Fission yeast Rad8/HLTF facilitates Rad52-dependent chromosomal rearrangements through PCNA lysine 107 ubiquitination

Abstract

Gross chromosomal rearrangements (GCRs), including translocation, deletion, and inversion, can cause cell death and genetic diseases such as cancer in multicellular organisms. Rad51, a DNA strand exchange protein, suppresses GCRs by repairing spontaneous DNA damage through a conservative way of homologous recombination, gene conversion. On the other hand, Rad52 that catalyzes single-strand annealing (SSA) causes GCRs using homologous sequences. However, the detailed mechanism of Rad52-dependent GCRs remains unclear. Here, we provide genetic evidence that fission yeast Rad8/HLTF facilitates Rad52-dependent GCRs through the ubiquitination of lysine 107 (K107) of PCNA, a DNA sliding clamp. In rad51Δ cells, loss of Rad8 eliminated 75% of the isochromosomes resulting from centromere inverted repeat recombination, showing that Rad8 is essential for the formation of the majority of isochromosomes in rad51Δ cells. Rad8 HIRAN and RING finger mutations reduced GCRs, suggesting that Rad8 facilitates GCRs through 3’ DNA-end binding and ubiquitin ligase activity. Mms2 and Ubc4 but not Ubc13 ubiquitin-conjugating enzymes were required for GCRs. Consistent with this, mutating PCNA K107 rather than the well-studied PCNA K164 reduced GCRs. Rad8-dependent PCNA K107 ubiquitination facilitates Rad52-dependent GCRs, as PCNA K107R, rad8, and rad52 mutations epistatically reduced GCRs. In contrast to GCRs, PCNA K107R did not significantly change gene conversion rates, suggesting a specific role of PCNA K107 ubiquitination in GCRs. PCNA K107R enhanced temperature-sensitive growth defects of DNA ligase I cdc17-K42 mutant, implying that PCNA K107 ubiquitination occurs when Okazaki fragment maturation fails. Remarkably, K107 is located at the interface between PCNA subunits, and an interface mutation D150E bypassed the requirement of PCNA K107 and Rad8 ubiquitin ligase for GCRs. These data suggest that Rad8-dependent PCNA K107 ubiquitination facilitates Rad52-dependent GCRs by changing the PCNA clamp structure.

Post-Translational Modifications of PCNA: Guiding for the Best DNA Damage Tolerance Choice

“…Focusing on alternative ubiquitination sites on yeast PCNA, K107 and K242, occurring as a response to a deficiency in Ligase I activity or in the presence of unligated Okazaki fragments ([ 254 , 255 , 257 ]), and as observed in Figure 4 , we can conclude that these positions are not well conserved among fungi. There is a very low conservation of the K242 position, although some of the fungal species in Figure 4 present a lysine at position 240, as human PCNA.…”

“…The ubiquitination of this alternative site is required to initiate the S phase checkpoint and promote a cell cycle delay when the maturation of Okazaki fragments is impaired. It depends on Rad5 (or Rad8 in fission yeast), together with the E2 partner formed by Mms2 and Ubc4, but not by Ubc13 [ 253 , 255 ]. K107 in yeast PCNA is positioned at the interface between PCNA subunits [ 255 ], suggesting that ubiquitination at this site might change the PCNA structure and the interaction between subunits, which would impair the correct function of PCNA.…”

“…It depends on Rad5 (or Rad8 in fission yeast), together with the E2 partner formed by Mms2 and Ubc4, but not by Ubc13 [ 253 , 255 ]. K107 in yeast PCNA is positioned at the interface between PCNA subunits [ 255 ], suggesting that ubiquitination at this site might change the PCNA structure and the interaction between subunits, which would impair the correct function of PCNA. Related to this, in fission yeast, K107 ubiquitination was proposed to contribute to increased non-allelic crossovers, leading to gross chromosomal rearrangements (GCRs) depending on Rad52 [ 255 ].…”

“…In human cells, the down-regulation of DNA ligase I (LIG1A) also induces PCNA monoubiquitination [ 253 ]. In this case, the alternative ubiquitinated lysine is not known, since the K107 position is not conserved in human PCNA, although it contains a close-by lysine at position 110 [ 255 ], which is also present in other metazoans. Position 110 has a more conserved composition in all the fungal species compared in Figure 4 , with arginine or lysine residues at this position.…”

“…Curiously, all the compared species that contain a lysine at position 106, also present a lysine at position 110. Both, K107 in yeast PCNA, and K110 in human PCNA, are placed at the interface between PCNA subunits [ 255 ], suggesting that ubiquitination at these sites could alter their interaction and promote the release of PCNA from DNA. Nevertheless, the methylation of K110 in human PCNA was described as mediating trimerization and stabilizing the interaction of Polδ [ 285 ].…”