Chapter Seven - The Many Roles of PCNA in Eukaryotic DNA Replication
Introduction
Since its discovery in the late 1970s, our view of proliferating cell nuclear antigen (PCNA) and its roles in DNA replication and genome maintenance has expanded considerably. PCNA was originally identified as the target of an autoimmune antibody derived from patients with systemic lupus erythematosus [1]. This protein was later shown to be one produced predominantly in proliferating and transformed cells [2], [3], [4]. By the middle of the 1980s, the involvement of PCNA in DNA replication was suggested based on its pattern of staining throughout the cell cycle [5].
Definitive evidence of a role for PCNA in DNA replication came a couple years later with the discovery that PCNA is required for the replication of simian virus 40 in vitro [6], [7]. It was soon realized that PCNA was an auxiliary protein for DNA polymerase delta (pol δ) that increases its activity by making it more processive [8], [9], [10]. PCNA was subsequently shown to be an auxiliary factor for DNA polymerase epsilon (pol ɛ) [11], [12], [13], [14]. By the early 1990s, the role of PCNA came to be viewed as being the processivity factor of eukaryotic replicative polymerases.
An understanding of how PCNA confers high processivity to DNA polymerases was achieved when the X-ray crystal structure of PCNA was determined [15]. PCNA was shown to be a ring-shaped trimer similar to the structure of the Escherichia coli beta clamp determined a couple years earlier [16]. By the middle of the 1990s, it was known that PCNA is loaded onto double-stranded DNA by replication factor C (RFC) [17], [18], where the PCNA functions as a sliding clamp that binds and anchors polymerases onto the DNA.
As more and more PCNA-interacting partners were identified, it became clear that PCNA is not simply a processivity factor for replicative polymerases. It interacts with and regulates the activities of many proteins involved in Okazaki fragment maturation [19], [20], mismatch repair [21], nucleotide excision repair [22], and translesion synthesis [23], [24], [25], [26]. It also interacts with proteins involved in other processes such as cell cycle control [27], [28], [29], sister chromatid cohesion [30], epigenetic inheritance [31], and S-phase-specific proteolysis [32]. By the early 2000s, PCNA came to be viewed as an important hub protein that is critical for organizing and orchestrating events at the replication fork and other sites of DNA synthesis.
Since the early 2000s, it has become clear that the regulation of several DNA metabolic processes is governed by posttranslational modifications of PCNA, most notably ubiquitylation and sumoylation [33], [34]. Ubiquitylation of PCNA promotes translesion synthesis via the recruitment of translesion synthesis polymerases to stalled replication forks [35]. Sumoylation of PCNA inhibits recombination via the recruitment of antirecombinases to sites of DNA synthesis [36], [37].
In this chapter, we will describe the many roles of PCNA in eukaryotic DNA replication and in replication-associated processes. We will begin by discussing the features of the structure and function of PCNA common to all of its roles. Then we will focus on its roles in normal DNA replication, translesion synthesis and error-free damage bypass, break-induced replication, mismatch repair, and replication-coupled nucleosome assembly.
Section snippets
PCNA Structure and Function
Sliding clamps are proteins that encircle double-stranded DNA and are found in all three domains of life. Although these proteins have different oligomeric states, they all possess a general pseudo-sixfold ring-shaped structure. Bacterial sliding clamps form homodimers, whereas archaeal and eukaryotic sliding clamps form homotrimers and heterotrimers. These sliding clamps function as platforms for recruiting and regulating various enzymes that function in DNA replication and repair, such as
The Role of PCNA in Normal DNA Replication
In eukaryotes, DNA replication is an extraordinarily complex, dynamic, multistage process that initiates at origins of replication [54], [55], [56], [57]. Before an origin can fire, it must be licensed. Origin licensing occurs during late M phase and early G1 phase, when the pre-replication complex (pre-RC) forms at the origin. The pre-RC includes Cdt1, Cdc6, and two hexamers of the Mcm2–7 helicase. Origin firing occurs at the onset of S phase. At this point, the origin is melted by the Mcm2–7
The Role of PCNA in Translesion Synthesis
DNA damage causes replication forks to stall, because classical DNA polymerases, such as pol δ and pol ɛ, are unable to efficiently incorporate deoxynucleotides opposite damaged DNA templates. Without a means of overcoming these replication blocks, replication forks collapse resulting in DNA strand breaks, chromosomal rearrangements, and cell death. Translesion synthesis is one of the means by which damaged DNA is bypassed during DNA replication. During translesion synthesis, one or more
The Role of PCNA in Error-Free Damage Bypass
In addition to translesion synthesis, which is mutagenic, another pathway for circumventing DNA lesions in the template strand during DNA replication is error-free damage bypass. The detailed mechanism of error-free damage bypass has yet to be elucidated, but it is believed to involve a template-switching event whereby the replicative DNA polymerases moves to the newly synthesized sister strand and uses it as a template [93], [94]. The model for how this template-switching event occurs is that
The Role of PCNA in Break-Induced Replication
If translesion synthesis and error-free damage bypass fail to allow the resumption of DNA replication, several other pathways may be used to restart the stalled replication fork. These pathways generally involve the use of the recombination machinery and are beyond the scope of this review; these pathways are described in detail elsewhere [98], [99], [100]. However, one such pathway, break-induced replication, does warrant attention. This is because PCNA is required for break-induced
The Role of PCNA in Mismatch Repair
Replicative polymerases make errors when synthesizing DNA that can lead to base–base mismatches or short insertions and deletions. These errors are recognized and corrected by mismatch repair [106], [107], [108], [109], [110], [111], [112]. The first step of mismatch repair is recognition of the mismatches or insertions/deletions in the newly synthesized DNA. This involves either the MutSα heterodimer composed of Msh2 and Msh6 or the MutSβ heterodimer composed of Msh2 and Msh3. These mismatch
The Role of PCNA in Replication-Coupled Nucleosome Assembly
Immediately following DNA replication, nucleosomes must be assembled on the newly synthesized daughter duplexes behind the replication fork. In transcriptionally silent, heterochromatic regions of the genome including the centromeric and telomeric regions of chromosomes, this is carried out in part by chromatin association factor-1 (CAF-1). CAF-1 is a heterotrimer comprising Cac1, Cac2, and Cac3 subunits. It functions as a histone H3–H4 chaperone that catalyzes the deposition of nucleosomes
Putting It All Together
One of the most important, unanswered questions regarding PCNA's role in DNA replication is how does it interact with and regulate the activity of so many binding partners. One widely discussed idea is that PCNA can function as a “tool belt” by binding several partners simultaneously. Because PCNA is a trimer, it can potentially bind up to three PIP motif-containing proteins at the same time. Although there is so far no direct evidence that eukaryotic PCNA forms such tool belts, archaeal PCNA
Acknowledgments
We thank Christine Kondratick, Kyle Powers, Brittany Ripley, Lynne Dieckman, and Maria Spies for their valuable discussions. This work was supported by National Institute of Health Grants R01 GM108027, R01 GM081433, and T32 GM067795.
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