Alignment of deduced amino acid sequences of hXPV and mXPV proteins. Identical and similar residues are boxed in dark and light gray, respectively. The conserved regions in human and other related translesion polymerases (Rad30, DinB, UmuC and Rev1) are boxed from A to G (9). Two putative nuclear localization signals are underlined (9).

CPD bypass activity of recombinant mXPV protein. (A) Recombinant hXPV and mXPV proteins tagged with hexa-histidine at their C-terminal end were expressed in the baculovirus system and purified by sequential chromatography on Hitrap DEAE, Ni–NTA agarose and MonoS. The purified fractions (0.27 ng) of both proteins were subjected to SDS–PAGE and silver stained. M, 10 kDa protein ladder marker (Gibco BRL). (B) DNA polymerase activity of recombinant proteins. Recombinant hXPV (open circles) or mXPV (open squares) proteins were incubated at 37°C with reaction mixtures containing single primed DNA templates and [α-³²P]dCTP. After 30 min incubation, acid-insoluble material was measured by scintillation counting. (C) The damage bypass DNA polymerase activity. The recombinant hXPV (0.05 ng in lanes 2, 8 and 14; 0.5 ng in lanes 3, 9 and 15) or mXPV (0.075 ng in lanes 4, 10 and 16; 0.75 ng in lanes 5, 11 and 17) protein or mouse DNA polymerase α (8.2 ng) was incubated at 37°C for 15 min with the 5′-³²P-labeled 13mer primer annealed with the 30mer DNA fragment containing a lesion [CPD, lanes 7–12; (6–4) photoproduct, lanes 13–18] or not containing a lesion (lanes 1–6). After incubation, the products were subjected to PAGE and detected by autoradiography. The position of the thymine dimer on the template is indicated by the bridge.

Complementation of UV sensitivities of XP-V cells by hXPV and mXPV cDNA. After UV irradiation, cells were cultured in the presence of 1 mM caffeine for 4 days. The number of proliferating cells was measured by [³H]thymidine incorporation. Open circle, WI38-VA13; closed circle, XP7TA; open square, XP7TA + hXPV cDNA; closed square, XP7TA + mXPV cDNA; open triangle, XP7TA + vector (pRC-CMV).

Expression of mXPV mRNA in adult mouse tissues. The mXPV cDNA probe was hybridized with 2 µg of poly(A)⁺ RNA from each tissue as indicated (upper). A β-actin cDNA was used as a control (lower). (A) Clontech. (B) OriGene.

Expression profiles of mXPV in Swiss 3T3 cells. (A and B) Serum-starved mouse Swiss 3T3 cells were stimulated with 10% serum and mRNA was isolated at the indicated times and subjected to northern blot analysis. (A) mRNA levels of mXPV, DNA polymerase α 68 kDa subunits (Polα 68K) and EF-1α in each fraction. EF-1α was used as an internal control. An asynchronous log phase (log) control is also shown. (B) Autoradiograms were quantitated and relative expression was normalized to EF-1α at each time point. The values were plotted relative to the values for 0 h. Closed circle, mXPV; open square, Polα 68K. DNA synthesis in Swiss 3T3 cells, measured by incorporation of [³H]thymidine into the acid-insoluble fractions, is plotted as a function of time (h) after the addition of serum to quiescent cells (open triangle). (C) Swiss 3T3 cells in log phase were exposed to UV light (10 J/m²) and poly(A)⁺ RNA was isolated at the indicated times after UV irradiation and subjected to northern blot analysis with the probes indicated. (D) Autoradiograms were quantified and relative expression was normalized to the level of EF-1α at each time point. The values were plotted relative to the values for 0 h. Closed circle, mXPV; open square, Polα 68K; closed triangle, p21.