Roles of PCNA-binding and ubiquitin-binding domains in human DNA polymerase eta in translesion DNA synthesis

Abstract

Treatment of yeast and human cells with DNA-damaging agents elicits Rad6-Rad18-mediated monoubiquitination of proliferating cell nuclear antigen (PCNA) at its Lys-164 residue [ubiquitin (Ub)-PCNA], and this PCNA modification is indispensable for promoting the access of translesion synthesis (TLS) polymerases (Pols) to PCNA. However, the means by which K164-linked Ub modulates the proficiency of TLS Pols to bind PCNA and take over synthesis from the replicative Pol has remained unclear. One model that has gained considerable credence is that the TLS Pols bind PCNA at 2 sites, to the interdomain connector loop via their PCNA-interacting protein (PIP) domain and to the K164-linked Ub moiety via their Ub-binding domain (UBD). Specifically, this model postulates that the UBD-mediated binding of TLS Pols to the Ub moiety on PCNA is necessary for TLS. To test the validity of this model, we examine the contributions that the PIP and Ub-binding zinc finger (UBZ) domains of human Poleta make to its functional interaction with PCNA, its colocalization with PCNA in replication foci, and its role in TLS in vivo. We conclude from these studies that the binding to PCNA via its PIP domain is a prerequisite for Poleta's ability to function in TLS in human cells and that the direct binding of the Ub moiety on PCNA via its UBZ domain is not required. We discuss the possible role of the Ub moiety on PCNA in TLS.

Fig. 1.

Requirement of PCNA-binding PIP domains of hPolη for its in vivo function in TLS. (A) Alignment of PIP domains of yeast (Sc) and human (Hs) Polη. In addition to the C-terminal PIP domain present in both yPolη and hPolη, hPolη harbors a PIP domain that is located just after the PAD domain. The highly conserved hydrophobic residues of the PIP domain are shown in bold. The positions of the 5 conserved Pol domains (I–V), the PAD domain, and the UBZ domain are also indicated. (B) Effects of PIP1 and PIP2 mutations on the UV sensitivity of hPolη. The survival of XPV cells transfected with different Polη constructs was examined after exposure to different doses of UV irradiation and incubation on growth media in the presence of 1 mM caffeine.

Fig. 2.

Requirement of PIP domains in hPolη for stimulation of its DNA synthetic activity by PCNA. DNA synthesis by WT hPolη (lanes 2 and 3), PIP1 mutant hPolη (lanes 4 and 5), PIP2 mutant hPolη (lanes 6 and 7), or PIP1, PIP2 mutant hPolη (lanes 8 and 9) protein (1 nM each) was examined on M13 circular DNA annealed to a radiolabeled primer (5 nM) in the absence or presence of PCNA (50 ng), RFC (15 ng), and RPA (200 ng). Lane 1, DNA substrate with no hPolη added.

Fig. 3.

PCNA binding by PIP domains is essential for accumulation of hPolη in replication foci. MRC5 cells were cotransfected with WT GFP PCNA and WT FLAG Polη (I) or PIP1 (F443A, L444A) (II), PIP2 (F707A, F708A) (III), or PIP1, PIP2 (F443A, L444A, F707A, F708A) (IV) mutant FLAG Polη. Twenty-four hours after transfection, cells were UV-irradiated with 40 J/m² followed by incubation for 6 h before fixation. FLAG-tagged hPolη proteins were imunostained with anti-FLAG mAb. (Magnification: 600×.)

Fig. 4.

Role of UBZ domain in hPolη function in vivo. (A) Sequence alignment of human (Hs) and yeast (Sc) Polη UBZ domains. Highly conserved residues are shown in bold. The positions of 2 short β-sheets and the α-helix as determined from the solution structure of hPolη UBZ are indicated above the sequences. (B) Complementation of the UV sensitivity of XPV cells by hPolη UBZ mutations. The survival of XPV cells transfected with WT or UBZ mutant Polη constructs was examined after exposure to 5 J/m² of UV irradiation followed by incubation on growth media in the presence of 1 mM caffeine.

Fig. 5.

hPolη UBZ mutant proteins are not defective in functional interaction with PCNA. DNA synthesis by WT hPolη (lanes 2–4) or the UBZ mutant hPolη proteins C635A (lanes 5–7), H650A (lanes 8–10), D652A (lanes 11–13), H654A (lanes 14–16), and F655A (lanes 17–19) (1 nM each) was examined by using M13 circular DNA annealed to a radiolabeled primer (5 nM) in the presence of each of the 4 dNTPs under standard reaction conditions. As indicated, the reactions were carried out in the presence or absence of PCNA or Ub-PCNA (50 ng), RFC (15 ng), and RPA (200 ng). Lane 1, DNA substrate with no hPolη added.

Fig. 6.

UBZ mutant hPolη proteins accumulate in replication foci. MRC5 cells were cotransfected with WT GFP PCNA and UBZ mutant FLAG Polη as indicated. Twenty-four hours after transfection, cells were UV-irradiated with 40 J/m² followed by incubation for 6 h before fixation. FLAG-tagged proteins were immunostained with anti-FLAG mAb. For the accumulation of WT hPolη, see Fig. 3. (Magnification: 600×.)