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

Abstract

Translesion DNA synthesis (TLS) is a process whereby specialized DNA polymerases are recruited to bypass DNA lesions that would otherwise stall high-fidelity polymerases. We provide evidence that TLS across cisplatin intrastrand cross-links is performed by multiple translesion DNA polymerases. First, we determined that PCNA monoubiquitination by RAD18 is necessary for efficient bypass of cisplatin adducts by the TLS polymerases eta (Pol), REV1, and zeta (Pol) based on the observations that depletion of these proteins individually leads to decreased cell survival, cell cycle arrest in S phase, and activation of the DNA damage response. Second, we showed that in addition to PCNA monoubiquitination by RAD18, the Fanconi anemia core complex is also important for recruitment of REV1 to stalled replication forks in cisplatin treated cells. Third, we present evidence that REV1 and Pol are uniquely associated with protection against cisplatin and mitomycin C-induced chromosomal aberrations, and both are necessary for the timely resolution of DNA double-strand breaks associated with repair of DNA interstrand cross-links. Together, our findings indicate that REV1 and Pol facilitate repair of interstrand cross-links independently of PCNA monoubiquitination and Pol, whereas RAD18 plus Pol, REV1, and Pol are all necessary for replicative bypass of cisplatin intrastrand DNA cross-links.Maintenance of genomic integrity involves the activation of cell cycle checkpoints coupled with DNA repair. Despite these sophisticated mechanisms to remove DNA lesions prior to DNA replication, replication forks may inevitably encounter nonrepaired lesions that block high fidelity polymerases, potentially leading to replication fork instability, gaps in replicated DNA, and the generation of DNA double-strand breaks (DSBs). In order to preserve replication fork stability by allowing replication through polymerase blocking lesions, template DNA containing a damaged base or abasic site can be replicated through the actions of specialized translesion DNA synthesis (TLS) polymerases (61). A key event in the regulation of TLS is the monoubiquitination of PCNA, a homotrimeric protein that functions as an auxiliary factor for DNA polymerases (28,31,57,60). The RAD6 (E2)-RAD18 (E3) complex specifically monoubiquitinates PCNA on Lys-164 in response to replication fork stalling. This event is thought to operate as a molecular switch from normal DNA replication to the TLS pathway based on the observations that association of Y-family TLS polymerases with monoubiquitinated PCNA is strengthened through the cooperative binding of one or more ubiquitin-binding domains (UBM or UBZ) plus a PCNA-interacting domain (6,25).Extensive biochemical evidence suggests that replication through a large variety of lesions requires the sequential action of two TLS polymerases (44). The Y-family polymerase eta (Pol) plays a key role in the efficient and error-free bypass of cyclobutane pyrimidine (TT) dimers, one of the major lesions resulting from exposure to UV radiation (...

A small ubiquitin binding domain inhibits ubiquitin-dependent protein recruitment to DNA repair foci

“…Lentivirus was generated as described. 48 Cells expressing mCherry were enriched by flow cytometry sorting prior to experimentation. Cells were irradiated at room temperature at a dose rate of 3 Gy/ min using a Pantak DXT300 orthovoltage unit.…”

“…Coverslips were stained for the indicated antibodies as described. 33,48 Briefly, the immunofluorescence staining protocol consisted of blocking for 30 min with 5% fetal bovine serum, 0.05% Triton X-100, and 1% goat serum, and then incubating coverslips with primary antibodies for 45 min. Coverslips were washed 3 times with PBS and then incubated with the appropriate secondary goat anti-rabbit or goat anti-mouse Alexa Fluor dye (488 or 595) conjugated secondary antibody (Molecular Probes) for 45 min, washed with PBS, counterstained with DAPI (Sigma) to visualize nuclear DNA, and then mounted onto slides with ProLong Gold antifade reagent (Invitrogen).…”

“…Coverslips were stained and imaged by immunofluorescence for S1981P-ATM and γ-H2AX as described. 48 U20S cells containing an integrated copy of the DR-GFP reporter 39 were transduced with lentivirus encoding mCherry-tagged RAD18 WT, RAD18 D221A, RAD18 UBD, or mCherry alone. Cells were then transduced with AdNGUS24i adenovirus encoding I-SceI.…”

“…48 Human RAP80 and RNF168 cDNA were subcloned into pLenti EV (University of Michigan vector core facility) such that EGFP was fused in frame at the N-terminal end of each protein. To create FLAG-tagged UBDs, primers for cDNA amplification were designed such that the coding frames for full-length RAD18, RAD18 UBD (191-238), RAP80 UBD (64-130), or Pol η UBD (615-670) were subcloned in frame with the Flag epitope in the vector SG5 (Invitrogen).…”

Chromatin Regulates Genome Targeting with Cisplatin

“…Cotreatment with SAHA and APPA led to mono‐ubiquitination of PCNA (Figure 4 B), a mark that can promote the recruitment of low‐fidelity polymerases involved in TLS,19 and cells cotreated with SAHA and cisplatin or APPA exhibited increased number of RAD18 foci (see Supporting Information). Furthermore, treatment with SAHA and cisplatin led to the focal accumulation of the TLS polymerase Polη20 that colocalized with PCNA (Figure 4 C and Supporting Information). Altogether, these data were consistent with the notion that SAHA and cisplatin derivatives synergistically activated TLS.…”