Lysine residue 185 of Rad1 is a topological but not a functional counterpart of lysine residue 164 of PCNA

Abstract

Monoubiquitylation of the homotrimeric DNA sliding clamp PCNA at lysine residue 164 (PCNA(K164)) is a highly conserved, DNA damage-inducible process that is mediated by the E2/E3 complex Rad6/Rad18. This ubiquitylation event recruits translesion synthesis (TLS) polymerases capable of replicating across damaged DNA templates. Besides PCNA, the Rad6/Rad18 complex was recently shown in yeast to ubiquitylate also 9-1-1, a heterotrimeric DNA sliding clamp composed of Rad9, Rad1, and Hus1 in a DNA damage-inducible manner. Based on the highly similar crystal structures of PCNA and 9-1-1, K185 of Rad1 (Rad1(K185)) was identified as the only topological equivalent of PCNA(K164). To investigate a potential role of posttranslational modifications of Rad1(K185) in DNA damage management, we here generated a mouse model with a conditional deletable Rad1(K185R) allele. The Rad1(K185) residue was found to be dispensable for Chk1 activation, DNA damage survival, and class switch recombination of immunoglobulin genes as well as recruitment of TLS polymerases during somatic hypermutation of immunoglobulin genes. Our data indicate that Rad1(K185) is not a functional counterpart of PCNA(K164).

Figure 1. Targeting strategy and genotyping Rad1 ^K185R mouse.

A) Targeting strategy Rad1 ^K185R mouse. LoxP recombination sites are represented by black triangles. Flpe recombination sites are represented by white triangles. PCR primers are represented by gray arrow heads. Please note that this figure is not drawn to scale. B) Genotyping PCRs for non-flipped (Primers G1 FWD, G1 REV and G2 REV) and flipped Rad1 ^K185R mice (Primers G1 FWD and G3 REV).

Figure 2. Rad1 ^K185R MEFs have normal Chk1 activation.

WT and Rad1 ^K185R MEFs were irradiated with 100 J/m² UV-C and harvested after 10, 40 and 70 minutes after irradiation. Subsequently, the Chk1 phosphorylation status at S345 (pChk1 S345) was investigated by Western blotting using pChk1 S345-specific antibodies. The results are representatives of two independent experiments.

Figure 3. Rad1 ^K185R B cells do not display sensitivity to various DNA damaging agents.

WT (blue) and Rad1 ^K185R (Red) B cells were stimulated with LPS and exposed to increasing amounts of UV-C (A), MMS (B), CisPt (C) and γ-irradiation (D). The percentage of survival is shown on the y-axis after four days of culture. Data represent the mean and SD of individual cultures (n = 3). The results are representatives of two independent experiments.

Figure 4. Normal SHM in Rad1 ^K185R GC B cells.

A) Mutated JH4 regions from WT and Rad1 ^K185R GC B cells. B) Rad1 ^K185R GC B cells display a normal nucleotide exchange pattern in hypermutated Ig genes. In the left panel, values are expressed as the total numbers of mutations. In the right panel, values are expressed as the percentage of total mutations. Chi square testing did not reveal any significant changes in the nucleotide exchange pattern (p<0.01). C) Relative contributions of A/T mutations, G/C transversions and G/C transitions in the different mouse strains. Values are expressed as the percentage of total mutations.

Figure 5. CSR is not altered in Rad1 ^K185R B cells.

WT (gray bars) and Rad1 ^K185R (black bars) B cells were tested for their ability to switch to either IgG3 or IgG1 by stimulation with LPS or LPS and IL-4, respectively. Data represent the mean and SD of individual B cell cultures from three independent mice. The results are representatives of two independent experiments.