue.

Fig. 4.

Rev1–Pol32 complex does not bind Rev7 or Rad30. Rev1–Pol32 complex was mixed with GST-Rev7 (lanes 1–4) or GST-Rad30 (lanes 5–8). Approximately 2 μg of each protein was used for this study. After incubation, samples were bound to glutathione-Sepharose beads, followed by washings and elution of the bound proteins by SDS-sample buffer. Aliquots of each sample before additions to the beads (L), the flow-through fraction (F), last washing fraction (W), and the eluted proteins (E) were analyzed on a SDS-12% polyacrylamide gel developed with Coomassie blue.