What is Dr GFP assay?
DR-GFP is composed of two differentially mutated green fluorescent protein (GFP) genes oriented as direct repeats (hence, “DR”): the upstream repeat contains the recognition site for the rare-cutting I-SceI endonuclease and the downstream repeat is a 5′ and 3′ truncated GFP fragment.
Can your body repair DNA?
Most damage to DNA is repaired by removal of the damaged bases followed by resynthesis of the excised region. Some lesions in DNA, however, can be repaired by direct reversal of the damage, which may be a more efficient way of dealing with specific types of DNA damage that occur frequently.
What enzymes are involved in homologous recombination?
Most recombination events in E. coli also require the RecBCD enzyme, which is a complex of three proteins (RecB, C, and D). The properties of RecBCD are consistent with the hypothesis that it initiates recombination by providing the single-stranded DNA to which RecA binds.
What triggers homologous recombination?
Meiosis-specific proteins In addition to research on Dmc1, studies on the Spo11 protein have provided information on the origins of meiotic recombination. Spo11, a type II topoisomerase, can initiate homologous recombination in meiosis by making targeted double-strand breaks in DNA.
Why is NHEJ used more than HR?
Nevertheless, when NHEJ and HR are compared across multiple integration sites, NHEJ is more efficient than HR. In unsynchronized proliferating cell populations, NHEJ of compatible DNA ends is twice as efficient as NHEJ of incompatible DNA ends, and NHEJ of incompatible DNA ends is three times more efficient than HR.
What are steps in the Holliday model?
The steps in the Holliday Model are illustrated in Figure 8.6.
- Two homologous chromosomes, each composed of duplex DNA, are paired with similar sequences adjacent to each other.
- An endonuclease nicks at corresponding regions of homologous strands of the paired duplexes.
Can GFP-based assays measure cnhej and non-canonical NHEJ?
Similar GFP-based assays have been developed to measure both cNHEJ and non-canonical NHEJ in cells. Most of these systems are based on two adjacent I-SceI sites, without a downstream homology template.
How long does it take to complete a HR assay?
A HR assay using DR-GFP takes only two days, whilst A HR assay using SCneo takes more than a week (time for neo selection). 4. Data from the DR-GFP assay vary substantially in individual experiments.
Can the Dr-GFP assay be integrated into U2OS cells?
The DR-GFP assay has been integrated into U2OS cells previously, and high levels of HR activity have been reported in this cell line ( 36 ), which we refer to as the U2OS DR cell line.
How does Dr-GFP work?
The DR-GFP reporter consists of two tandem, inactive copies of the GFP gene, which can be integrated into the cellular genome or expressed transiently. The first GFP copy (SceGFP) has a promoter and contains the I-SceI restriction site with an in-frame stop codon and the second copy (iGFP) harbours truncations at both ends [8, 9].