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. 2008 Dec;19(12):5193-202.
doi: 10.1091/mbc.e08-07-0724. Epub 2008 Sep 17.

Effect of proliferating cell nuclear antigen ubiquitination and chromatin structure on the dynamic properties of the Y-family DNA polymerases

Simone Sabbioneda  1 , Audrey M Gourdin, Catherine M Green, Angelika Zotter, Giuseppina Giglia-Mari, Adriaan Houtsmuller, Wim Vermeulen, Alan R Lehmann
Affiliations

Effect of proliferating cell nuclear antigen ubiquitination and chromatin structure on the dynamic properties of the Y-family DNA polymerases

Simone Sabbioneda et al. Mol Biol Cell. 2008 Dec.

Abstract

Y-family DNA polymerases carry out translesion synthesis past damaged DNA. DNA polymerases (pol) eta and iota are usually uniformly distributed through the nucleus but accumulate in replication foci during S phase. DNA-damaging treatments result in an increase in S phase cells containing polymerase foci. Using photobleaching techniques, we show that poleta is highly mobile in human fibroblasts. Even when localized in replication foci, it is only transiently immobilized. Although ubiquitination of proliferating cell nuclear antigen (PCNA) is not required for the localization of poleta in foci, it results in an increased residence time in foci. poliota is even more mobile than poleta, both when uniformly distributed and when localized in foci. Kinetic modeling suggests that both poleta and poliota diffuse through the cell but that they are transiently immobilized for approximately 150 ms, with a larger proportion of poleta than poliota immobilized at any time. Treatment of cells with DRAQ5, which results in temporary opening of the chromatin structure, causes a dramatic immobilization of poleta but not poliota. Our data are consistent with a model in which the polymerases are transiently probing the DNA/chromatin. When DNA is exposed at replication forks, the polymerase residence times increase, and this is further facilitated by the ubiquitination of PCNA.

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Figures

Figure 1.
Figure 1.
Dynamics of eGFP-polη in living cells. (A) Western blot of the XP30RO-eGFP-polη cell line used in this study (lane 3), compared with MRC5 (lane 1) and XP30RO (lane 2). (B) Comparison of FRAP curves (relative fluorescence recovery plotted against time) of eGFP-polη uniformly distributed in untreated XP30RO-eGFP-polη cells and in foci in S phase cells from untreated, UV-treated, and HU-treated cells. (C) Fluorescence recovery of “pol-dead” mutant (blue) and wild-type polη (red) in foci (7 h after 15 Jm−2 UV-C). Also shown is the FRAP curve for eGFP-PCNA in foci (orange), showing large immobile fraction. (D) FLIP-FRAP analysis of eGFP-polη. Cells were not irradiated (no UV, mean of 63 cells) or globally irradiated with 12 (mean of 50 cells) and 16 Jm−2 (mean of 27 cells). Five hours later, half-nucleus bleaching associated with FLIP-FRAP analysis was performed. The data were normalized as described in Materials and Methods. The error bars represent the SE of the mean. (E) eGFP-polη accumulated at site of local irradiation. (F) Five hours after local irradiation, the area of local damage was entirely bleached, the recovery of fluorescence was measured in the bleached area and normalized to the level of fluorescence in the whole nucleus. Control cells represent cells in which no local damage was inflicted, but in which a square of the same size as irradiated cells was bleached.
Figure 2.
Figure 2.
Ubiquitination of PCNA and polη mobility. (A) MRC5 cells were UV-irradiated (15 Jm−2) and incubated for 6 h with epoxomicin. PCNA was analyzed by Western blotting. (B) MRC5 cells transfected with eGFP-polη and mRFP-PCNA were UV irradiated and incubated either in the presence or absence of epoxomicin. Six hours later, the cells were fixed and analyzed by autofluorescence. (C) MRC5 cells were transfected with empty vector, wild-type Rad18, or C28F mutant together with His-tagged PCNA, treated with or without UV, and then incubated for 6 h before analysis by Western blotting. (D) XP30RO-eGFP-polη cells were cotransfected with either wild-type or C28F mutant Rad18 together with mRFP-tubulin to identify the transfected cells. The following day, they were unirradiated or UV irradiated, and the mobility of eGFP-polη was measured using FRAP. (E) Western blot showing increased ubiquitination of PCNA in XP30RO-eGFP-polη cells in which USP1 was depleted by siRNA (lanes 3 and 4). Nontargeting control (siNTC, lanes 1 and 2). (F) Effect of siUSP1 on mobility of eGFP-polη in foci in HU-treated cells.
Figure 3.
Figure 3.
Polι is more mobile than polη. (A) Western blot with anti-polι of lysates from stable cell lines expressing eGFP-polι. (Note that the slow mobility band is the previously reported ubiquitinated form of polι; Bienko et al., 2005.) (B) Comparison of mobilities of eGFP-polι in stable cell lines of MRC5 and XP30RO expressing GFP-polι. (C) Comparison of mobilities of polι distributed uniformly in unirradiated cells and in foci in irradiated cells. Data for polη from Figure 1B are also shown (as dotted curves) for comparison.
Figure 4.
Figure 4.
Fractionation of polη and polι from cell lysates. (A) Lysates from unirradiated or UV-irradiated MRC5 cells were fractionated on a Superdex 200 gel filtration column, and fractions were analyzed by immunoblotting for polη and polι. L, load. (B) Equivalent lysates were centrifuged on glycerol gradients. (C) Molecular weight calculations from data obtained in A and B.
Figure 5.
Figure 5.
Effects of DRAQ5 on the mobilities of polη and ι. (A) Effect of DRAQ5 on the mobility of eGFP-histone H2B, eGFP-pol η, and eGFP-polι. Cells were treated with or without DRAQ5 for 3 min and then subjected to FRAP analysis. (B) MRC5 cells transfected with either eGFP-polη or eGFP-polι were UV irradiated, incubated for 6 h, and either fixed immediately or extracted with Triton X-100 before analysis by epifluorescence.
Figure 6.
Figure 6.
Model for dynamics of polη and ι. (A) In undamaged cells, polη and polι probe the chromatin, but the residence time of polη is greater than that of polι, implying either a higher Kon or lower Koff rate. The double-headed arrow signifies weak interaction between the two polymerases. (B) In damaged S phase cells, where there is a replication fork blocked by damage and resulting ubiquitination of PCNA, there are two dynamic processes, transport into the focus and association with the blocked fork.
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