The human intra-S checkpoint response to UVC-induced DNA damage

Abstract

The intra-S checkpoint response to 254 nm light (UVC)-induced DNA damage appears to have dual functions to slow the rate of DNA synthesis and stabilize replication forks that become stalled at sites of UVC-induced photoproducts in DNA. These functions should provide more time for repair of damaged DNA before its replication and thereby reduce the frequencies of mutations and chromosomal aberrations in surviving cells. This review tries to summarize the history of discovery of the checkpoint, the current state of understanding of the biological features of intra-S checkpoint signaling and its mechanisms of action with a focus primarily on intra-S checkpoint responses in human cells. The differences in the intra-S checkpoint responses to UVC and ionizing radiation-induced DNA damage are emphasized. Evidence that [6-4]pyrimidine-pyrimidone photoproducts in DNA trigger the response is discussed and the relationships between cellular responses to UVC and the molecular dose of UVC-induced DNA damage are briefly summarized. The role of the intra-S checkpoint response in protecting against solar radiation carcinogenesis remains to be determined.

Fig. 1.

Cell cycle-dependent initiation of carcinogenesis. Replication of damaged DNA is required to induce mutations and chromosomal aberrations as permanent heritable changes in the genome that can initiate carcinogenesis. Repair of DNA damage before DNA replication reduces the frequencies of mutations and chromosomal aberrations and thereby reduces the risk of initiation of carcinogenesis.

Fig. 2.

Velocity sedimentation analysis of intermediates of DNA replication in UV-damaged human fibroblasts. The velocity sedimentation method involves separating ³H-thymidine-labeled nascent DNA molecules according to size by sedimentation in alkaline sucrose gradients. Incorporation of ³H-thymidine is normalized to DNA content, so profiles represent the specific activities of DNA synthesis in various size classes of nascent DNA. DNA size is a measure in part of replicon age. Cells were incubated with ³H-thymidine for 15 min beginning 30 min after sham or UV treatment. Sedimentation was from right to left. The peak of ³H radioactivity at fractions 16 and 17 in the no-UV control corresponds to nascent molecules of about half the average replicon size (2 × 10⁷ Da) that initiated synthesis after the sham treatment. The selective inhibition of DNA synthesis in half-replicon-size intermediates seen 30 min after 0.5 or 1 J/m² reflects the inhibition of replicon initiation. The inhibition of DNA synthesis in multi-replicon-size intermediates (fractions 5–12) reflects the UV dose-dependent inhibition of DNA chain elongation in replicons that had initiated synthesis before UV treatment. Note the abnormally small nascent intermediates that appear in fractions 18–21 after 5 and 10 J/m². These probably arise by DNA synthesis between sites of UV-induced DNA damage.

Fig. 3.

Working model of intra-S checkpoint response to UV-induced DNA damage. (A) Schematic structure of a replicon with UV-induced CPDs and 6-4PPs in template strands blocking leading-strand and lagging-strand DNA polymerases. (B) Rapid NER excision repair of 6-4PPs ahead of DNA growing points reduces the blocking of replicative polymerases and DNA growing points. Rapid TLS of TT CPDs by DNA pol eta suppresses intra-S checkpoint signaling. Sustained blockage at 6-4PPs and uncoupling of the replicative helicase and polymerases stimulate Rad17/RFC2–5-dependent loading of 9-1-1 complexes and generate RPA-coated single-stranded templates that attract ATR–ATRIP and Tim–Tipin–claspin complexes. Binding of TopBP1 to phospho-Rad9 and Chk1 to Tim and claspin completes the assembly of the RFPC to allow ATR to phosphorylate and activate Chk1. XPA and Cep164 appear to stimulate Chk1 phosphorylation. Activated Chk1 diffuses to dissociate MCM–GINS–Cdc45 complexes to inhibit replicon initiation and DNA chain elongation. Scission of the 6-4PP-containing template strand at a blocked DNA growing point produces a DNA dsb, thereby triggering the ATM-dependent checkpoint signaling pathway and recruiting DNA dsb repair pathways. See the Supplementary data at Carcinogenesis Online for an animated version of the working model.