Joining of linear plasmid DNA is reduced and error-prone in Bloom's syndrome cells.

Abstract

A linearized, replicating, shuttle vector plasmid, pZ189, was used to measure in vivo DNA joining ability of cells from patients with the cancer‐prone, immunodeficient, chromosome breakage disorder, Bloom's syndrome (BS). The BS cell lines we studied were reported to contain reduced in vitro activity of DNA ligase I. We assessed in vivo joining ability by transfecting linear plasmids with overlapping or blunt ends (produced by EcoRI or StuI) into BS and normal fibroblast or lymphoblast host cells and measuring the amount of re‐joined, replicated plasmids by their ability to transform bacteria. With plasmids having either overlapping or blunt ends we found a 1.3‐ to 3‐fold lower (P less than 0.05) joining efficiency in BS cells than in the normal cells. The mutation frequency of the recovered plasmids was measured by screening for function of the suppressor tRNA contained in pZ189, for plasmid size, for presence of restriction sites, or by DNA sequencing. The spontaneous mutation frequency with the circular plasmid was 0.05‐0.08% with both BS cell lines, values 2‐ to 21‐fold higher (P less than 0.03) than with the normal cell lines. The mutation frequency with the linear plasmid passaged through both BS cell lines was 21‐52%, values 1.4‐ to 5.4‐fold higher (P less than 0.001) than with the normal lines. Detailed analysis of 210 recovered plasmids revealed an increase (P less than or equal to 0.001) in deletions, insertions or complex mutations at the joining sites, and in point mutations with the EcoRI cut plasmid with the BS cells in comparison to the normal cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Fidelity of Primate Cell Repair of a Double-strand Break within a (CTG)·(CAG) Tract

“…An assessment of stability was then made through electrophoretic comparison of the BamHI-HindIII and BamHI-BamHI fragments to the starting material. As previously observed, several rescued plasmids contained insertions of either genomic or plasmid DNA (27) and yielded aberrant restriction digestion patterns. The occurrence of these recombinants did not correlate with either repeat tract length or the presence of slipped structures (data not shown).…”

“…This is the first time this phenomenon has been observed for a DSB located within a repeat tract in primate cells. Similar double-strand break repair assays that have not concerned repeating sequences have also found deletions to be the most common result of improper DSB repair (27,32). It has been proposed that this bias toward deletions is likely a result of fraying/ unwinding at the two free ends of the double-strand break, thereby becoming suitable substrates for repair/recombination enzymes (33).…”

“…This is essentially a modification of a previously described assay for the fidelity of DNA ligation and DSB repair (27). Linear SV40 templates are incapable of DNA replication, and only those templates that are re-circularized by DSB repair are capable of primate cell-mediated replication.…”

“…In this study, we have used an established primate DSB repair system (27) to investigate the fidelity of repair of a DSB within a (CTG)⅐(CAG) repeat tract. Following repair of the DSB substrates in primate cells, products were characterized for sequence alterations within the repeat tract length and the flanking sequence.…”

Drosophila mus301/spindle-C Encodes a Helicase With an Essential Role in Double-Strand DNA Break Repair and Meiotic Progression

“…For instance, Bloom's syndrome is a rare disorder in humans caused by mutations in the RecQ helicase BLM that results in a predisposition to cancers of all types (Hickson 2003). Evidence for a role of this helicase in DSB metabolism comes from its involvement in homologous recombination-dependent repair of damaged replication forks (Wu and Hickson 2003) and from the fact that mending of dsDNA breaks in the absence of BLM results in defective products with large deletions (Runger and Kraemer 1989;Gaymes et al 2002). The ortholog of BLM in Drosophila is encoded by mus309 (Kusano et al 2001), a gene identified in a screen for hypersensitivity to chemical DNA-damaging agents such as nitrogen mustard, a mutagen that induces interstrand DNA crosslinks, and MMS (Boyd et al 1981).…”