Defects in DNA ligase I trigger PCNA ubiquitylation at Lys 107 - PubMed (original) (raw)

. 2010 Jan;12(1):74-9; sup pp 1-20.

doi: 10.1038/ncb2007. Epub 2009 Dec 13.

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Defects in DNA ligase I trigger PCNA ubiquitylation at Lys 107

Sapna Das-Bradoo et al. Nat Cell Biol. 2010 Jan.

Abstract

In all eukaryotes, the ligation of newly synthesized DNA, also known as Okazaki fragments, is catalysed by DNA ligase I (ref. 1). An individual with a DNA ligase I deficiency exhibits growth retardation, sunlight sensitivity and severe immunosuppression, probably due to accumulation of DNA damage. Surprisingly, not much is known about the DNA damage response (DDR) in DNA ligase I-deficient cells. As DNA replication and DDR pathways are highly conserved in eukaryotes, we used Saccharomyces cerevisiae as a model system to address this issue. We uncovered a new pathway, which facilitates ubiquitylation at Lys 107 of proliferating cell nuclear antigen (PCNA). Unlike ubiquitylation at Lys 164 of PCNA in response to UV irradiation, which triggers translesion synthesis, modification of Lys 107 is not dependent on the ubiquitin conjugating enzyme (E2) Rad6 (ref. 4) nor the ubiquitin ligase (E3) Rad18 (ref. 5), but requires the E2 variant Mms2 (ref. 6) in conjunction with Ubc4 (ref. 7) and the E3 Rad5 (Refs 8, 9). Surprisingly, DNA ligase I-deficient S. cerevisiae cdc9-1 cells that carry a PCNAK107R mutation are inviable, because they cannot activate a robust DDR. Furthermore, we show that ubiquitylation of PCNA in response to DNA ligase I deficiency is conserved in humans, yet the lysine residue that is modified remains to be determined. We propose that PCNA ubiquitylation provides a 'DNA damage code' that allows cells to categorize different types of defects that arise during DNA replication.

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Figures

Figure 1

Figure 1

DNA ligase I is required for S phase progression. (a) Asynchronous cultures of ABy010 (GAL-UBR1 CDC9) and ABy008 (GAL-UBR1 cdc9-td) were induced with galactose at 28°C for 30 min and subsequently shifted to 37°C. (b) Strains were arrested in G1 phase at 28°C and shifted to 37°C in 2% galactose and α-factor. After 90 min, cells were released from G1 phase either at 37°C in galactose or at 28°C in glucose. (c) Strains were arrested in G1 and released into S phase at 28°C in the presence of HU. Once arrested in S phase, cultures were shifted to 37°C in the presence of 2% galactose and HU. After 90 min, cells were transferred into nocodazole either at 37°C or 28°C. (d) Asynchronous cultures of SSL204 (CDC9), SSL612 (cdc9-1), and SSL613 (cdc9-2) were shifted to 35°C. In a-d, DNA content was monitored by flow cytometry and the vertical line indicates a 2C DNA content.

Figure 2

Figure 2

S. cerevisiae PCNA is mono-ubiquitinated in cdc9 mutants. (a) Asynchronous cultures of SSL204 (CDC9), SSL612 (cdc9-1), and SSL613 (cdc9-2) were shifted to 35°C for 3 h. Total protein was TCA-precipitated, diluted and probed with a yeast specific PCNA antibody (S871). (b) Cells were shifted to 35°C for 3 h in the presence of copper sulfate to induce expression of Myc-tagged ubiquitin. Whole cell extracts were immunoprecipitated with an anti-Myc antibody and PCNA was detected with anti-PCNA antibody (S871). The right panel shows the beads control with or without the cell lysate. 5% of the input was loaded. (c) PCNA is mono-ubiquitinated in cdc9 mutants. Wild-type or mutant ubiquitin expression was induced in asynchronously growing cultures. Total protein was TCA-precipitated and diluted. Unmodified and mono-ubiquitinated PCNA was detected by PCNA antibody (S871). (d) PCNA poly-ubiquitination is linked through lysine 29 in cdc9 mutants. Undiluted TCA-precipitated protein samples from c were probed with anti-PCNA antibody (S871) to detect poly-ubiquitinated PCNA. In b, d, asterisks indicate non-specific bands. In d, filled circles (right side of the band) indicate poly-ubiquitinated forms of PCNA. In a, c, d, α-tubulin served as a loading control.

Figure 3

Figure 3

PCNA mono-ubiquitination in cdc9 mutants is mediated by Mms2, Rad5 and Ubc4 but not Ubc13. (a) Cultures were grown asynchronously and shifted to 35°C for 3 h. (b) Asynchronous cultures of SSL204 (CDC9), SSL612 (cdc9-1) and ABy579 (cdc9-1 ubc5Δ UBC4-NES-3HA) were grown at 25°C and shifted to 35°C for 3 h. In a-b, unmodified and mono-ubiquitinated PCNA was detected using PCNA antibody (S871). α-tubulin served as a loading control.

Figure 4

Figure 4

MMS2 but not UBC13 is required for S phase checkpoint activation in cdc9 mutants. (a) Cells were arrested in G1 phase at 25°C and released at 35°C for 3 h. DNA content was monitored by flow cytometry. The vertical line indicates a 2C DNA content. (b) Cells were grown asynchronously at 25°C and then shifted to 35°C for the indicated time period. Total protein was TCA-precipitated and Rad53 was detected by an anti-Rad53 antibody. (c) Asynchronous cultures were split and shifted to the non-permissive temperature of 35°C in the presence of methyl methane sulfonate (MMS) for 90 and 180 min or left untreated for 180 min. Rad53 was detected as described in b. In b-c, α-tubulin served as a loading control.

Figure 5

Figure 5

PCNA mono-ubiquitination occurs at lysine 107 in DNA ligase I mutants and is required for Rad53 activation. (a) PCNA lysine mutants in DNA ligase I deficient cells (cdc9-1) were grown asynchronously and then shifted to 35°C for 3 h. (b) Cultures of SSL204 (CDC9), ABy685 (CDC9 pol30-K107R), SSL612 (cdc9-1), ABy782 (cdc9-1* pol30-K107R) were grown asynchronously and then shifted to 30°C for 3 h. (c) Asynchronous cultures were grown at 25°C and shifted to 30°C in the presence of MMS for the indicated time. Cultures not treated with MMS served as negative controls. (d) Cultures were induced with galactose at 28°C for 30 min and subsequently shifted to 37°C for 3 h. In a-d, TCA-precipitated protein samples were diluted and PCNA was detected with anti-PCNA antibody (S871). (e) Undiluted TCA-precipitated samples from d were run on SDS-PAGE and poly-ubiquitinated PCNA was detected with anti-PCNA antibody (S871). (f) Cells were grown asynchronously at 28°C and then shifted to 37°C in the presence or absence of MMS for 3 h. Rad53 and Cdc9-td-HA were detected with anti-Rad53 and anti-HA antibodies, respectively. In b, c, e, f, α-tubulin served as a loading control. In e-f, the asterisks indicate non-specific bands. (g) U2OS cells were either untreated or treated with 60 J/m2 UV and harvested 2 h later along with control shRNA (shCON) and DNA Ligase I shRNA (shLIG1 A) cell lines. Chromatin fractions were prepared, fractionated on SDS-PAGE and analyzed with the indicated antibodies. Samples on the PCNA and ubiquitin blots were analyzed side by side. Unmodified PCNA served as a loading control for the PCNA blot and histone H3 served as a loading control for the ubiquitin blot.

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