Ubc4 and Not4 Regulate Steady-State Levels of DNA Polymerase-α to Promote Efficient and Accurate DNA Replication

Abstract

The accurate duplication of chromosomal DNA is required to maintain genomic integrity. However, from an evolutionary point of view, a low mutation rate during DNA replication is desirable. One way to strike the right balance between accuracy and limited mutagenesis is to use a DNA polymerase that lacks proofreading activity but contributes to DNA replication in a very restricted manner. DNA polymerase-␣ fits this purpose exactly, but little is known about its regulation at the replication fork. Minichromosome maintenance protein (Mcm) 10 regulates the stability of the catalytic subunit of pol-␣ in budding yeast and human cells. Cdc17, the catalytic subunit of pol-␣ in yeast, is rapidly degraded after depletion of Mcm10. Here we show that Ubc4 and Not4 are required for Cdc17 destabilization. Disruption of Cdc17 turnover resulted in sensitivity to hydroxyurea, suggesting that this pathway is important for DNA replication. Furthermore, overexpression of Cdc17 in ubc4 and not4 mutants caused slow growth and synthetic dosage lethality, respectively. Our data suggest that Cdc17 levels are very tightly regulated through the opposing forces of Ubc4 and Not4 (destabilization) and Mcm10 (stabilization). We conclude that regular turnover of Cdc17 via Ubc4 and Not4 is required for proper cell proliferation. INTRODUCTIONThe accurate duplication of the genome is crucial for the prolonged health of eukaryotic organisms. Inaccurate DNA replication and/or replication of any portion of the genome more than once can result in genomic instability, which is a consistently observed hallmark of cancer cells (Vaziri et al., 2003;Venkatesan et al., 2007). It is crucial, therefore, to understand the entire process of DNA replication. The initiation of DNA replication requires the coordinated recruitment of several proteins. Prereplicative complexes (Diffley et al., 1994), including the core helicase Mcm2-7 (Bochman and Schwacha, 2008), form at origins of replication, are converted to pre-initiation complexes (Zou and Stillman, 1998), and DNA is subsequently unwound (reviewed in Bell and Dutta, 2002). Once the DNA is unwound, the accuracy of DNA replication depends in large part upon DNA polymerases (pol)-␣, -␦, and -, all of which coordinate to synthesize the nascent copy of DNA during replication in eukaryotes (Burgers, 2009). Highlighting the importance of accurate DNA replication, 43% of mice homozygous for a proofreading-deficient allele of pol-␦ develop cancer, primarily lymphoma, but also squamous-cell carcinoma (Goldsby et al., 2001). Interestingly, pol-␣, the only enzyme that can synthesize DNA de novo, naturally lacks proofreading activity (Morrison et al., 1991). Unlike pol-␦, pol-␣ is foremost a replicative -and not a repair -polymerase (Wu et al., 2001;Wang et al., 2004). The lack of proofreading activity likely reflects one mechanism by which nature enforces evolution. However, this raises the question of how humans use a potentially mutagenic polymerase responsible for the initiation of more than 30 million Okazaki fragmen...

Mcm10 deficiency causes defective-replisome-induced mutagenesis and a dependency on error-free postreplicative repair

“…21,22,[24][25][26][27][28][29][30][31] Therefore, our data are consistent with the notion that inefficient chromatin assembly of pol-α/primase complexes in both pol1-1 and mcm10-1 mutants may be responsible for the impairment of DNA synthesis, which triggers DRIM. However, we cannot exclude that Mcm10 depletion interferes with DNA synthesis in a manner unrelated to priming.…”

“…21,23,25,28 Importantly, we also demonstrate that disruption of Mcm10 function leads to pol-ζ-dependent DRIM. This is particularly relevant to cancer biology, because dysregulation of translesion polymerases is strongly associated with tumor formation [68][69][70][71][72][73] and negative clinical outcomes.…”

“…[21][22][23][24][25][26][27][28][29][30] Moreover, Mcm10 is post-translationally modified during G 1 and S phase, resulting in non-proteolytic ubiquitination at 2 distinct lysines. This modification is a prerequisite for Mcm10's interaction with PCNA, which is essential for cell proliferation.…”

The Rix1 (Ipi1p-2p-3p) complex is a critical determinant of DNA replication licensing independent of their roles in ribosome biogenesis

“…This kind of synthetic dosage interaction has been shown to reflect functional relationships and physical interactions among the replication proteins examined. 29,42,43 The report on the fission yeast Noc3p has been discussed recently in reference 9.…”

Proteolytic Degradation of the Yap1 Transcription Factor Is Regulated by Subcellular Localization and the E3 Ubiquitin Ligase Not4

“…The DNA polymerase ␣ encoded by CDC17 is degraded in a Not4-dependent manner (49). Strikingly, degradation of the histone demethylase Jhd2 is regulated by Not4 and required to support normal levels of methylation of histone H3 (50).…”