Inhibition of Homologous Recombination by the PCNA-Interacting Protein PARI

Abstract

Summary Inappropriate homologous recombination (HR) causes genomic instability and cancer. In yeast, the UvrD family helicase Srs2 is recruited to sites of DNA replication by SUMO-modified PCNA, where it acts to restrict HR by disassembling toxic RAD51 nucleofilaments. How human cells control recombination at replication forks is unknown. Here, we report that the protein PARI, containing a UvrD-like helicase domain, is a PCNA interacting partner, required for preservation of genome stability in human and DT40 chicken cells. Using cell-based and biochemical assays, we show that PARI restricts unscheduled recombination by interfering with the formation of RAD51-DNA HR structures. Finally, we show that PARI knockdown suppresses the genomic instability of Fanconi Anemia/BRCA pathway-deficient cells. Thus, we propose that PARI is a long sought-after factor that suppresses inappropriate recombination events at mammalian replication forks.

Regulation of Recombination and Genomic Maintenance

“…Also, direct biochemical evidence supports the similarity between Srs2 and FBH1, showing that human FBH1 displays translocase-dependent disruption of RAD51-ssDNA filaments (Simandlova et al 2013). Another UvrD-helicase domaincontaining protein, PARI, has been found to destabilize RAD51-ssDNA filaments and, like Srs2, interacts with PCNA (Papouli et al 2005;Pfander et al 2005;Moldovan et al 2012). Although PARI itself lacks translocase activity and appears to act by competitive binding with RAD51 (Moldovan et al 2012), it may act in conjunction with another motor protein.…”

“…Another UvrD-helicase domaincontaining protein, PARI, has been found to destabilize RAD51-ssDNA filaments and, like Srs2, interacts with PCNA (Papouli et al 2005;Pfander et al 2005;Moldovan et al 2012). Although PARI itself lacks translocase activity and appears to act by competitive binding with RAD51 (Moldovan et al 2012), it may act in conjunction with another motor protein. Human BLM has been shown to dissociate RAD51 from ssDNA, specifically after ATP turnover was triggered by the chelation of Ca 2þ , which renders the filament nonfunctional for homology search and DNA-strand invasion (Bugreev et al 2007).…”

DNA damage tolerance pathway involving DNA polymerase ι and the tumor suppressor p53 regulates DNA replication fork progression

“…p53 inhibits the helicase and the branch-migrating activities of Bloom syndrome protein (BLM) and Werner syndrome protein (WRN) helicases, which are involved in the regulation of HR and in the bypass of replication barriers (32,33), whereas RAD51 and breast cancer 2 (BRCA2) are involved in HR-dependent postreplication repair (34,35). Proliferating cell nuclear antigen (PCNA)-associated recombination inhibitor (PARI) associates with DNA damage sites via SUMOylated PCNA and blocks recombination by inhibition of RAD51-DNA filament formation (36). Surprisingly, BLM, WRN, RAD51, BRCA2, and PARI were not required for the p53(WT)-mediated stimulation of recombination, hence suggesting an insignificant contribution of any RAD51-dependent pathway to this recombination event (Fig.…”

“…Alternative routes mediating a replicative lesion bypass are known to be triggered by PCNA ubiquitination, namely, TLS or template switching. The latter can further be subdivided into strand invasion or fork reversal mechanisms (36,60). The stimulation of recombination by p53(WT) was fully epistatic with the E3-ubiquitin ligase RAD18, which monoubiquitinates PCNA in conjunction with the E2-conjugating enzyme RAD6 (60), and with POLι, which recognizes monoubiquitinated PCNA (48).…”

FBH1 influences DNA replication fork stability and homologous recombination through ubiquitylation of RAD51

“…Many of these belong to the UvrD family of proteins, such as Srs2 in yeast and PARI in mammals [12][13][14] . Mammalian cells express another UvrD family member, FBH1 (F-box DNA helicase 1), which has been reported previously to suppress HR 15 .…”