Effect of Proliferating Cell Nuclear Antigen Ubiquitination and Chromatin Structure on the Dynamic Properties of the Y-family DNA Polymerases

Abstract

Y-family DNA polymerases carry out translesion synthesis past damaged DNA. DNA polymerases (pol) and 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 pol 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 pol in foci, it results in an increased residence time in foci. pol is even more mobile than pol, both when uniformly distributed and when localized in foci. Kinetic modeling suggests that both pol and pol diffuse through the cell but that they are transiently immobilized for ϳ150 ms, with a larger proportion of pol than pol immobilized at any time. Treatment of cells with DRAQ5, which results in temporary opening of the chromatin structure, causes a dramatic immobilization of pol but not pol. 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.

Epstein-Barr Virus BPLF1 Deubiquitinates PCNA and Attenuates Polymerase η Recruitment to DNA Damage Sites

“…3B). Pol focus formation in cells expressing inactive BPLF1 C61S was essentially the same as that observed in untransfected cells after UV damage (6,32,48). Formation of Pol foci in cells expressing BPLF1 1-246 was almost completely abolished, whereas cells expressing the inactive mutant had little effect on foci (approximately 25-fold reduction as quantitated in Fig.…”

“…We used fluorescence microscopy to examine the effects of BPLF1 on the UV-inducible redistribution of Pol to nuclear foci. UV treatment induces an increase in the number of cells containing YFP-Pol foci (48). In cultures coexpressing Pol and active BPLF1 1-246, but not inac- tive BPLF1 C61S, there was a striking reduction of focus formation (Fig.…”

Differential Roles for DNA Polymerases Eta, Zeta, and REV1 in Lesion Bypass of Intrastrand versus Interstrand DNA Cross-Links

“…Consistent with this model, Pol and REV1 form distinct foci in response to cisplatin treatment which colocalize with PCNA and focus formation is dependent upon monoubiquiti- nation of PCNA. Whether or not TLS polymerase focus formation truly represents an increase in actual TLS activity at stalled replication forks is not known; however, these visible foci likely represent increased residency times of Y-family TLS polymerases associated with monoubiquitinated PCNA (49). The fact that cisplatin-induced PCNA foci also colocalized with ␥-H2AX strongly suggests that these sites coincide with a replication stress response that effectively results in the localized phosphorylation of H2AX.…”

Human DNA Polymerase η and Its Regulatory Mechanisms

“…More confusingly, some point mutations in the UBZ domain severely aŠected the ability of XP-V cells to cope with UV-induced DNA damage, while a version of Polh lacking its C-terminus (including the UBZ sequence) but retaining an intact PIP-like motif was able to promote cellular survival in response to UV (30). The interaction of Polh with monoubiquitinated PCNA via these domains could be important for its function, although some recent reports suggest that cells are able to activate and relocate Polh independently of PCNA monoubiquitination (35)(36)(37)(38). Thus, the role of these domains and the relevance of the interaction with monoubiquitinated PCNA still need to be elucidated.…”