Defects in DNA ligase I trigger PCNA ubiquitylation at Lys 107

Abstract

In all eukaryotes, the ligation of newly synthesized DNA, also known as Okazaki fragments, is catalyzed by DNA ligase I1. An individual with a DNA ligase I deficiency exhibited growth retardation, sunlight sensitivity and severe immunosuppression2, likely due to accumulation of DNA damage. Surprisingly, not much is known about the DNA damage response (DDR) in DNA ligase I-deficient cells. Because DNA replication and DDR pathways are highly conserved in eukaryotes, we utilized Saccharomyces cerevisiae as a model system to address this question. We uncovered a novel pathway, which facilitates ubiquitination of lysine 107 of proliferating cell nuclear antigen (PCNA). Unlike ubiquitination at lysine 164 of PCNA in response to UV irradiation, which triggers translesion synthesis3, modification of lysine 107 is not dependent on the ubiquitin conjugating enzyme (E2) Rad64 nor the ubiquitin ligase (E3) Rad185, but requires the E2 variant Mms26 in conjunction with Ubc47 and the E3 Rad58,9. Surprisingly, DNA ligase I-deficient cdc9-1 cells that carry a PCNAK107R mutation are inviable, because they cannot activate a robust DDR. Furthermore, we show that ubiquitination of PCNA in response to DNA ligase I-deficiency is conserved in humans, yet the lysine that mediates this modification remains to be determined. We propose that PCNA ubiquitination provides a “DNA damage code” that allows cells to categorize different types of defects that arise during DNA replication.

Okazaki Fragment Processing-independent Role for Human Dna2 Enzyme during DNA Replication

“…Although protein extracts from these cells are deficient in OF ligation during SV40 DNA replication in vitro, they support incorporation of [␣-32 P] dATP into plasmid DNA with similar kinetics as extracts from control fibroblasts (56). In addition, yeast deficient in LigI completely replicate their genomes despite being unable to ligate OF (54,55). Together these studies suggest that replication fork progression is unperturbed in the absence of LigI.…”

Damage-specific modification of PCNA

“…However, regardless of their outcome, it is important to mention that ubiquitination of PCNA is conserved in human cells that are depleted for DNA ligase I, even though lysine 107 of PCNA is not evolutionarily conserved. 12 This underscores the significance of our findings.…”

“…46 All of these findings were consistent with the notion that DNA ligase I was not required until the triggered mono-and poly-ubiquitination of PCNA. 12 Mutation of lysine 164 of PCNA in cdc9 mutants had no effect on cell viability, arguing that neither error-prone translesion synthesis 19,21,[60][61][62] nor error-free replication by template switch [63][64][65][66] played a role in this process. Instead, we observed synthetic lethality between cdc9-1 and a mutation in lysine 107 of PCNA, leading us to propose that ubiquitin is conjugated to lysine 107 in DNA ligase I-deficient cells.…”

“…However, our recent analysis of the cdc9-1 mutation in a different strain background suggested that the cells do not move through S phase with normal kinetics, but rather appeared to accumulate in mid-S phase. 12 Further exploration of a temperature-sensitive degron mutant (cdc9-td) verified that DNA replication was extensively delayed in the absence of DNA ligase I (Fig. 1).…”

“…Mono-ubiquitinated PCNa is visible in darker exposures around 42kDa in cdc9 mutants with Ha-tagged PCNa (only visible as a faint band in this exposure). cdc9-1 is a more stringent allele than cdc9-2, 12 and thus sumoylation of PCNa is more prominent in this mutant.…”