Role of human Pso4 in mammalian DNA repair and association with terminal deoxynucleotidyl transferase

Abstract

Terminal deoxynucleotidyl transferase (TdT; EC 2.7.7.31) adds nucleotides to DNA ends generated during V(D)J recombination that are subsequently processed by proteins involved in general doublestrand break (DSB) repair pathways. We report an association between TdT and a 55-kDa protein in lymphoid cells. This protein, identified as hPso4, is a homolog of the protein encoded by the PS04͞PRP19 gene in Saccharomyces cerevisiae that has pleiotropic functions in DNA recombination and error-prone repair. Purified hPso4 binds double-stranded DNA in a sequence-nonspecific manner but does not bind single-stranded DNA. hPso4 protein is induced 15-to 30-fold in cells by ␥ radiation and chemical mutagens but not by UV treatment. Loss of hPso4 expression induced by siRNA results in accumulation of DSBs, apoptosis, and decreased cell survival after DNA damage. We conclude that hPso4 plays a major and previously undefined role in mammalian DNA DSB repair.double-strand breaks ͉ end joining C ells encounter genomic lesions in the form of double-strand breaks (DSBs). In mammalian cells, DSB repair (DSBR) is mediated either by the error-free homologous recombination (HR) or the more predominant error-prone nonhomologous end joining (NHEJ) pathways (1). Agents that induce other types of DNA lesions, such as DNA interstrand crosslinks and adducts that proceed via DSB intermediates, are thought to invoke more than one type of DNA repair, including HR, nucleotide excision repair, and, to a lesser extent, the NHEJ pathway (2). The NHEJ repair proteins Ku70͞86, DNA PKcs, and Xrcc4͞ligase IV are ubiquitously expressed and function both in generalized DSBR and in the repair of ends generated during V(D)J recombination, a programmed gene rearrangement process that leads to the assembly of Ig and T cell receptors (3). Although these proteins seem sufficient for end joining in vitro, recent studies suggest the requirement of additional unknown factors for end joining in vivo (3, 4). One such recently identified human protein is artemis, mutations in which result in ionizing radiation sensitivity and severe combined immunodeficiency (5). Artemis protein has an inherent 5Ј to 3Ј singlestrand specific exonuclease activity. However, its association with and phosphorylation by DNA PKcs alter its activity to enable endonucleolytic processing of overhangs and cleavage of hairpin substrates such as those generated during V(D)J recombination (6).Terminal deoxynucleotidyl transferase (TdT; EC 2.7.7.31), a template-independent DNA polymerase, is unique in its expression and function, because it is expressed only in lymphoid cells undergoing V(D)J recombination (3) and has the ability to add nucleotides to the recombining V, (D), and J coding ends, thereby promoting antibody and receptor diversity (7). Our earlier studies revealed the association of TdT with the NHEJ protein Ku via the TdT BRCA1 C terminus (BRCT) region (8). Experiments performed with Ku86-deficient mice and cell lines have indeed demonstrated an absolute requirement of Ku86 for TdT-med...

The Prp19/Pso4 core complex undergoes ubiquitylation and structural alterations in response to DNA damage

“…Previous reported findings including ours have indicated that Prp19 plays a direct role in DNA repair processing [1][2][3][4]20,21], although the nature of this role remains undefined. To examine whether hPrp19 self-association is affected by exposure of cells to DNA damaging agents, we expressed GFP-hPrp19, and treated the transfected cells with methyl-methane sulfonate (MMS).…”

“…However, an analysis of splicing of the intron containing RAD14 gene in the pso4-1 mutant showed that the contribution of Prp19/Pso4 in the repair of UV damage is independent of RAD14 pre-mRNA processing [19]. Furthermore, the human protein has also been shown to interact with terminal deoxynucleotidyl transferase, and to be involved in mediating cell survival after DNA damage [20]. In addition, we have recently demonstrated a biochemical role for the core complex in processing of ICLs in vitro, and showed a direct physical interaction between Cdc5L and WRN [21].…”

Using peripheral blood mononuclear cells to determine proteome profiles in human cardiac failure

“…In PBMC from CHF patients we also detected PRP19/ PSO4, another stress response protein which is not normally expressed in PBMC, but which has been shown to be induced following DNA damage in other cell systems [26]. CHF induced expression of this protein may reflect an attempt to increase cell survival, since in different tumour cell lines (HL60, HeLa, Jurkat, Molt4) abolition of this protein by RNA interference was associated with impaired ability to survive DNA damage [26].…”

“…Protein N. 16 was identified as NADH-ubiquinone oxidoreductase, a multiprotein complex located in the inner mitochondrial membrane involved in the transport of electrons from NADH to ubiquinone [25]. Protein N. 18 was identified as PRP19/PSO4, a ubiquitary protein involved in stress response and DNA repair, which is induced 15 to 30-fold in cells by gamma radiation and chemical mutagens [26]. Loss of hPRP19/PSO4 expression induced by siRNA results in accumulation of double-strand breaks, apoptosis, and decreased cell survival after DNA damage [26].…”

“…Protein N. 18 was identified as PRP19/PSO4, a ubiquitary protein involved in stress response and DNA repair, which is induced 15 to 30-fold in cells by gamma radiation and chemical mutagens [26]. Loss of hPRP19/PSO4 expression induced by siRNA results in accumulation of double-strand breaks, apoptosis, and decreased cell survival after DNA damage [26].…”