Spatiotemporal recruitment of human DNA polymerase delta to sites of UV damage.
Human DNA polymerase δ (Pol δ) is involved in various DNA damage responses in addition to its central role in DNA replication. The Pol δ4 holoenzyme consists of four subunits, p125, p50, p68 and p12. It has been established that the p12 subunit is rapidly degraded in response to DNA damage by UV leading to the in vivo conversion of Pol δ4 to Pol δ3, a trimeric form lacking the p12 subunit. We provide the first analysis of the time-dependent recruitment of the individual Pol δ subunits to sites of DNA damage produced by UV irradiation through 5 μm polycarbonate filters by immunofluorescence microscopy and laser scanning cytometry (LSC). Quantitative analysis demonstrates that the recruitments of the three large subunits was near complete by 2 h and did not change significantly up to 4 h after UV exposure. However, the recruitment of p12 was incomplete even at 4 h, with about 70% of the Pol δ lacking the p12 subunit. ChIP analysis of Pol δ after global UV irradiation further demonstrates that only p125, p50 and p68 were present. Thus, Pol δ3 is the predominant form of Pol δ at sites of UV damage as a result of p12 degradation. Using LSC, we have further confirmed that Pol δ was recruited to CPD damage sites in all phases of the cell cycle. Collectively, our results show that Pol δ at the DNA damage site is the Pol δ trimer lacking p12 regardless of the cell cycle phase.
Department of Biochemistry and Molecular Biology; New York Medical College; Valhalla, NY USA.
Jennifer
J
Chea
Sufang
S
Zhang
Hong
H
Zhao
Zhongtao
Z
Zhang
Ernest Y C
EY
Lee
Zbigniew
Z
Darzynkiewicz
Marietta Y W T
MY
Lee
D002453:Cell Cycle; D045744:Cell Line, Tumor; D004249:DNA Damage / Q000235:genetics*; D004258:DNA Polymerase III / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D006801:Humans; D021122:Protein Subunits / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering; D014466:Ultraviolet Rays
D021122:Protein Subunits; D034741:RNA, Small Interfering; C435720:POLD3 protein, human; C521101:POLD4 protein, human; D004258:DNA Polymerase III