REV1 and DNA polymerase zeta in DNA interstrand crosslink repair

Abstract

DNA interstrand crosslinks (ICLs) are covalent linkages between two strands of DNA, and their presence interferes with essential metabolic processes such as transcription and replication. These lesions are extremely toxic, and their repair is essential for genome stability and cell survival. In this review, we will discuss how the removal of ICLs requires interplay between multiple genome maintenance pathways and can occur in the absence of replication (replication-independent ICL repair) or during S phase (replication-coupled ICL repair), the latter being the predominant pathway used in mammalian cells. It is now well recognized that translesion DNA synthesis (TLS), especially through the activities of REV1 and DNA polymerase zeta (Polζ), is necessary for both ICL repair pathways operating throughout the cell cycle. Recent studies suggest that the convergence of two replication forks upon an ICL initiates a cascade of events including unhooking of the lesion through the actions of structure-specific endonucleases, thereby creating a DNA double-stranded break (DSB). TLS across the unhooked lesion is necessary for restoring the sister chromatid before homologous recombination repair. Biochemical and genetic studies implicate REV1 and Polζ as being essential for performing lesion bypass across the unhooked crosslink, and this step appears to be important for subsequent events to repair the intermediate DSB. The potential role of Fanconi anemia pathway in the regulation of REV1 and Polζ-dependent TLS and the involvement of additional polymerases, including DNA polymerases kappa, nu, and theta, in the repair of ICLs is also discussed in this review.

Figure 1. Physical representation of the functional domains of the human REV3, REV1 and REV7 proteins

REV3 has a region in the N-terminus that is homologous to Polδ, a REV7 binding domain, and a conserved Zinc finger motif in the catalytic domain. REV1 possesses a BRCT domain that interacts with PCNA or DNA, two ubiquitin binding motifs that mediate localization to stalled replication forks, and a C-terminal TLS polymerase and REV7-binding domain. REV7 is almost entirely composed of a HORMA domain that is thought to mediate interactions with chromatin.

Figure 2. A model for human ICL repair

Nonproliferating cells are capable of removing ICLs in a manner that is independent of DNA replication. Depending upon the type of ICL or whether the ICL has interfered with RNA transcription, the XPC/HR23B (global genome NER) or CSB (transcription-coupled NER) protein recognizes the lesion and stimulates ‘unhooking’ by the XPF-ERCC1 endonuclease. A single stranded DNA gap is formed that attracts Replication Protein A (RPA) which in turn promotes RAD18/RAD6-dependent monoubiquitination of PCNA. Monoubiquitinated PCNA recruits REV1/Polζ and/or Polκ to the site where they perform TLS opposite the unhooked lesion, the latter presumably removed by a second set of NER reactions to return the DNA duplex to its original state. If the lesion is not removed in G1 (left panel), convergence of DNA polymerases on an unrepaired ICL during DNA replication initiates the Fanconi anemaia (FA) pathway involving detection of the ICL by the FA core complex followed by ubiquitination of the FANCD2/FANCI heterodimer in the late S/G2 phase of the cell cycle (right panel). Ubiquitinated FANCD2/FANCDI promotes excision or ‘unhooking’ of the ICL by structure-dependent endonucleases and likely promotes TLS opposite the ‘unhooked’ ICL in preparation for homologous recombination (HR). MUS81-EME1, SNM1A, FAN1, XPF-ERCC1 and SLX1-SLX4 are structure specific endonucleases thought to be involved in unhooking the ICL, generating ICL-associated double strand breaks (DSBs) and nucleolytic processing of the ICL during repair. SLX4 has also been shown to be an endonuclease scaffold protein and mutations in the SLX4 gene cause Fanconi anemia. FAAP20 interacts with the FA core complex and directly binds to REV1. The mismatch repair protein MSH2 is implicated in ICL repair through an unknown mechanism. The FA pathway and REV1/Polζ can promote HR repair of a direct DSB. We speculate that REV1 and Polζ are not only involved in TLS across the ‘unhooked ICL’ but also perform DNA synthesis during HR repair when the template is incompatible for normal replicative DNA polymerases.