Dissecting PCNA function with a systematically designed mutant library in yeast

Abstract

Proliferating cell nuclear antigen (PCNA), encoded by POL30 in Saccharomyces cerevisiae, is a key component of DNA metabolism. Here, a library consisting of 304 PCNA mutants was designed and constructed to probe the contribution of each residue to the biological function of PCNA. Five regions with elevated sensitivity to DNA damaging reagents were identified using high-throughput phenotype screening. Using a series of genetic and biochemical analyses, we demonstrated that one particular mutant, K168A, has defects in the DNA damage tolerance (DDT) pathway by disrupting the interaction between PCNA and Rad5. Subsequent domain analysis showed that the PCNA-Rad5 interaction is a prerequisite for the function of Rad5 in DDT. Our study not only provides a resource in the form of a library of versatile mutants to study the functions of PCNA, but also reveals a key residue on PCNA (K168) which highlights the importance of the PCNA-Rad5 interaction in the template switching (TS) pathway.

Introduction

The accurate and efficient propagation of genetic information to descendants is the central task for dividing cells. Nevertheless, DNA is highly vulnerable to many types of genotoxic challenges, which can lead to DNA damage and replication fork stalling, termed DNA replication stress (Hoeijmakers, 2009). Inappropriate molecular management of replication stress may result in genetic variation, replication fork collapse or cell death. Proliferating cell nuclear antigen (PCNA) plays central roles in DNA replication and repair by guaranteeing replisome fluidity (Kurth and O'Donnell, 2013). This ring-shaped homotrimer is structurally superimposable across eukaryotes, which is consistent with the evolutionary conservation of its role as a sliding clamp (Stoimenov and Helleday, 2009). Based on a common mode of action, PCNA was reported to interact with various partners to orchestrate different DNA metabolic functions in harmony (Moldovan et al., 2007; Stoimenov and Helleday, 2009).

Several established mechanisms of DNA damage tolerance (DDT), which are vital for relieving replication fork stalling and ensuring complete duplication of the genome even in the presence of bulky DNA lesions, are regulated by distinct post-translational modifications (PTMs) of PCNA (Chang and Cimprich, 2009; Stoimenov and Helleday, 2009; Branzei and Szakal, 2016). The monoubiquitination of PCNA on the K164 site by the Rad6-Rad18 complex triggers an error-prone bypass mechanism through the recruitment of dedicated translesion polymerases. These enzymes can replicate across lesions (translesion synthesis; TLS) (Freudenthal et al., 2010). The polyubiquitination of PCNA on the K164 site by the Mms2/Ubc13-Rad5 complex is involved in a recombination-related mechanism that can switch the replication template from the damaged strand to the undamaged sister chromatid and thus is named the template switching (TS) pathway (Boiteux and Jinks-Robertson, 2013). A typical symbol of this pathway is the sister chromatid junction (SCJ), which is shaped like an “X molecule” similar to the Holliday junction (Giannattasio et al., 2014). Several important genes involved in this pathway have been identified by detecting the presence of SCJs (Minca and Kowalski, 2010; Vanoli et al., 2010), leading to a “sketch map” of this pathway. Another type of modification of PCNA is SUMOylation at K127 and K164 by Ubc9 and Siz1, respectively, which act as inhibitors of homologous recombination (HR) by recruiting Srs2 to remove Rad51 from chromatin and thus keep certain potentially deleterious HR pathways in check (Pfander et al., 2005).

Although PCNA modifications by SUMO and ubiquitin are known to be crucial for DDT, the regulation or choice among these three pathways remains poorly understood. Structural analysis of the PCNA-Siz1-Srs2 and PCNA-polη complexes has shed some light on the working mechanisms of the HR and TLS pathways (Lau et al., 2015; Streich Jr and Lima, 2016), whereas the TS pathway remains largely elusive. It is unclear how Rad5, a key component that functions both upstream (as a ubiquitin ligase) and downstream (as a helicase lacking a canonical PCNA-interacting peptide (PIP) box) of the polyubiquitination-related DDT process, participates in this pathway (Minca and Kowalski, 2010). In addition, how polyubiquitination on PCNA is correlated with the TS pathway and why some well-known HR-related proteins are involved in TS (Vanoli et al., 2010) are still unclear.

On the other hand, not all functions of PCNA rely on its modification states, and indeed, previous mutagenesis studies have identified many unmodifiable residues which are crucial for the participation of PCNA in DNA replication and repair (Ayyagari et al., 1995; Amin and Holm, 1996; Eissenberg et al., 1997; Chen et al., 1999; Zamir et al., 2012; Goellner et al., 2014), chromatin dynamics, and cell cycle regulation (Zhang et al., 2000; Miller et al., 2010). Several functional regions, such as the interdomain-connecting loop (IDCL) (Amin and Holm, 1996), the trimer interface (Hishiki et al., 2008; Dieckman and Washington, 2013; Kondratick et al., 2016), and the DNA interface helix, have been identified, leading to the present model involving the exchange of PCNA partners and the loading and sliding mechanism of the PCNA clamp. Currently, mutational analysis of PCNA structure-function relationships is limited to defined subsets of residues (Dieckman et al., 2012).

In this study, we generated a PCNA mutant library consisting of 304 alleles to cover every residue within this protein based on a carefully designed synthetic construct, which could be generally useful and serve as a flexible resource. We systematically substituted each non-alanine residue with alanine and replaced each native alanine residue with serine. In the IDCL region, a more comprehensive mutagenesis strategy was applied, including swapping the charge status of each residue and substituting each modifiable residue with another amino acid residue to mimic either modified or unmodified status, when possible. We screened the library for individual mutants that made yeast vulnerable to DNA damaging reagents such as methyl methanesulfonate (MMS), hydroxyurea (HU) and UV to reveal important regions within PCNA. We then focused our study on a particular mutant, K168A, which showed severe sensitivity to DNA lesions and revealed the importance and the intrinsic action mechanism of this site for PCNA during DDT.