Review ArticleTolerating DNA damage during eukaryotic chromosome replication
Introduction
DNA damage is inevitable and constitutes a threat to the maintenance of genome integrity. Cells can cope with this challenge thanks to surveillance mechanisms (checkpoints) that detect the problem, and to different pathways that repair the damaged DNA [1], [2]. Although these systems are highly efficient, some DNA lesions may escape repair, and those remaining at the moment of DNA replication may dangerously interfere with the progression of replication forks. Faithful transmission of genetic information requires accurate and complete genome replication, and therefore it is crucial to overcome unrepaired DNA lesions that cause fork blocks. Indeed, cells use strategies that allow them to effectively deal with replication-blocking damage without removing the DNA lesions. These are collectively referred to as DNA damage tolerance (DDT) mechanisms (also known as DNA damage bypass or post-replication repair processes) [3], which act on DNA lesions in single-stranded DNA that block the replicative polymerases. In eukaryotic cells, the principal mediator of DDT is the evolutionarily conserved RAD6/RAD18 pathway, which is subdivided into two branches that define two modes of DDT: translesion DNA synthesis and DNA damage avoidance. Translesion DNA synthesis uses specialised DNA polymerases that replicate directly across the damaged template in a process that is frequently error-prone and a major source of mutagenesis. In contrast, in the DNA damage avoidance mechanism, the blocked nascent DNA strand uses the undamaged strand of the sister chromatid as a template for replication over the DNA lesion, and the result is error-free. DDT mechanisms are controlled by post-translational modifications of PCNA (Proliferating Cell Nuclear Antigen), the sliding clamp that is essential for the processivity of DNA polymerases. Additionally, new proteins have been recently identified as modulators of DDT. In this review, we summarise our present knowledge of the processes that allow tolerance of DNA damage during eukaryotic chromosome replication, a topic of growing interest in which considerable progress has been made in recent years.
Section snippets
Activation of DNA damage tolerance mechanisms
DNA damage tolerance mechanisms respond to the presence of DNA lesions, and a key issue is how the DDT pathway is initiated. When replication forks stall because of DNA damage or replicative stress, the actions of the DNA polymerases and the replicative helicase can partially uncouple. This uncoupling leads to the formation of long stretches of single-stranded DNA (ssDNA) that are coated by the replication protein A (RPA). The exposed RPA-coated ssDNA acts as a signal for the recruitment of the
Bypassing DNA lesions by translesion DNA synthesis
Translesion DNA synthesis (TLS) is a direct mechanism of bypassing unrepaired DNA lesions (Fig. 2). Although it contributes to the resistance to genotoxic agents and is important for genome stability, it is however a significant source of damage-induced mutagenesis. During TLS, the stalled replicative polymerase is replaced by a specialised polymerase that is able to replicate over the DNA lesion (Fig. 1). Unlike replicative DNA polymerases, TLS polymerases are non-processive low-fidelity
Avoiding DNA damage by template switching
The DNA damage avoidance sub-pathway is an alternative, error-free mode of DDT that is triggered by PCNA polyubiquitylation at Lys164. Although this process is not yet completely understood at the molecular level, it is thought to mediate lesion bypass by a transient “template switching” in which the stalled nascent DNA strand utilises the newly synthesised, undamaged strand of the sister chromatid as a template for replication across the DNA lesion (Fig. 2). Initially, it was proposed that
The choice between translesion DNA synthesis and template switching for damage bypass
TLS and template switching are different strategies for DNA damage bypass, and a key question to address is which mechanisms regulate the choice between them when cells are required to tolerate DNA damage. This choice is important because it can determine an error-free or error-prone outcome; therefore, an optimal balance between the two modes of DDT needs to be established. Thus far, what prompts the switch from monoubiquitylation to polyubiquitylation of PCNA and, consequently, from TLS to
When and where DNA damage tolerance mechanisms operate
A long-standing problem in the field has been to determine exactly when and where DNA damage bypass takes place: coupled to on-going replication forks or post-replicatively (Fig. 2). Although DNA damage tolerance was initially considered as a fork-independent, “post-replicative repair” process, a prevailing idea was that damage bypass occurred directly at replication forks. However, several studies carried out in yeast strongly support the notion that the RAD6-RAD18 pathway functions behind
Concluding remarks
DNA damage tolerance is crucial for the maintenance of genome integrity. The diverse modes of bypassing damaged DNA constitute a robust and versatile cellular system that allows unrepaired DNA lesions to be efficiently overcome, contributing decisively to the completion of chromosome replication in every cell cycle. Recent works have provided novel insights into how DNA damage tolerance mechanisms operate. The identification of new functional modulators of this cellular response demonstrates
Acknowledgements
We apologize for any omission of articles that we were unable to cite. Work in JAT׳s lab is supported by the Spanish Ministry of Economy and Competitiveness (MINECO), grants BFU2010-16989 and Consolider Ingenio CSD2007-00015. M.O.B. is the recipient of a pre-doctoral fellowship from MINECO. The Centro de Biología Molecular Severo Ochoa receives an institutional grant from the Fundación Ramón Areces.
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S Phase
2022, Encyclopedia of Cell Biology: Volume 1-6, Second EditionHLTF Promotes Fork Reversal, Limiting Replication Stress Resistance and Preventing Multiple Mechanisms of Unrestrained DNA Synthesis
2020, Molecular CellCitation Excerpt :PCNA polyubiquitination, mediated by the E3 ligase Rad5 in yeast, promotes template switching, which uses the sister chromatid as a template for error-free lesion bypass (Branzei and Szakal, 2017; Hoege et al., 2002). In mammalian cells, the E3 ubiquitin ligases HLTF and SHPRH contribute to PCNA polyubiquitination, although polyubiquitination is still observed upon the loss of both proteins (Saugar et al., 2014; Unk et al., 2010). This implies that additional factors are likely involved and that DDT processes are more complex in mammalian cells.
Divalent Cations Alter the Rate-Limiting Step of PrimPol-Catalyzed DNA Elongation
2019, Journal of Molecular BiologyCitation Excerpt :Unfortunately, DNA lesions or non-canonical DNA structures constantly challenge DNA replication [1,2]. The stressed replication can be rescued by one or more of the following mechanisms: (i) DNA damage avoidance [3], (ii) specialized DNA polymerases (pols) copying past the obstructive DNA structures, and (iii) replication restart after the roadblock [4,5]. The lesion bypass (also known as translesion synthesis) is coordinated by specialized DNA polymerases and accessory proteins, and can occur at the fork or post-replicatively [6–11].
Role of specialized DNA polymerases in the limitation of replicative stress and DNA damage transmission
2018, Mutation Research - Fundamental and Molecular Mechanisms of MutagenesisRole of recombination and replication fork restart in repeat instability
2017, DNA RepairCitation Excerpt :Given the ability of TNRs to form secondary structures that impair fork progression, its not surprising that proteins required for template switch have been shown to be involved in the stability of repetitive sequences. Repair of post-replication gaps is dependent on ubiquitination of PCNA, and can be subdivided into two categories: translesion synthesis (TLS) and error-free post-replication repair (PRR) or template switching (reviewed by [68,69]). The TLS branch is dependent on the PCNA ubiquitin ligases, Rad6 and Rad18, which together monoubiquitinate Lys164 of PCNA.