Understanding Site-Specific Recombination: Key Concepts and
School
The University of Hong Kong**We aren't endorsed by this school
Course
BBMS 2007
Subject
Biology
Date
Dec 10, 2024
Pages
8
Uploaded by lokman03
BBMS2007Recombination - Tutorial
Q1There are two site-specific recombination sites on one DNA molecule as shown above. Assume that the recombinase cleaves one nucleotide away from the recombinase binding site:1) What is the sequence of the recombinase binding site?2) What is the core sequence of the site-specific recombination site?3) What are the possible outcomes of the site-specific recombination?
ACCATAGTTTATT AAGTACTC AATAAACTATGGTSite-Specific Recombination Siteinverted repeatscore region
ExcisionInversion
Q2A 8.5-kb linear plasmid, pBMS, is introduced into yeast. The ends of the linearized plasmid are homologous to the his3 gene from yeast (Figure 1). After transformation, genomic DNA is extracted from the yeast and digested with EcoRI. The resulting DNA fragments are analysed by hybridization with a probe complementary to the his3 gene (see Orr-Weaver TL and Szostak JW (1983) PNAS 80:4417-21).Assuming that one copy of the plasmid is integrated into the yeast chromosome by homologous recombination, what are the expected sizes of DNA fragments detected by the probe? Please explain your prediction using the double-strand break repair model for recombination.Figure 1: Map of plasmid pBMS and the chromosome region around his3 gene.Homologous DNA on plasmid and chromosome.E: EcoRI site.
5kb9.2kbAssuming one copy of the repaired duplex is integrated (after resolution of the Holliday junctions). After digestion with EcoRI (E), a 5kb and a 9.2kb DNA fragment containing the his3gene can be detected.
Double-strand break repair model of recombinationCraig et al (2010) Molecular Biology. OUP
The same as:↓Remember we are using a gapped duplex!The gapped duplex is repaired and integrated into the chromosome