DNA Replication And Telomere Maintenance Require Pcna-K164 Ubiquitination

Abstract

Genome integrity relies on a robust DNA replication program to ensure faithful duplication of genetic material, free from sequence mutations, deletions or rearrangements. There is an estimated 10 quadrillion (1x1016) cell divisions that occur in the average lifetime of a human being (Weinberg 2014). Thus, cells rely on a global DNA damage response (DDR) network to sense and repair errors that occur during replication to prevent the perpetuation of mutations (Ciccia and Elledge 2010). Although the DDR is highly efficient, some errors may escape repair and interfere with the progression of replication forks. In this scenario, cells utilize DNA damage tolerance (DDT) pathways to bypass errors/lesions encountered during replication and promote replication fork restart (Friedberg 2005, Chang and Cimprich 2009, Ghosal and Chen 2013). A major regulator of DDT pathways is proliferating cell nuclear antigen (PCNA) (Hoege et al. 2002). Ubiquitin modification at the conserved lysine residue 164 (K164) is crucial to DDT pathway choice – mono-ubiquitination activates error-prone translesion synthesis (TLS), while poly-ubiquitination activates error-free template switching (TS) (Shcherbakova and Fijalkowska 2006, Lehmann et al. 2007, Branzei 2011, Sale et al. 2012). However, whether PCNA ubiquitination regulates other genome maintenance mechanisms is unclear. The ends of chromosomes, known as telomeres, are origin-poor and present multiple challenges for the replication machinery including the propensity to form guanine (G)-quadruplexes and RNA-DNA hybrids (Sfeir et al. 2009, Maestroni et al. 2017). Because telomeres are intrinsically “difficult to replicate”, these regions are particularly sensitive to replication stress (Özer and Hickson 2018). In addition to the canonical replication machinery, additional proteins are needed to properly replicate the telomeric duplex. One of these proteins, the TLS polymerase η, functions to alleviate telomeric replication stress (Pope-Varsalona et al. 2014, Garcia-Exposito et al. 2016). The recruitment of TLS polymerases, including Pol η, to DNA lesions occurs through the direct interaction with mono-ubiquitinated PCNA (Bienko et al. 2005). These observations suggest a direct role for PCNA ubiquitination in the replication of telomeres. However, several reports have suggested that TLS can operate in the absence of PCNA ubiquitination (Haracska et al. 2006, Acharya et al. 2007, Parker et al. 2007, Edmunds et al. 2008, Nikolaishvili-Feinberg et al. 2008, Hendel et al. 2011, Krijger et al. 2011), thus it is not clear whether this modification is involved in telomere maintenance. While the role of PCNA-K164 ubiquitination for normal DNA replication and DDT pathway activation has been extensively studied in model systems of yeast, chicken, and mouse, how this modification functions in maintaining human genome stability is still not understood. This thesis addresses several critical functions of K164 ubiquitination in human cells. Studies in PCNAK164R mutants reveal that PCNA ubiquitination is required for gap-filling on the lagging strand behind progressing replication forks (Thakar et al. 2020). Additionally, we provide evidence that K164 ubiquitination functions to resolve late replicating intermediates (LRIs) through mitotic DNA synthesis (MiDAS) and promote efficient origin licensing in the subsequent G1 phase. Finally, we find that post-translational modification of PCNA at K164 regulates telomere maintenance specifically in transformed cells. Together, these studies show that the functions of PCNA-K164 go well beyond progressive DNA synthesis and DDT activation and extend to MiDAS and telomere maintenance.