High-fidelity DNA ligation enforces accurate Okazaki fragment maturation during DNA replication

Jessica S Williams,Thomas A Kunkel,R Scott Williams,Percy P Tumbale,Mercedes E Arana,Julian A Rana

doi:10.1038/s41467-020-20800-1

Abstract

DNA ligase 1 (LIG1, Cdc9 in yeast) finalizes eukaryotic nuclear DNA replication by sealing Okazaki fragments using DNA end-joining reactions that strongly discriminate against incorrectly paired DNA substrates. Whether intrinsic ligation fidelity contributes to the accuracy of replication of the nuclear genome is unknown. Here, we show that an engineered low-fidelity LIG1Cdc9 variant confers a novel mutator phenotype in yeast typified by the accumulation of single base insertion mutations in homonucleotide runs. The rate at which these additions are generated increases upon concomitant inactivation of DNA mismatch repair, or by inactivation of the Fen1Rad27 Okazaki fragment maturation (OFM) nuclease. Biochemical and structural data establish that LIG1Cdc9 normally avoids erroneous ligation of DNA polymerase slippage products, and this protection is compromised by mutation of a LIG1Cdc9 high-fidelity metal binding site. Collectively, our data indicate that high-fidelity DNA ligation is required to prevent insertion mutations, and that this may be particularly critical following strand displacement synthesis during the completion of OFM.

Highlights

The most abundant DNA transaction in eukaryotic cells that affects the fidelity of nuclear DNA replication is the nucleotide selectivity of DNA polymerases (Pols) α, δ, and ε during incorporation into a growing DNA chain during leading and lagging strand synthesis[1]
To test whether high-fidelity ligation is critical for genome maintenance in vivo, we constructed a low-fidelity ligation yeast strain containing alanine substitutions of the conserved Glu[206] and Glu[443] residues in the CDC9 gene that encodes Saccharomyces cerevisiae DNA ligase 1 (Fig. 1a)
Highresolution mapping of Okazaki fragments from S. cerevisiae showed that their size is roughly consistent with the nucleosome repeat, which averages 165 bp in size[24,25], and DNA ligase 1 is able to efficiently seal a nick on a nucleosomal substrate[26]

Summary

Introduction

The most abundant DNA transaction in eukaryotic cells that affects the fidelity of nuclear DNA replication is the nucleotide selectivity of DNA polymerases (Pols) α, δ, and ε during incorporation into a growing DNA chain during leading and lagging strand synthesis[1]. Okazaki fragment processing involves replacement of RNA–DNA primers made by Pol α-primase This occurs by nick-translation/ strand displacement DNA synthesis catalyzed by Pol δ to create DNA substrates that are cleaved by Fen[1] and/or by DNA2 nucleases[5,6,7,8,9,10]. Expression of a Cdc9EE/AA variant in yeast that compromises ligation fidelity results in a high rate of +1 base insertion events in short homonucleotide runs These mutagenic events are exacerbated upon loss of DNA mismatch repair (MMR) or loss of Fen1-dependent nuclease activity, demonstrating that highly accurate DNA ligation is a previously underappreciated critical determinant of faithful replication of the nuclear genome. Biochemical and structural dissection of the mutagenic ligation reaction performed by the low-fidelity mutant DNA ligase provides a molecular framework for this insertion mutagenesis and solidifies the importance of accurate DNA ligation during DNA synthesis

Methods

Results

Conclusion