Specific complex formation between proteins encoded by the yeast DNA repair and recombination genes RAD1 and RAD10.

Abstract

The RADI and RADIO genes of Saccharomyces cerevisiae are required for excision repair of ultraviolet light-damaged DNA, and they also function in a mitotic recombination pathway that is distinct from the double-strandbreak recombination pathway controlled by RAD52. Here, we show that the RAD1 and RADi1 proteins are complexed with each other in vivo. Immunoprecipitation of yeast cell extracts with either anti-RAD1 antibody or anti-RAD10 antibody coprecipitated quantitative amounts of both RAD1 and RADiO proteins. The level of coprecipitable RAD1 and RADiO increased when both proteins were overproduced together, but not if only one of the proteins was overproduced. The RAD1/RAD10 complex is highly stable, being refractory to 1 M NaCl and to low concentrations of SDS. By hydroxylamine mutagenesis, we have identified a radi mutant allele whose encoded protein fails to complex with RADiO. The interactiondefective radi mutant resembles the radi or radiO null mutant in defective DNA repair and recombination, implying that complex formation is essential for the expression of biological activities controlled by RADI and RADIO.In humans, the genetic disorder xeroderma pigmentosum (XP) causes extreme sensitivity to sunlight and XP individuals suffer from a high incidence of skin cancers. XP cells exhibit defects in the incision step of excision repair of DNA damaged by ultraviolet (UV) light. Cell fusion studies with skin fibroblasts from XP patients have identified seven XP complementation groups, A-G (1, 2). Eight complementation groups have been identified among UV-sensitive Chinese hamster ovary (CHO) cell lines, and mutants from five of these groups are incision-defective (3). Cross-complementation of UV sensitivity of mutant CHO cell lines by human DNA has led to the isolation of the ERCCI, ERCC2, and ERCC3 genes (4-6), and ERCC3 and XP-B represent the same gene (6). Recently, the XPAC cDNA was cloned by using as hybridization probe a mouse gene that corrects the excision repair defect of XP-A cell lines (7).The yeast Saccharomyces cerevisiae resembles humans in the genetic complexity of excision repair, and at least 10 genes-RADJ, RAD2, RAD3, RAD4, RADIO, RADI4, RAD7, RAD16, RAD23, and MMSJ9-are involved in this process. The first 6 of these genes are indispensable for the incision step of repair and they very likely encode proteins that collectively mediate the recognition and endonucleolytic scission of damaged DNA (8, 9). Characterization ofgenomic deletion mutations has revealed a role of some of these genes in cellular processes unrelated to excision repair. Specifically, RAD3 is also essential for cell viability (10, 11), and RADI and RADIO function in a mitotic recombination pathway that is distinct from the RAD52 double-strand-break recombination pathway (12).The proteins encoded by the human ERCCI, ERCC2, and XPAC genes share remarkable homology with the products of the yeast RADIO, RAD3, and RADI4 genes, respectively (4,5,9), and a yeast gene homologous to the human ERCC3 (XPBC) gene has also been ...

DNA Repair Defects Channel Interstrand DNA Cross-links into Alternate Recombinational and Error-prone Repair Pathways

“…DSB repair efficiency was unaffected by loss of RAD3 or RAD4 function but was lower in rad1 and rad10 strains (Table I). Rad1 and Rad10 form a heterodimeric protein that makes the 5Ј side incisions during the excision stage of NER (32,33). In addition, the Rad1⅐Rad10 complex acts in recombination to cleave at double strand-single strand junctions and functions in DSB-induced plasmid integration (34, 35) as we observe here (Table II).…”

Correction of xeroderma pigmentosum repair defect by basal transcription factor BTF2 (TFIIH).

“…Thus these results rule out the theoretical possibility raised by these and other remarkable observations with respect to the ERCC3 gene in the yeast and Drosophila systems, namely that the ERCC3 -NER connection could be indirect, i.e. ERCC3 might control the expression of one or more (real) NER genes without being involved itself in this process (Gulyas and Donahue, 1992;Mounkes et al, 1992;Schaeffer et al, 1993 (Schiestl and Prakash, 1990;Bailly et al, 1992;Bardwell et al, 1992;Biggerstaff et al, 1993;van Vuuren et al, 1993). Using the in vitro NER assay Wood and coworkers have shown that the replication factors PCNA and HSSB (RPA) also participate in the post-incision stages of the NER reaction (Coverley et al, 1992;Shivji et al, 1992) Serizawa et al, 1993a,b) and a helicase activity (Schaeffer et al, 1993 (Gerard et al, 1991).…”

Functional domains within FEN-1 and RAD2 define a family of structure-specific endonucleases: implications for nucleotide excision repair.

“…If this is true, it suggests that the S region is important for the repair activity of RAD2. As mentioned above, there is increasing evidence for the presence of a multiprotein repair complex involv-ing many of the RAD proteins (Bailly et al 1992;Feaver et al 1993;van Vuuren et al 1993;Bardwell et al 1994). One likely function of the S region, therefore, may be that it participates in protein-protein interactions with other RAD proteins.…”

“…Because RAD1, RAD2, RAD3, RADIO, and RAD25 show no preference for damaged DNA, it is thought that they are localized to the site of damage through interactions with a damage recognition protein such as RAD14. Experimental evidence for interactions among RAD proteins now exists (Sung et al 1987;Bailly et al 1992;Bardwell et al 1992Bardwell et al , 1994Feaver et al 1993;van Vuuren et al 1993). However, it is not clear how these enzymes work together to incise and remove the oligonucleotide excision tract containing the damaged bases.…”