Werner syndrome protein interacts functionally with translesion DNA polymerases

Abstract

Werner syndrome (WS) is characterized by premature onset of age-associated disorders and predisposition to cancer. The WS protein, WRN, encodes 3' --> 5' DNA helicase and 3' --> 5' DNA exonuclease activities, and is implicated in the maintenance of genomic stability. Translesion (TLS) DNA polymerases (Pols) insert nucleotides opposite replication-blocking DNA lesions and presumably prevent replication fork stalling/collapse. Here, we present in vitro and in vivo data that demonstrate functional interaction between WRN and the TLS Pols, Poleta, Polkappa, and Poliota. In vitro, WRN stimulates the extension activity of TLS Pols on lesion-free and lesion-containing DNA templates, and alleviates pausing at stalling lesions. Stimulation is mediated through an increase in the apparent V(max) of the polymerization reaction. Notably, by accelerating the rate of nucleotide incorporation, WRN increases mutagenesis by Poleta. In vivo, WRN and Poleta colocalize at replication-dependent foci in response to UVC irradiation. The functional interaction between WRN and TLS Pols may promote replication fork progression, at the expense of increased mutagenesis, and obviate the need to resolve stalled/collapsed forks by processes involving chromosomal rearrangements.

Fig. 1.

WRN stimulates the activity of Y-family TLS Pols. (A) A 5′-end-labeled 28-nt DNA primer, hybridized to a 36-nt DNA template (oligonucleotides 1 and 2; SI Table 2), was extended by Polη (0.375 fmol), Polκ (1 fmol), or Polι (0.4 fmol) in the absence (−) or presence (+) of WRN (30 fmol) at 37°C for 10 min. Reaction aliquots were electrophoresed through 14% polyacrylamide-urea gels; extension products were visualized and quantified by PhosphorImager analysis. (B) Extension of the 28-nt DNA primer was carried out as described in A except that human Polα and Polε were assayed in parallel with Polη. S, (−) enzyme; W, WRN alone.

Fig. 2.

TLS polymerase stimulation is specific to WRN. Extension of the 28/36 P/T DNA substrate (Fig. 1) by Polη (0.375 fmol) was monitored in the absence (−) or presence of increasing, equimolar amounts (3–30 fmol) of hWRN, hBLM or E. coli RecQ. S, (−) enzyme.

Fig. 3.

WRN stimulates lesion bypass activity of TLS Pol. (A) A 14-nt primer (oligo 8), hybridized to a DNA template with a site-specific cyclobutane pyrimidine dimer (oligo 10), was extended by 0.75 fmol Polη, 4 fmol Polκ, or 0.4 fmol Polι in the absence or presence of a fixed amount of WRN (30 fmol) as described. A lesion-free DNA template (oligo 9) served as a control. (B) A 20-nt DNA primer (oligo 11) was annealed either to a DNA template containing a single BPDE-dG adduct (oligo 13) or lacking the lesion (oligo 12) and extended by either Polκ (4 fmol, lanes 1 and 2; 16 fmol, lanes 3 and 4) or Polη (1.5 fmol, lanes 1 and 2; 6 fmol, lanes 3 and 4) with or without WRN (40 fmol) as indicated. Extension reactions were as described except that the dNTP concentration was increased to 0.2 mM. (C) A 28-nt primer (oligo 1), annealed to a 36-nt DNA template (oligo 2) containing a site-specific abasic site analog (AB), O⁶-methylguanine (O⁶mG), O⁴-methylthymine (O⁴mT), 1, N⁶-etheno adenine (eA), or 8-oxoguanine (8-oxdG), was extended by Polη (0.375 fmol) or Polκ (2 fmol) in the absence (−) or presence (+) of WRN (30 fmol). Sequences of DNA templates are indicated at the left of each panel.

Fig. 4.

WRN increases the extent of nucleotide misinsertion and misextension by Polη. Polη (1.5 fmol) was incubated with 0.1 pmol of a 16/30 P/T (oligo 14/oligo 9) (A) or a 14/30 P/T (oligo 8/oligo 9) (B) and either a single dNTP (A) or three of four dNTPs (B). Extension reactions were carried out at 37°C for 10 min ± 15 fmol WRN. N, reactions with all four dNTPs; S, DNA P/T (−) polymerase. Sequences of DNA templates are indicated at the left of each panel.

Fig. 5.

WRN increases mutagenesis by Polη. A 407-nt gap within the lacZ α-complementation sequence of bacteriophage M13mp2 was copied by Polη (12 fmol) ± an equimolar amount of exonuclease-deficient WRN. Reaction aliquots were transformed and plated on minimal medium containing X-gal. DNA isolated from colorless or light blue plaques was sequenced to score alterations in the target region. The frequencies at which DNA from mutant plaques displayed single or multiple mutations within the first 100 nucleotides of the gap in reactions lacking (open bars) or containing WRN (filled bars), are presented.

Fig. 6.

WRN colocalizes with Polη after UVC irradiation. (A) HeLa cells expressing EGFP-WRN and DsRed-Polη were irradiated with 20 J/m² UVC light, +UVC. After 6 h incubation in growth media, cells were fixed and examined for foci formation. DNA was stained with DAPI; −UVC, unirradiated controls. (B) WRN mutants used for transfection. K577M, helicase-defective; ΔExo, deletion of exonuclease domain; HRDC (helicase and ribonuclease D C-terminal domain), amino acids 1021–1432; C-ter (C-terminal) amino acids 1229–1432. HeLa cells, transfected with one of these EGFP-WRN constructs and DsRed-Polη, were irradiated and processed as described.