Active transcription promotes single-stranded oligonucleotide mediated gene repair.
The lambda-Red-mediated recombination has been exploited as an efficient means for DNA manipulation. We previously reported that replication plays a pivotal role during this process. Replication direction dictates strand bias, such that single stranded oligonucleotide (SSO) with sequence corresponding to the nascent lagging strand directs higher levels of recombinant formation compared to its complementary SSO. In addition, the Escherichia coli methyl-directed mismatch repair system impedes efficient SSO-mediated site-specific gene repair. However, the role of transcription in determining strand bias and recombination efficiency is unclear. To address the potential role of transcriptional processes, we constructed plasmid substrates that harbor a mutant antibiotic reporter under the control of an inducible promoter. We found that transcription activation can promote recombinant formation to more than 10-folds whilst it has negligible effect on strand bias. Our findings provide evidence for a role of transcription in SSO-mediated gene repair process.
Huen
Michael S Y
MS
Department of Biochemistry, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong SAR, China.
Lu
Lin-Yu
LY
Liu
De-Pei
DP
Huang
Jian-Dong
JD
D004249:DNA Damage / Q000235:genetics*; D004260:DNA Repair / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D011995:Recombination, Genetic / Q000235:genetics*; D052416:Targeted Gene Repair / Q000379:methods*; D015533:Transcriptional Activation / Q000235:genetics*
D004269:DNA, Bacterial