Differential Requirement for SUB1 in Chromosomal and Plasmid Double-Strand DNA Break Repair

Abstract

Non homologous end joining (NHEJ) is an important process that repairs double strand DNA breaks (DSBs) in eukaryotic cells. Cells defective in NHEJ are unable to join chromosomal breaks. Two different NHEJ assays are typically used to determine the efficiency of NHEJ. One requires NHEJ of linearized plasmid DNA transformed into the test organism; the other requires NHEJ of a single chromosomal break induced either by HO endonuclease or the I-SceI restriction enzyme. These two assays are generally considered equivalent and rely on the same set of NHEJ genes. PC4 is an abundant DNA binding protein that has been suggested to stimulate NHEJ. Here we tested the role of PC4's yeast homolog SUB1 in repair of DNA double strand breaks using different assays. We found SUB1 is required for NHEJ repair of DSBs in plasmid DNA, but not in chromosomal DNA. Our results suggest that these two assays, while similar are not equivalent and that repair of plasmid DNA requires additional factor(s) that are not required for NHEJ repair of chromosomal double-strand DNA breaks. Possible roles for Sub1 proteins in NHEJ of plasmid DNA are discussed.

The Sub1 nuclear protein protects DNA from oxidative damage

“…Sub1 family of proteins have been proposed to function in many processes: they are recruited to transcription complexes [8, 9], DNA repair complexes [11, 21], double-strand DNA breaks [22, 23], and replication complexes [24, 25]. Since Sub1 binds to DNA with a strong preference for unpaired double-stranded DNA regions and single-stranded DNA[9,10, 11, 20], and our results indicate that it prevents oxidative DNA damage, this suggests a common function for this family of proteins in all of these processes.…”

The non-homologous end-joining pathway of S. cerevisiae works effectively in G1-phase cells, and religates cognate ends correctly and non-randomly

“…These experiments are done in the absence of a second, uncuttable copy of MAT , so that homologous recombination cannot be used for repair. In these experiments, unlike the gamma ray experiments cited above, all workers seem to find that a defect in NHEJ gives a high degree of sensitivity (Bonetti et al 2013; Mahaney et al 2014; Shim et al 2005; Yu and Volkert 2013). Breaks made by HO, unlike breaks made by gamma rays, leave a specific structure in the DNA that may be favorable for ligation.…”

Identification of the ssDNA-binding protein of bacteriophage T5

“…14 Apart from its well-established activities in eukaryotic transcription regulation, which seem to depend to a large extent on protein-protein interactions, 12 a number of recent reports have suggested that PC4 plays additional roles in replication, recombination and repair (3R) via the ssDNA-binding capacity of its core domain. [15][16][17][18][19] Interestingly, the gene encoding the PC4 homolog in T5-like bacteriophages is highly conserved and invariably occupies the same position within the gene array of the T5 replication module. 10 This is a strong indication that the PC4 homolog plays a critical role in phage DNA replication.…”

“…20 The unwound DNA in the complex thus seems poised for sequence-specific recognition of its bases, the purpose of which may well be to facilitate processes such as DNA repair and recombination. Although activity of eukaryotic PC4 in homologous recombination remains to be investigated, recent reports show that the protein is able to stimulate double-stranded break repair by nonhomologous end-joining (NHEJ), 18,19 presumably through a mechanism that depends on interaction with DNA. Thus, it seems tempting to speculate that the RDR mechanism of T5 is evolutionarily related to PC4-mediated DNA repair in eukaryotes, just like RDR in T4 is highly similar to eukaryotic repair mechanisms that involve Rad51.…”