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 pol eta 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 eta in foci, it results in an increased residence time in foci. pol iota is even more mobile than pol eta, both when uniformly distributed and when localized in foci. Kinetic modeling suggests that both pol eta and pol iota diffuse through the cell but that they are transiently immobilized for similar to 150 ms, with a larger proportion of pol eta than pol iota immobilized at any time. Treatment of cells with DRAQ5, which results in temporary opening of the chromatin structure, causes a dramatic immobilization of pol eta but not pol iota. 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.
Effect of Proliferating Cell Nuclear Antigen Ubiquitination and Chromatin Structure on the Dynamic Properties of the Y-family DNA Polymerases
Sabbioneda Simone;
2008
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 pol eta 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 eta in foci, it results in an increased residence time in foci. pol iota is even more mobile than pol eta, both when uniformly distributed and when localized in foci. Kinetic modeling suggests that both pol eta and pol iota diffuse through the cell but that they are transiently immobilized for similar to 150 ms, with a larger proportion of pol eta than pol iota immobilized at any time. Treatment of cells with DRAQ5, which results in temporary opening of the chromatin structure, causes a dramatic immobilization of pol eta but not pol iota. 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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
