Integrating S-phase checkpoint signaling with trans-lesion synthesis of bulky DNA adducts

Abstract

Bulky adducts are DNA lesions generated in response to environmental agents including benzo[a]pyrene (a combustion product) and solar ultraviolet radiation. Error-prone replication of adducted DNA can cause mutations, which may result in cancer. To minimize the detrimental effects of bulky adducts and other DNA lesions, S-phase checkpoint mechanisms sense DNA damage and integrate DNA repair with ongoing DNA replication. The essential protein kinase Chk1 mediates the S-phase checkpoint, inhibiting initiation of new DNA synthesis and promoting stabilization and recovery of stalled replication forks. Here we review the mechanisms by which Chk1 is activated in response to bulky adducts and potential mechanisms by which Chk1 signaling inhibits the initiation stage of DNA synthesis. Additionally, we discuss mechanisms by which Chk1 signaling facilitates bypass of bulky lesions by specialized Y-family DNA polymerases, thereby attenuating checkpoint signaling and allowing resumption of normal cell cycle progression.

Fig. 1. S-phase checkpoint signaling results from uncoupling of replicative helicase and polymerase activities

Bulky adducts and other DNA lesions (shown in black) cause stalling of the replicative DNA polymerase (illustrated here in association the PCNA trimer). Continued unwinding of DNA by the Mcm2–7 replicative helicase complex generates excessive ssDNA which is coated by RPA. ATRIP-ATR and the 9-1-1 complex (not shown) are recruited independently to the stalled replication fork. TopBP1 stimulates ATR kinase activity (no association between TopBP1 and RPA-ssDNA is implied). Chk1 is an ATR substrate and is activated by phosphorylation. ATR-mediated Chk1 activation also requires the mediator protein Claspin (not shown). Chk1 signaling inhibits initiation of DNA synthesis at late-firing origins of replication, as described in the text and in Fig. 2.

Fig. 2. Model describing effect of Polk status on BPDE-induced S-phase checkpoint signaling

In Polk^+/+ cells (left panel) BPDE-adducted DNA (shown in black) uncouples the replicative DNA polymerase (Pol) and helicase activities thereby generating ssDNA and initiating ATR/Chk1 signaling (A). A polymerase switch replaces the stalled DNA polymerase with Polκ (B). Polκ-mediated bypass of the adduct lesion allows recovery of the stalled replication fork and attenuates S-phase checkpoint signaling. In Polk^−/− cells (right panel), failure to bypass BPDE-adducted DNA results in persistently stalled forks (B). Eventual fork collapse generates DSB (C) and elicits ATM/Chk2 signaling.

Fig. 3. Hypothetical mechanisms for checkpoint-dependent PCNA mono-ubiquitination and polymerase switching in mammalian cells

Chk1 signaling could promote PCNA mono-ubiquitination and TLS via Rad18 recruitment or activation, negative regulation of USP1 (or other DUBs), or by a combination of both mechanisms. Although not shown in the figure, 9-1-1 represents an alternative sliding clamp for TLS polymerases.

Fig. 4. Hypothetical role of REV1 in lesion-specific recruitment of Y-family polymerases in mammalian cells

According to this model, Polη is the first TLS polymerase recruited to forks stalled by any DNA lesion. For thymine dimers (TT-CPD), Polη is sufficient to perform the insertion and extension steps of TLS and no additional polymerases are necessary. For (6-4) photoproducts (TT (6-4)PP), Polη inserts one of the 4dXMPs opposite 3'-T while Polι inserts the correct A, but Polζ is required for extension and completion lesion bypass. REV1 may facilitate the exchange of Polη for Polζ. For BPDE adducts (BPDE-dG), Polκ carries out both insertion and extension steps of TLS, whereas Polη cannot perform lesion bypass. If Polη is recruited to BPDE-stalled forks, REV1 may facilitate the exchange of Polη for Polκ, thereby enabling lesion bypass.