Eukaryotic translesion synthesis: Choosing the right tool for the job

Abstract

Normal DNA replication is blocked by DNA damage in the template strand. Translesion synthesis is a major pathway for overcoming these replication blocks. In this process, multiple non-classical DNA polymerases are thought to form a complex at the stalled replication fork that we refer to as the mutasome. This hypothetical multi-protein complex is structurally organized by the replication accessory factor PCNA and the non-classical polymerase Rev1. One of the non-classical polymerases within this complex then catalyzes replication through the damage. Each non-classical polymerase has one or more cognate lesions, which the enzyme bypasses with high accuracy and efficiency. Thus, the accuracy and efficiency of translesion synthesis depends on which non-classical polymerase is chosen to bypass the damage. In this review article, we discuss how the most appropriate polymerase is chosen. In so doing, we examine the structural motifs that mediate the protein interactions in the mutasome; the multiple architectures that the mutasome can adopt, such as PCNA tool belts and Rev1 bridges; the intrinsically disordered regions that tether the polymerases to PCNA and to one another; and the kinetic selection model in which the most appropriate polymerase is chosen via a competition among the multiple polymerases within the mutasome.

Introduction

DNA damage in the template strand blocks DNA synthesis by classical DNA polymerases – those involved in normal DNA replication and repair. Consequently, when replication forks encounter DNA damage, they stall. Without a means of replicating through damaged DNA, genome instability, chromosome instability, and cell death can occur. Thus, several damage bypass pathways have evolved that allow replication to proceed through damaged DNA. Translesion synthesis (TLS) is a major damage bypass pathway in eukaryotes. In TLS, one or more non-classical DNA polymerases – those involved in damaged DNA replication – catalyze DNA synthesis through the DNA lesion [[1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11]]. Unfortunately, the bypass of DNA damage by non-classical DNA polymerases is often error-prone and mutagenic.

Cells possess a variety of non-classical polymerases for replicating through DNA damage. These non-classical polymerases differ from classical DNA polymerases in that they can utilize DNA lesions as templates for nucleotide incorporation. Each of these polymerases has one or more DNA lesions that it bypasses with relatively high accuracy and efficiency as determined by steady state or pre-steady state kinetics (for a recent discussion of this topic, see [12]). These lesions are known as the cognate lesions of the non-classical polymerases (Table 1). For example, the cognate lesions of DNA polymerase eta (pol η) include thymine dimers and 8-oxoguanines [[13], [14], [15]]. The cognate lesions of Rev1 include minor-groove and exocyclic guanine adducts [16,17].

The accuracy and efficiency of TLS depends on which of the available non-classical polymerases is chosen to bypass the DNA damage. If the damage is a cognate lesion for the chosen polymerase, the accuracy and efficiency of TLS will be relatively high. If the damage is not a cognate lesion for the polymerase, the accuracy and efficiency will be much lower. Thus, the central question in trying to understand the accuracy and efficiency of TLS is: how is a non-classical polymerase chosen to bypass DNA damage? Answering this question will involve investigating the structures and functions of the individual proteins involved in TLS as well as the structures and functions of any protein complexes that they form.

These non-classical polymerases interact with each other and with several other replication-associated proteins. This has led to the general working hypothesis that these polymerases function within a large, multi-protein complex that forms at stalled replication forks [[18], [19], [20]]. In this article, we are adopting this theoretical framework and are using the term “mutasome” (i.e., mutagenic replisome) to refer to this complex. These multiple non-classical polymerases contain intrinsically disordered regions that act as flexible tethers allowing the polymerases to sample a wide range of conformational states without dissociating from the mutasome [21,22]. This conformational flexibility presumably allows the multiple polymerases within the mutasome to compete with one another for binding the DNA substrate. Ultimately, such a competition would maximize the accuracy and efficiency of TLS via a kinetic selection model [12].

In this review article, we will examine this emerging paradigm. Following in the footsteps of Aristotle, we will first discuss the composition of the mutasome – its material cause. This will include an examination of the intrinsically disordered regions of the non-classical polymerases and the structural motifs that mediate the protein-protein interactions within the mutasome. We will next discuss the structural organization of the mutasome – its formal cause. This will include an examination of both the architectures and high-resolution structures that the mutasome can adopt. We will then discuss the assembly of the mutasome – its efficient cause. This will include an examination of the potential roles that PCNA ubiquitylation plays in TLS. We will lastly discuss how the mutasome functions to maximize the accuracy and efficiency of TLS – its final cause. This will include an examination of the kinetic selection model for choosing the most appropriate non-classical polymerase to bypass the lesion.