Work

563. Analysis of DNA Repair Pathway Choice Upon Induction of Double-Strand Breaks by Engineered Nuclease

Year: 2016

Type: article

Abstract: DNA double-strand breaks (DSBs) are among the most dangerous classes of DNA damage. Cells have evolved complex DNA damage response pathways to repair DSBs. In mammalian cells, two major and mechanistically distinct DSB repair pathways are non-homologous end-joining (NHEJ) and homologous recombination (HR). NHEJ and HR can be regulated by multiple factors including position in the cell cycle. For single-copy loci, HR is thought to be restricted to late S/G2 phase, based on the need for a sister chromatid as a repair template. It remains unclear whether this is also true for multi-copy loci. Here, we have investigated DSB pathway choice by developing multi-copy and single-copy DSB reporter human cell systems. Using these systems we initiated a DSB with a specific zinc finger nuclease and then visualized DSB repair as it relates to cell cycle phase. We show that both the NHEJ and HR pathways can simultaneously repair DSBs at the reporter locus. The frequency of recruitment of the HR repair protein Rad51 is higher with the multi-copy DSB substrate than with the single-copy. This suggests that, availability of a homologous template near the DSB repair site influences repair pathway choice. Consistent with this, molecular analysis of repair at the single-copy reporter by SMRT sequencing revealed an increase in gene editing frequency by HR with increasing amounts of transfected exogenous donor template. Together, results indicate that HR can occur in G1 phase of the cell cycle if donor template is available for repair. Our findings suggest a competition between the HR and NHEJ repair machineries throughout interphase of the cell cycle, which could be a critical factor for the stability of mammalian genomes that are highly enriched with repeat sequences. Insights from these studies will help in understanding genomic instability disorders and to improve gene therapy approaches.

Source: Molecular Therapy

DOI: 10.1016/s1525-0016(16)33371-8

Authors: R. Ileng Kumaran, Jingjing Li, William S. Dynan, Matthew H. Porteus, David L. Spector

Institutions: Cold Spring Harbor Laboratory, Emory University, Stanford Medicine, Stanford University

Language: English

Topic: DNA Repair Mechanisms

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

SDG: Good health and well-being

Open Access status: hybrid

Estimated APC paid: $4,500