Nonhomologous DNA End Joining in Cell-Free Extracts

Figure 3

Comparison of efficiencies of NHEJ in rat and mice testis. Cell-free extract was prepared from age-matched rat and mice testes and protein profile was normalized between both animals. 5 𝜇g of protein was incubated with 4 nM of 5^-end labelled with [𝛾⁻³²P] DNA containing both compatible and blunt termini against each lane (a) which are represented schematically along with buffer containing 30 mM HEPES-KOH, pH 7.9, 7.5 mM MgCl₂, 1 mM DTT, 2 mM ATP, 50 𝜇M dNTPs, and 0.1 𝜇g BSA. End joining reaction using (b) compatible end and (c) blunt end DNA. Lane 1 shows negative control that contains substrate alone, Lane 2 shows heat-inactivated control which is mice testis cell-free extract-boiled for 10 min and used for the reaction, and Lanes 3 and 4 are the end joining reactions with cell-free extract from mice testis. Lane 5 is the heat-inactivated control which is boiled rat testicular extract as described previously. Lanes 6 and 7 are the end joining reactions with rat testicular cell-free extracts. “M” indicates 5^-end-labelled 50 bp ladder. The efficiency of joining is similar in both mice and rat testicular extracts. Different types of end-joined products formed are indicated.