The USP1-UAF1 complex interacts with RAD51AP1 to promote homologous recombination repair

<p>USP1 deubiquitinating enzyme and its stoichiometric binding partner UAF1 play an essential role in promoting DNA homologous recombination (HR) repair in response to various types of DNA damaging agents. Deubiquitination of FANCD2 may be attributed to the key role of USP1-UAF1 complex in regulating HR repair, however whether USP1-UAF1 promotes HR repair independently of FANCD2 deubiquitination is not known. Here we show evidence that the USP1-UAF1 complex has a FANCD2-independent function in promoting HR repair. Proteomic search of UAF1-interacting proteins revealed that UAF1 associates with RAD51AP1, a RAD51-interacting protein implicated in HR repair. We show that UAF1 mediates the interaction between USP1 and RAD51AP1, and that depletion of USP1 or UAF1 led to a decreased stability of RAD51AP1. Protein interaction mapping analysis identified some key residues within RAD51AP1 required for interacting with the USP1-UAF1 complex. Cells expressing the UAF1 interaction-deficient mutant of RAD51AP1 show increased chromosomal aberrations in response to Mitomycin C treatment. Moreover, similar to the RAD51AP1 depleted cells, the cells expressing UAF1-interaction deficient RAD51AP1 display persistent RAD51 foci following DNA damage exposure, indicating that these factors regulate a later step during the HR repair. These data altogether suggest that the USP1-UAF1 complex promotes HR repair via multiple mechanisms: through FANCD2 deubiquitination, as well as by interacting with RAD51AP1.</p

FACS Analysis of Ig Hypermutation Activity

<p>(A) FACS profiles of representative subclones derived from a sIgM (+) cell after staining with a monoclonal antibody to IgM. (B) The average percentages of events falling into sIgM (−) gates based on the measurement of 24 subclones are shown by graph.</p

Colony Survival Curves after Exposure to DNA-Damaging Agents

<p>The values of DNA damaging agents, which give 10% cell viability, are also summarized (<i>D</i>₁₀ values).</p

Mutation Spectrum

<p>(A) Frequencies of particular nucleotide substitutions within light-chain gene. (B) A graphical view showing the frequencies of different types of mutations per hundred sequences.</p

Site-Directed Mutagenesis of the <i>PCNA</i> Locus

<div><p>(A) Alignment of the human, mouse, chicken, <i>Schizosaccharomyces pombe,</i> and S. cerevisiae PCNA amino acid sequences. Amino acid 164 serving as the attachment site for ubiquitination in S. cerevisiae is marked by an asterisk.</p> <p>(B) A physical map of the <i>PCNA</i> locus and the <i>PCNA</i> mutagenesis construct, <i>pPcna^K164RBsr</i>. The targeting strategy of <i>PCNA</i> locus and the genealogy of the mutant clones are shown below and to the right, respectively.</p> <p>(C) Sequence chromatographs covering the <i>PCNA</i> codon 164 which was changed from AAA in the <i>AID^R</i>ψ<i>V⁻</i> clone to AGA in the <i>PCNA^K164R/K164R</i> clone.</p></div

Ubiquitination and SUMOylation of PCNA

<div><p>(A) Cells were treated with or without MMS and were analyzed by immunoblotting using an monoclonal antibody to PCNA. The asterisk denotes a band reactive with PCNA antibodies, possibly corresponding to a PCNA modification independent of K164 and Rad18.</p> <p>(B) Analysis of clones stably transfected with His-tagged ubiquitin or SUMO-1 expression vectors. Whole cell lysates (left) and lysates after NiNTA chromatography (right) are shown. The positions expected for unmodified, mono-ubiquitinated, and SUMOylated PCNA are indicated by lines. Due to the low residual level of PCNA ubiquitination in the <i>RAD18</i> mutant, this modification could not be detected by pull-downs. The bands at the bottom represent low levels of unmodified PCNA unspecifically bound to the beads.</p> <p>(C) Quantification of mono-ubiquitinated and SUMOylated PCNA, histone H3, and AID by immunoblotting. Cells were treated with or without MMS, and immunoblotted using monoclonal antibodies to PCNA (left upper), histone H3 (left middle), and AID (left lower). The values for mono-ubiquitinated and SUMOylated PCNA given in the right hand graphs were calculated as described in the Materials and Methods.</p></div

Ig Hypermutation of the <i>PCNA^K164R/K164R</i> Clone

<p>Ig light-chain sequence variation in the <i>PCNA^K164R/K164R</i> clone. All sequence differences in the region from the first intron to the J-C intron are shown relative to the rearranged light-chain consensus sequence of the <i>AID^R</i>ψ<i>V⁻</i> precursor clone. The position of complementary determining regions CDR1, CDR2, and CDR3 and that of Jλ are indicated.</p